Methods and systems for modifying motility of engineered multicellular organisms

Abstract

Disclosed are methods and systems for modifying the motility of engineered multicellular organisms. The engineered multicellular organisms include an aggregate of cells, e.g., ciliated cells. The methods may include exposing the engineered multicellular organism to an acoustic stimulation, e.g., sound at a frequency of about 5 Hz to about 1000 Hz.

Description

METHODS AND SYSTEMS FOR MODIFYING MOTILITY OF ENGINEERED MULTICELLULAR ORGANISMSCROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims priority to U. S. Provisional Patent Application No.63 / 717,707 that was filed November 7, 2024, and U. S. Provisional Patent Application No.63 / 773,732, that was filed March 18, 2025, the entire contents of each which are hereby incorporated by reference.SEQUENCE LISTING

[0002] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “166118 01579. xml” which is 1,771 bytes in size and was created on October 29, 2025. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] Not applicable.BACKGROUND

[0004] The standard research paradigm of developmental and synthetic biology seeks to discover how gene expression drives specific anatomical and behavioral outcomes. However, it is also well-understood that transcriptional machinery itself is sensitive to external cues. Rapid changes of gene expression can be induced by exposures to genetic, biochemical, biomechanical, bioelectrical, and materials-mediated influences. On a much longer timescale, the properties of gene expression profiles in vivo are thought to be determined by evolutionary selection, optimizing fitness to specific environment, life history and lived experiences. Here, we sought novel aspects of the responsiveness of gene expression, in a setting in which a non-canonical multicellular, functional form is achieved without transgenes, chemical signals, foreign nanomaterials, or other added influence.

[0005] A fascinating set of questions concerns the origins of species-specific transcriptomic profiles, normally shaped by eons of selection over the functionality of those forms in a specific environmental context. Unique aspects of these questions can be addressed in synthetic systems in which the entities have not had a history of selection in their current multicellular embodiment. Numerous synthetic life forms have recently been produced; however, many questions remain to be answered, via analysis of transcriptomic profiling in these synthetic living configurations.SUMMARY

[0006] Disclosed are methods and systems for modifying the motility of engineered multicellular organisms. The disclosed methods and systems comprise exposing engineered multicellular organisms to an acoustic stimulation.

[0007] In an aspect of the current disclosure, methods of modifying motility of an engineered multicellular organism are provided. In some embodiments, the methods comprise (a) exposing the engineered multicellular organism to an acoustic stimulation.

[0008] In an aspect of the current disclosure, systems for modifying motility of an engineered multicellular organism are provided. In some embodiments, the systems comprise (a) an acoustic stimulator; and (b) an engineered multicellular organism.

[0009] In an aspect of the current disclosure, methods comprising emitting sound from the acoustic stimulator of a system are provided. In some embodiments, the system comprises (a) an acoustic stimulator; and (b) an engineered multicellular organism.

[0010] In an aspect of the current disclosure, methods of conferring sound sensitivity to an engineered multicellular organism are provided. In some embodiments, the methods comprise introducing a heterologous polynucleotide to the cells of the engineered multicellular organism, wherein the heterologous polynucleotide comprises a sequence encoding a polypeptide, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.BRIEF DESCRIPTION OF THE FIGURES

[0011] FIGs. 1A and IB show major transcriptional changes in Xenobots compared to age-matched stage 35 / 36 Xenopus embryos. (A) A live Xenobot and schematic of the experimental setup with three replicates for each Xenobot pool and age-matched stage 35 / 36 Xenopus embryo pool with each replicate having fifty and ten samples respectively. (B) Volcano plot for differential expression between Xenobots and age-matched stage 35 / 36 Xenopus embryos. Significantly changed genes are highlighted in red.

[0012] FIGs. 2A, 2B, 2C, and 2D show inter-individual variation in gene counts is greater in Xenobots than in age-matched Xenopus embryos for most genes. (A) Schematic of the analysis method. All genes were ranked by their mean count value across all 6 data pools, after which the standard deviations of the gene counts amongst individual Xenobots and individual age-matched Xenopus embryos were estimated (Est. GX and Est. oE) and used to calculate the coefficients of variation (CVs) for each (CVX and CVE). (B) Histograms of the normalized distributions of CVs for Xenobots and age-matched embryos genes. Xenobots have significantly higher CVs and wider CVs distribution than age-matched embryos (Wilcoxon rank sum test, p=0). Where the CVE distribution had a mean of 1.2537 and standard deviation of 0.9716, and the CVX distribution had a mean of 2.6115 and standard deviation of 1.3609. (C) Comparison of gene expression variation between Xenobots and age-matched embryos across expression levels. The genes for which CVX > CVE were noted, and of all the included genes, 96.06% had greater CVX than CVE. The gene list was then divided into 100 equal-size bins (percentiles) and the fraction of each bin for which CVX > CVE was plotted. Each bar represents one bin of genes ranked from lowest to highest gene counts, and its height indicates the fraction of genes in the bin for which CVX > CVE. The black line marks 0.5. For all bins, most genes in the bin had a greater CV for Xenobots than age-matched Xenopus embryos. The red line shows the overall fraction of genes for which CVX> CVE (0.9606), and the dark blue bins were found to be significantly different from this value (p<0.05), while light blue bins were not (p>0.05) according to a permutation test, indicating that the trend of increasing bin value with increasing gene count was not due to chance. (D) Xenobots top 10 most variable genes.

[0013] FIGs. 3A, 3B, and 3C show functional enrichment and network clustering analysis of high stringency transcripts uniquely upregulated in Xenobots compared to age-matched Xenopusembryos. (A) Functional enrichment analysis showing enrichment of 7 different biological categories. (B-C) Network clustering analysis identified 10 clusters (Supplemental Dataset 6) including cluster for sensory perception of sound and mechanical stimuli (B) and immune / stress response (C).

[0014] FIGs. 4A, 4B, 4C, 4D, 4E, 4F, and 4G show Xenobots respond to acoustic vibrations by changing motion behavior. (A) Experimental setup for exposure of subjects to 300Hz acoustic vibration and time lapse recording of motion behavior for 10 mins before exposure, 10 mins during exposure, and lOmins after exposure. (B-G) Time lapse recording of motion behavior of day 1 non-ciliated, non-motile Xenobots, age-matched stage 35 Xenopus embryos, and day 7 autonomously motile Xenobots along with motion tracking of their behavior and quantification of change in peak velocity from baseline during interval of 300Hz vibration stimulus. (B, D, and F) Representative image of day 1 non-motile Xenobots, age-matched stage 35 Xenopus embryos treated with tricaine to inhibit muscle movement while leaving cilia-based motion intact, and day 7 autonomously motile Xenobots, respectively. Scale bar = 5mm. (C, E, and G) Representative tracking of day 1 non-motile Xenobots, age-matched stage 35 embryos, and day 7 autonomously motile Xenobots respectively, across the time intervals of before, during and after 300Hz vibration stimulus. (H) Quantification of change in peak velocity (millimeters / min) between the time intervals of before and during 300Hz vibration stimulus. n>5, ns-non-significant, **-p<0.01, ****-p<0.0001, repeated measures One-Way ANOVA with Tukey’s multiple comparison test.

[0015] FIG. 5 shows that Xenobot transcripts are enriched in evolutionary older strata compared to control transcripts. Control transcripts are all the Xenopus genes expressed in epidermal progenitor cells + multiciliate cells + alpha and beta ionocyes+ goblet cells (3374). Phylostratigraphic analysis of controls transcripts, all Xenobot upregulated transcripts (1812) and transcripts uniquely upregulated in Xenobots (1450) showing Xenobot transcripts more enriched in Bilateria, Eumetazoa, and Metazoa compared to controls and the unique Xenobot upregulated transcripts more enriched than all Xenobot upregulated transcripts in these evolutionarily older strata.

[0016] FIGs. 6 A, 6B, 6C, 6D, 6E, and 6F show that no major transcriptional changes in stage 35 / 36 Xenopus embryos raised in 0.1X MMR and 0.75X MMR. (A) Schematic of the experimentalsetup with three replicates for each 0.1X MMR and 0.75X MMR rearing condition and each replicate having ten stage 35 / 36 embryos. (B) Principal component analysis plot of RNA-sequencing data from stage 35 / 36 Xenopus embryos raised in 0.1X MMR and 0.75X MMR showing no clustering. (C) Principal component analysis plot using surrogate variable adjusted data. (D) Histogram of p-value significance. If no genes are associated with phenotype the - value histogram is expected to be relatively flat. Also, proportion of true null hypothesis (non-significant genes) is shown (E) Histogram of FDR significance. If no genes are associated with phenotype all the FDRs will be near one. (F) Volcano plot for differential expression between embryos raised in 0.1X MMR and 0.75X MMR. Significantly changed genes are highlighted in red.

[0017] FIG. 7 shows a principal component analysis plot of RNA-sequencing data using surrogate variable adjusted data for Xenobots and age-matched stage 35 / 36 Xenopus embryos showing distinct separation of the two groups.

[0018] FIGs. 8A, 8B and 8C show a network clustering analysis of high stringency transcripts uniquely upregulated in Xenobots compared to age-matched Xenopus embryos. (A-C) Network clustering analysis identified 10 clusters (Supplemental Dataset 6) including clusters for cilia and cytoskeleton (A), Ketone metabolism and EGF / EGFR signal (B), and ECM / proliferation / multicellular organization (C).

[0019] FIGs. 9A, 9B, and 9C show Xenobots have relatively similar total cell numbers. (A) Schematic of the Xenobot total cell count pipeline. Xenobots were fixed, their axial dimensions measured, cut through the middle into two halves, stained with nuclear stain followed by imaging nuclei in the interior, counting and calculation to obtain total nuclei per Xenobot. (B) Representative image of stained nuclei in the interior of Xenobot. (C) Table showing actual nuclei per unit volume imaged and estimated total cell count in Xenobots.

[0020] FIGs. 10A, 10B, 10C, 10D, 10E, and 10F show Xenobots have overlapping cilia characteristics with age-matched embryos. Immunostaining for cilia in Xenobots and age-matched embryos. (A & B) Representative images at the base of cilia of multiciliated cells showing each cilium as a punctate spot (magenta arrows). (C) Quantification of number of cilia per multiciliated cell n=8, *-p=0.04, unpaired t-test. (D & E) Representative images of cilia length in multiciliatedcells (orange arrows). (F) Quantification of cilia length n>13, ns-non-significant, unpaired t-test. Data represented as mean + SD.

[0021] FIG. 11 shows a list of upregulated genes after klein tools epidermal sub.

[0022] FIG. 12 shows a list of Mesodermal -Endodermal and Axis patterning genes.DETAILED DESCRIPTION

[0023] The following discussion is presented to enable a person skilled in the art to make and use embodiments of the disclosure. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the generic principles herein can be applied to other embodiments and applications without departing from embodiments of the disclosure. Thus, embodiments of the disclosure are not intended to be limited to embodiments shown but are to be accorded the widest scope consistent with the principles and features disclosed herein. The following detailed description is to be read with reference to the figures. The figures, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of embodiments of the disclosure. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of embodiments of the disclosure.Methods of modifying the motility of engineered multicellular organisms

[0024] The inventors discovered that engineered multicellular organisms derived from xenopus embryos express a variety of transcripts associated with sensory perception of sound and mechanical stimuli (FIG. 3). The inventors demonstrated that exposing the engineered multicellular constructs to an acoustic stimulus modified the motility or behavior of the engineered multicellular organisms (FIGs. 4F and 4G) but did not modify the motility of age matched embryos (FIGs. 4D and 4E) or immature multicellular organisms that had not yet acquired motility (FIGs.4B and 4C).

[0025] The standard paradigm of developmental and synthetic biology focuses on gene expression driving changes in morphology. Here, we investigated the reverse relationship: would transcriptomes change if cell collectives acquired a novel morphogenetic and behavioralphenotype in the absence of genomic editing, transgenes, heterologous materials, or drugs? We investigated the effects of morphology and nascent emergent life history on gene expression in the basal (no engineering, no sculpting) form of Xenobots - autonomously motile constructs derived from Xenopus embryo ectodermal cell explants. We compared transcriptomes of these basal Xenobots with age-matched Xenopus embryos. Basal Xenobots showed significantly larger interindividual gene variability than age-matched embryos, suggesting increased exploration of the transcriptional space. We identified at least 537 (non-epidermal) transcripts uniquely upregulated in these Xenobots. Pathway analyses indicated transcriptomic shifts in the categories of motility machinery, multicellularity, stress and immune response, metabolism, and sensory perception of sound and mechanical stimuli. We experimentally confirmed that basal Xenobots respond to acoustic stimuli via observable changes in behavior, in ways that age-matched embryos do not. Phylostrati graphic analysis showed that the majority of transcriptomic shifts in the basal Xenobots were towards evolutionarily ancient transcripts and systems. Lastly, we found enrichment of thanatotranscriptomic genes, shedding light on the distinction between death of an organism and that of its cells. These data on the relationship between genotype and phenotype may have implications for evolution, biomedicine, and synthetic morphoengineering.

[0026] In an aspect of the current disclosure, methods of modifying motility of an engineered multicellular organism are provided. In some embodiments, the methods comprise (a) exposing the engineered multicellular organism to an acoustic stimulation.

[0027] As used herein, “engineered multicellular organism” comprises an aggregate of cells, e.g., ciliated cells. In some embodiments, the organisms move when the ciliated cells are actuated.

[0028] Referring now to FIGs. 4F and 4G, exposing engineered multicellular organisms, derived from xenopus embryos, to an acoustic stimulus altered the trajectory of the organisms. As used herein, “acoustic stimulation” comprises sound. The acoustic stimulation may comprise sound at about 5 Hz to about 1000 Hz, or any value or subrange therein, including the endpoints. Acoustic stimulation may comprise sound at a frequency of about 50 Hz to about 500 Hz, about 50 Hz to about 300 Hz, or about 300 Hz.

[0029] The engineered multicellular organisms may be exposed to the acoustic stimulation for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute,at least about 5 minutes, at least about 10 minutes, or at least about 10 hours, or any value therein, including the endpoints. The engineered multicellular organisms may be exposed to the acoustic stimulation for at least about 1 second and less than about 10 seconds, at least about 1 second and less than about 1 minute, at least about 1 second and less than about 10 minutes, or at least about 1 second and less than about 10 hours, or any value or subrange therein, including the endpoints.

[0030] The acoustic stimulation may be ceased for a period of time, e.g., at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours, or any value therein, including the endpoints. The acoustic stimulation may be resumed, e.g., for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours, or any value therein, including the endpoints, or for at least about 1 second and less than about 10 seconds, at least about 1 second and less than about 1 minute, at least about 1 second and less than about 10 minutes, or at least about 1 second and less than about 10 hours, or any value or subrange therein, including the endpoints.

[0031] The acoustic stimulation may be resumed, e.g., for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours, or any value therein, including the endpoints or for at least about 1 second and less than about 10 seconds, at least about 1 second and less than about 1 minute, at least about 1 second and less than about 10 minutes, or at least about 1 second and less than about 10 hours, or any value or subrange therein, including the endpoints. The acoustic stimulation may be initiated and ceased any number of times n, where n is an integer. The acoustic stimulation may be static, i.e., may comprise a single wavelength of sound, or a combination of wavelengths of sound, for the duration of stimulation, or the acoustic stimulation may be variable, i.e., may comprise a single changing wavelength of sound or a changing combination of wavelengths of sound.

[0032] Modifying the motility of the engineered multicellular organisms may be performed in a suitable environment, e.g., in an aquatic or aqueous environment, in an aquatic environment in a vessel, e.g., a dish, a tube, a well, a plate, a beaker, a flask, a tank, etc., in an organism (in vivo), on the surface of tissue of an organism, e.g., mucus membrane.

[0033] The engineered multicellular organisms may be genetically modified, e.g., the engineered multicellular organisms may comprise an antisense oligo or a morpholino. The engineered multicellular organisms may comprise a heterologous polynucleotide. As used herein, a “heterologous polynucleotide” refers to a polynucleotide that is not found in the cells, absent introduction by an external source. The heterologous polynucleotide may be introduced into cells, e.g., a single cell, a plurality of cells, each of the cells, by, e.g., transduction with a virus, electroporation, transfection, through use of a gene gun, etc. The heterologous polynucleotide may comprise a sequence encoding a transcript. The heterologous polynucleotide may further comprise a regulatory sequence operably linked to the sequence encoding a transcript. The transcript may encode a polypeptide.

[0034] As used herein, “operably linked” refers to a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s). For example, an operable linkage between a polynucleotide of interest, e.g., a sequence encoding transcript, and a regulatory element (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest. In some embodiments, the transcript encodes a polypeptide.

[0035] The transcript may encode a sound responsive protein or a mechanical stimulus responsive protein. The sound or mechanical stimulus protein may comprise one or more of the following exemplary proteins: Prestin, TRP channels (like TRPC, TRPV, TRPA), PMC-1, PIEZO, PIEZO2, MET, TMC1, TMIE, TMHS, TMC2, GJB2, GJB4, GJB6, TPC, MY07A, CDH23, PCDH15, OTOF, SLC26A4, KCNQ4, COL11A2, TECTA, KCNJ10, SLC26A5, MT-RNR1, MT-TS1, USH1C, CLRN1, SLC12A2, TRPA1, TRPV4, FOS, OTOF, FOXP2, ATOH1, FGF20, KCNJ10, GCLC, HSP70, NPTX2, LHFPL5, CFAP206, OTOG, STRC, SCNN1G, NMUR2, BSND, STPG2, and SLITRK6. The full names of the aforementioned sound or mechanical stimulus proteins are: TRP - Transient Receptor Potential, PMC-1 - Plasma Membrane Calcium channel 1, Piezo, MET - Hepatocyte growth factor receptor protein, TMC1 and 2 - TransMembrane Channel like protein 1 and 2, TMIE - TransMembrane Inner Ear expressed protein, TMHS - tetraspan subfamily member 5 protein, GJB 2, 4, 6 - Gap Junction Beta 2,4, and 6, TPC - two pore channel protein, MY07A - Myosin 7A, CDH23 - Cadherin 23, PCDH15 - Protocadherin 15, OTOF -otoferlin, SLC26A4 - Pendrin, KCNQ4 - Potassium voltage-gated channel subfamily KQTmember 4, COL11A2 - Collagen type 11 alpha 2, TECTA - alpha tectorin, KCNJ10 - ATP sensitive potassium channel 10, USH1C - Usher syndrome 1C protein, CLRN1 - Clarin 1, FOS -c-Fos protein, FOXP2 - Fork head Box family transcription factors, ATOH1 - Atonal 1, FGF -Fibroblast growth factor, GCLC - Glutamate-Cysteine Ligase catalytic subunit, HSP70 - Heat Shock Protein 70, OTOG - otogelin, STRC - sterocilin, SCNN1G - Epithelial sodium channel subunit gamma, NMUR2 - Neuromedin - U receptor 2, BSND - Barttin, STPG2 - Sperm tail PG rich repeat containing protein 2, SLITRK6 - SLIT and NTRK like protein 6.

[0036] The engineered multicellular organisms typically comprise an aggregate of ciliated cells. The aggregate of cells may comprise, consist essentially of, or consist of epidermal cells, such as ciliated epidermal cells. Suitable ciliated cells may include, but are not limited to, ciliated cells derived from ectoderm (e.g., differentiated ectodermal cells).

[0037] Ciliated cells for use in preparing the disclosed engineered multicellular organisms may include ciliated cells which are non-motile in their native condition or tissue but which are motile in the engineered multicellular organisms. The cilia of the ciliated cells utilized for forming the disclosed engineered multicellular organisms may be motile cilia, in contrast to non-motile primary cilia. Motile cilia of ciliated cells utilized to form the disclosed engineered multicellular organisms may include an axoneme as known in the art to actuate motility.

[0038] The engineered multicellular organisms may meet at least one of the following criteria: (i) the organism comprises less than about 1000 total cells, or less than about 900, 700, 600, 500, 400, 300, 200, or 100 cells (or the organism comprises a number of cells within a range bounded by any of these values (e g., 100-1000 cells); and (ii) the organism has an effective diameter of less than about 2 mm, or less than about 1.5 mm, 1.0 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm (or the organism has an effective diameter within a size range bounded by any of these values (e.g., 0.1 - 0.5 mm).

[0039] The engineered multicellular organisms preferably are self-motile and move when the cilia of the organisms are actuated. In some embodiments, the cilia of the organisms may be actuated by electrical stimulation or optogenetics where the cilia have been genetically modified to express light-sensitive ion channels.

[0040] In some embodiments, the engineered multicellular organisms move when the cilia of the organisms are actuated. The organisms may move at a rate of at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, or 1000 microns / second or faster when the cilia of the organisms are actuated.

[0041] The engineered multicellular organisms may have a self-limiting life span when placed in a physiologically suitable environment of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. However, the life span of the engineered multicellular organisms may be extended by, e.g., providing exogenous nutrients, e.g., carbohydrates, e.g., glucose, to the engineered multicellular organisms.

[0042] The engineered multicellular organisms may comprise an aggregate of cells which may be referred to as a plurality of living cells that are cohered to one another. The aggregate of cells forms a three-dimensional shape which shape may change over time.

[0043] The aggregate of cells of the disclosed organism may comprise selected cell types. Suitable cell types may include, but are not limited to, ciliated cells such as ciliated epidermal cells. Suitable ciliated cells may include, but are not limited to ciliated cells of derived from ectoderm (e.g., differentiated ectodermal cells). Suitable cell types may include epithelial cells of the bronchi and oviducts.

[0044] Suitable cells may comprise animal cells. Suitable animal cells may comprise amphibian cells (e.g., frog cells and the like) or mammalian cells (e.g., human cells, mouse cells, rat cells, and the like). Suitable cells may include cells that have been cultured in vitro. Suitable cells may comprise, e.g., ciliated respiratory epithelial cells. See, e.g., WO2022115790A1, which is incorporated by reference herein in its entirety.

[0045] The aggregate of cells may comprise, consist essentially of, or consist of ciliated cells. In some embodiments, the aggregate of cells may comprise or may not comprise additional non-ciliated cell types.

[0046] In some embodiments, the engineered multicellular organisms may comprise an aggregate of cells comprising passive cells and contractile cells. See, e.g., US20230235296A1 and US20220220437A1 which are incorporated by reference herein in their entirety.

[0047] In some embodiments, the engineered multicellular organisms are non-innervated (e.g., the organisms do not comprise neural cells or neural tissue) and / or or non-cartilaginous. The engineered multicellular organisms may be innervated.

[0048] The multicellular organisms may be described as "engineered" because they are different from naturally occurring organism that arise without the guidance of human ingenuity and modifications. In other words, the multicellular organisms are synthetic and non-naturally occurring, albeit the multicellular organism may utilize endogenous cell:cell signaling and morphogenesis to adopt a particular form.

[0049] The aggregate of cells of the engineered multicellular organisms may comprise cells that have been engineered to express a heterologous molecule. In some embodiments, the cells of the organisms are engineered to express a heterologous protein or secrete specific desired molecules.

[0050] In embodiments in which the aggregate of cells comprises cells that have been engineered to express a heterologous molecule, suitable heterologous molecules that are expressed may include enzymes. Suitable enzymes may include enzymes that metabolize a target substrate, which may include toxins. Other suitable heterologous molecules may include receptors for a target ligand (e.g., a target ligand sensed by the organism), or sensors of light, heat, and other physical properties in the environment. Other suitable heterologous molecules may include reporter molecules (e.g., photoconvertible fluorescent reporter molecules), nucleic acid-guided nucleases, e.g., Cas enzymes, e.g., Cas9, guide nucleic acids, e.g., gRNAs, therapeutic peptides, antibodies, single chain variable fragments (scFvs), sensor proteins for light, magnetic fields, and chemical molecules / odorants, sensors of mechanical pressure, sensors of vibration.

[0051] The disclosed engineered multicellular organisms may be self-repairing. In some embodiments, if the aggregate of cells is subjected to deaggregation e.g., physical damage that disrupts aggregation of the cells), the cells will reaggregate and / or remodel to re-form the aggregate of cells having the original shape or a new shape.

[0052] The engineered multicellular organisms may be configured in order to perform tasks. For example, the engineered multicellular organisms may be configured structurally and / or genetically.

[0053] In some embodiments, the organism is configured for moving a target object (e.g., by pushing a target object). In further embodiments, the organism is configured for moving target objects (e.g., by pushing target objects) and collecting the moved target objections (i.e., aggregating the target objects).

[0054] The engineered multicellular organisms may be configured to have a cavity. In some embodiments, the engineered multicellular organisms are configured to have a cavity for capturing and / or transporting a target object.

[0055] The engineered multicellular organisms may be utilized in a number of applications. In some embodiments, the engineered multicellular organisms are utilized in methods for removing a target substrate from an environment (e.g., a toxin from an environment). The methods may comprise engineering the organisms to express an enzyme that metabolizes the target substrate and placing the organism in the environment to remove the target substrate from the environment.

[0056] In other embodiments, the engineered multicellular organisms are utilized in methods for detecting a target ligand in a sample. The methods may comprise engineering the organisms to express a receptor for the target ligand and place the organism in the sample, where the organism generates a signal after the receptor binds the target ligand.

[0057] In other embodiments, the engineered multicellular organisms may express a photoconvertible fluorescent reporter molecule, and the organism generates a fluorescent signal when the photoconvertible fluorescent reporter molecule is exposed to light. As such, the activity and movement of the organisms may be monitored by placing the organisms in an environment and providing a light source for activating the photoconvertible fluorescent reporter molecule.

[0058] Also included in this disclosure are methods for preparing the engineered multicellular organisms. In some embodiments, the methods comprising explanting cells from tissue and culturing the explanted cells under conditions in which the cultured, explanted cells form the engineered multicellular organisms.

[0059] The methods may comprise using sound to have the engineered multicellular organisms navigate to a particular area within the body for drug delivery applications, e.g., by applying an acoustic stimulation ex vivo, while the engineered multicellular organisms are present on or in thebody. The methods may be comprise using sound to have the engineered multicellular organisms navigate to hard-to-reach aquatic environments, e.g., in nature to either test or achieve certain tasks in that area. The methods may comprise using sound to have the engineered multicellular organisms go to a toxic aquatic environment and perform cleanup or sampling. Further, the methods may comprise inducing the following changes in the organisms from the acoustic stimulation: change their exploratory behavior profile, change how they bind molecules of interest for sensing or collection of important cargo, change how they process information about what they have experienced, change the speed of motion or preferences about how they move in gradients, change their life-span, or change their morphology or shape in response to a sensor or target. Methods of conferring sound sensitivity to an engineered multicellular organism

[0060] In an aspect of the current disclosure, methods of conferring sound sensitivity to an engineered multicellular organism are provided. In some embodiments, the methods comprise introducing a heterologous polynucleotide to the cells of the engineered multicellular organism, wherein the heterologous polynucleotide comprises a sequence encoding a polypeptide, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.

[0061] In some embodiments, the heterologous polynucleotide further comprises a regulatory element operably linked to the nucleic acid sequence encoding the polypeptide. In some embodiments, the cells, e.g., a single cell, a plurality of cells, each of the cells, of the engineered multicellular organism express the polypeptide. In some embodiments, the engineered multicellular organisms comprise or consist of ciliated cells. In some embodiments, the engineered multicellular organisms comprise vertebrate cells. The engineered multicellular organisms may comprise ciliated epithelial cells, e.g., ciliated respiratory epithelial cells.

[0062] ‘ ‘Introduction” of a polynucleotide may comprise transfection, transduction, or any other suitable modality to allow the polynucleotide access through the cellular membrane to be expressed in the cell or to be incorporated into the genome of the cell.

[0063] The sound responsive or mechanical stimulus responsive protein may comprise sensor proteins for light, magnetic fields, and chemical molecules / odorants, sensors of mechanical pressure, sensors of vibration.Systems

[0064] In an aspect of the current disclosure, systems for modifying motility of an engineered multicellular organism are provided. In some embodiments, the systems comprise (a) an acoustic stimulator; and (b) an engineered multicellular organism. The systems may comprise a plurality of multicellular organisms.

[0065] The acoustic stimulator may comprise a sound component, e.g., a mechanical sound component or an electronic sound component. Sound components are divided into mechanical and electrical types. Mechanical type sound components produce sound with the application of mechanical force to a sound source, e.g., in bicycle bells, other types of bells, gongs, and similar devices. Electrical type sound components produce sound by vibrating a sound source with an application of electrical signals such as voltage and current. The electric type is further divided into piezoelectric, electromagnetic (magnetic), and electrodynamic (dynamic) types of electric sound components. The acoustic stimulator may comprise or consist of a speaker.

[0066] The acoustic stimulator may be configured to emit a frequency of about 5 Hz to about 1000 Hz, or any value or subrange therein. The acoustic stimulator may be configured to emit a frequency of about 50 Hz to about 500 Hz. The acoustic stimulator may be configured to emit a frequency of about 50 Hz to about 300 Hz. The acoustic stimulator may be configured to emit a frequency of about 300 Hz.

[0067] The vessel housing the engineered multicellular construct may be in close proximity to the acoustic stimulator, e.g., may be physically connected to the acoustic stimulator. The vessel may be about 1 meter or less from the acoustic stimulator, or about 1 centimeter or less from the acoustic stimulator, or any value or subrange therein, inclusive of the endpoints.

[0068] The disclosed systems may further comprise (c) one or more controller, processor, or memory. The one or more controller, processor, or memory may be connected, e.g., electronically connected, to a vessel, the acoustic stimulator, or a vessel housing the engineered multicellular organisms.

[0069] In an aspect of the current disclosure, methods are provided, the methods comprising emitting sound from the acoustic stimulator of the disclosed systems. Engineered multicellularorganisms may be exposed to the emitted sound. The emitted sound may modify the motility of the engineered multicellular organisms.

[0070] Further Definitions and Terminology

[0071] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a cell” should be interpreted to mean “one or more cells." As used herein, the term “plurality” means “two or more.”

[0072] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0073] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0074] The phrase “such as” should be interpreted as “for example, including.” Moreover the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0075] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C”would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0076] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0077] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.” Illustrative embodiments1. A method of modifying motility of an engineered multicellular organism, the method comprising (a) exposing the engineered multicellular organism to an acoustic stimulation.2. The method of embodiment 1, wherein the engineered multicellular organism comprises ciliated cells.3. The method of embodiment 2, wherein the ciliated cells are ciliated epithelial cells.4. The method of any one of the preceding embodiments, wherein the engineered multicellular organism comprises vertebrate cells.5. The method of any one of the preceding embodiments, wherein the engineered multicellular organism comprises amphibian cells.6. The method of any one of the preceding embodiments, wherein the engineered multicellular organism comprises Xenopus cells.7. The method of any one of the preceding embodiments, wherein the acoustic stimulation comprises sound at a frequency of about 5 Hz to about 1000 Hz.8. The method of any one of the preceding embodiments, wherein the acoustic stimulation comprises sound at a frequency of about 50 Hz to about 500 Hz.9. The method of any one of the preceding embodiments, wherein the acoustic stimulation comprises sound at a frequency of about 50 Hz to about 300 Hz.10. The method of any one of the preceding embodiments, wherein the acoustic stimulation comprises sound at a frequency of about 300 Hz.11. The method of any one of the preceding embodiments, wherein the engineered multicellular organism is exposed to the acoustic stimulation for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours.12. The method of any one of the preceding embodiments, wherein the method further comprises (b) ceasing the acoustic stimulation.13. The method of embodiment 12, wherein the acoustic stimulation is ceased for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours.14. The method of embodiment 12 or 13, wherein the method further comprises (c) exposing the engineered multicellular organisms to an acoustic stimulation.15. The method of embodiment 14, wherein the engineered multicellular organism is exposed to the acoustic stimulation for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours.16. The method of embodiment 14 or 15, wherein the acoustic stimulation of (c) is the same as the acoustic stimulation of (a).17. The method of embodiment 14 or 15, wherein the acoustic stimulation of (c) is different from the acoustic stimulation of (a).18. The method of any one of the preceding embodiments, wherein the method comprises modifying the motility of the engineered multicellular organisms in an aquatic environment.19. The method of any one of the preceding embodiments, wherein the engineered multicellular organisms are genetically modified.20. The method of any one of the preceding embodiments, wherein the engineered multicellular organisms comprise an antisense oligo or a morpholino.21. The method of any one of the preceding embodiments, wherein the engineered multicellular organisms comprise a heterologous polynucleotide.22. The method of embodiment 21, wherein the heterologous polynucleotide comprises a sequence encoding a transcript.23. The method of embodiment 21 or 22, wherein the heterologous polynucleotide further comprises a regulatory sequence operably linked to the sequence encoding a transcript.24. The method of embodiment 22 or 23, wherein the transcript encodes a polypeptide, optionally, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.25. A system for modifying motility of an engineered multicellular organism, the system comprising (a) an acoustic stimulator; and (b) an engineered multicellular organism.26. The system of embodiment 25, wherein the acoustic stimulator comprises a sound component.27. The system of embodiment 26, wherein the sound component is a mechanical sound component or an electronic sound component.28. The system of embodiment 25, wherein the acoustic stimulator is a speaker.29. The system of any one of embodiments 25-28, wherein the acoustic stimulator is configured to emit a frequency of about 5 Hz to about 1000 Hz.30. The system of any one of embodiments 25-29, wherein the acoustic stimulator is configured to emit a frequency of about 50 Hz to about 500 Hz.31. The system of any one of embodiments 25-30, wherein the acoustic stimulator is configured to emit a frequency of about 50 Hz to about 300 Hz.32. The system of any one of embodiments 25-31, wherein the acoustic stimulator is configured to emit a frequency of about 300 Hz.33. The system of any one of embodiments 25-32, wherein the engineered multicellular organism comprises ciliated cells.34. The system of embodiment 33, wherein the ciliated cells are ciliated epithelial cells.35. The system of any one of embodiments 25-34, wherein the engineered multicellular organism comprises vertebrate cells.36. The system of any one of embodiments 25-35, wherein the engineered multicellular organism comprises amphibian cells.37. The system of any one of embodiments 25-36, wherein the engineered multicellular organism comprises Xenopus cells.38. The system of any one of embodiments 25-37, wherein the engineered multicellular organism is in a liquid medium in a vessel.39. The system of any one of embodiments 25-38, wherein the engineered multicellular organisms are genetically modified.40. The system of any one of embodiments 25-39, wherein the engineered multicellular organisms comprise an antisense oligo or a morpholino.41. The system of any one of embodiments 25-40, wherein the engineered multicellular organisms comprise a heterologous polynucleotide.42. The system of embodiment 41, wherein the heterologous polynucleotide comprises a sequence encoding a transcript.43. The system of embodiment 42, wherein the heterologous polynucleotide further comprises a regulatory sequence operably linked to the sequence encoding a transcript.44. The system of embodiment 42 or 43, wherein the transcript encodes a polypeptide, optionally, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.45. The system of embodiment 38, wherein the vessel is physically connected to the acoustic stimulator.46. The system of embodiment 38, wherein the vessel is about 1 meter or less from the acoustic stimulator.47. The system of embodiment 38, wherein the vessel is about 1 centimeter or less from the acoustic stimulator.48. The system of any one of embodiments 25-47, wherein the system further comprises (c) one or more controller, processor, or memory.49. A method comprising emitting sound from the acoustic stimulator of the system of any one of embodiments 25-48.50. The method of embodiment 49, wherein the method modifies the motility of the engineered multicellular organism.51. A method of conferring sound sensitivity to an engineered multicellular organism, the method comprising introducing a heterologous polynucleotide to the cells of the engineered multicellular organism, wherein the heterologous polynucleotide comprises a sequence encoding a polypeptide, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.52. The method of embodiment 51, wherein the heterologous polynucleotide further comprises a regulatory element operably linked to the sequence encoding the polypeptide.53. The method of embodiment 51 or 52, wherein the cells of the engineered multicellular organism express the polypeptide.54. The method of any one of embodiments 51-53, wherein the engineered multicellular organisms comprise or consist of ciliated cells.55. The method of any one of embodiments 51-54, wherein the engineered multicellular organism comprises vertebrate cells.EXAMPLES

[0078] The following examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - Basal Xenobot Transcriptomics: Gene Expression Changes in wild-type cells comprising one form of biobot

[0079] Reference is made to Vaibhav Pai et al. “Basal Xenobot transcriptomics reveals changes and novel control modality in cells freed from organismal influence” Commun Biol. 2025 Apr 22;8(1):646, the content of which is incorporated herein by reference in its entirety.

[0080] The standard paradigm of developmental and synthetic biology focuses on gene expression driving changes in morphology. Here, we investigated the reverse relationship: would transcriptomes change if cell collectives acquired a novel morphogenetic and behavioral phenotype in the absence of genomic editing, transgenes, heterologous materials, or drugs? We investigated the effects of morphology and nascent emergent life history on gene expression in the basal (no engineering, no sculpting) form of Xenobots - autonomously motile constructs derived from Xenopus embryo ectodermal cell explants. We compared transcriptomes of these basal Xenobots with age-matched Xenopus embryos. Basal Xenobots showed significantly larger interindividual gene variability than age-matched embryos, suggesting increased exploration of the transcriptional space. We identified at least 537 (non-epidermal) transcripts uniquely upregulated in these Xenobots. Pathway analyses indicated transcriptomic shifts in the categories of motility machinery, multicellularity, stress and immune response, metabolism, and sensory perception of sound and mechanical stimuli. We experimentally confirmed that basal Xenobots respond to acoustic stimuli via observable changes in behavior, in ways that age-matched embryos do not. Phylostratigraphic analysis showed that the majority of transcriptomic shifts in the basal Xenobots were towards evolutionarily ancient transcripts and systems. Lastly, we found enrichment of thanatotranscriptomic genes, shedding light on the distinction between death of an organism and that of its cells. These data on the relationship between genotype and phenotype may have implications for evolution, biomedicine, and synthetic morphoengineering.

[0081] Introduction:

[0082] The standard research paradigm of developmental and synthetic biology seeks to discover how gene expression drives specific anatomical and behavioral outcomes2’3’4’5’6. However, it is also well-understood that transcriptional machinery itself is sensitive to external cues7’8’9‘10, 1112, 13. Rapid changes of gene expression can be induced by exposures to genetic, biochemical, biomechanical, bioelectrical, and materials-mediated influences. On a much longer timescale, the properties of gene expression profiles in vivo are thought to be determined by evolutionaryselection, optimizing fitness to specific environment, life history and lived experiences14 14 14 1418. Here, we sought novel aspects of the responsiveness of gene expression, in a setting in which a non-canonical multicellular, functional form is achieved without transgenes, chemical signals, foreign nanomaterials, or other added influence.

[0083] A fascinating set of questions concerns the origins of species-specific transcriptomic profiles, normally shaped by eons of selection over the functionality of those forms in a specific environmental context. Unique aspects of these questions can be addressed in synthetic systems in which the entities have not had a history of selection in their current multicellular embodiment. Numerous synthetic life forms have recently been produced14242422, 24 24‘2424242429; however, many questions remain to be answered, via analysis of transcriptomic profiling in these synthetic living configurations. In this study we utilize one such living system to understand how synthetic morphology might result in transcriptomic changes. Our goal was to characterize unique transcriptomic changes in this synthetic living system in comparison to its native (wild-type) embryo context.

[0084] Multiple different kinds of autonomously moving biobots34 34 32can be derived from Xenopus embryonic cells. They offer self-organization (a kind of developmental morphogenesis), as they need no scaffold in order to form and mature into functional constructs. They can form from a single tissue (prospective skin / epidermis) or from a combination of multiple tissues (e.g., skin / epidermis and muscles), can be of multitude of shapes and be actuated by either the muscle contraction or coordinated beating of cilia33’343436. They show self-healing and emergent group behaviors including kinematic self-replication3435. This overall class is referred to as “Xenobots”37to emphasize the fact that we see their utility as potentially programmable living materials - a model system in which to learn to control the plasticity of living active matter toward applications that shed light on life-as-it-can-be34 39, 44 44 42, 44 44. In this study we use the basal (spherical) Xenobots33derived from completely wild-type ectodermal explant cells with no drugs, synthetic biology circuits, nanomaterials, sculpting, or anything else added. Hence, we can observe latent behaviors of these cellular collectives that are released by lifting signaling constraints present when they were part of the larger organism4444444449, 50.

[0085] The basal Xenobots are autonomously motile, self-assembling constructs derived from frog (Xenopus laevis) embryonic ectodermal explants (colloquially referred to in developmental biology studies as “animal caps”) as starting material34. The Xenopus ectodermal explants (animal caps) have been studied for several decades and serve as an excellent model for studying epidermal cell fate and patterning as well as testing the plasticity and responsiveness of these cells to various inducers and inhibitors51, 52‘53, 54'55,?6, 57. Also, mucociliary organoids developed from these Xenopus ectodermal explants have served as excellent models for understanding dynamics of mucociliary epidermis formation and maintenance58, 59, 60, 61, 62, 63. However, we cultured these ectodermal explants until they formed autonomously-moving atypical entities aka the basal Xenobots used in this study. We were particularly interested in studying the transcriptomic changes in these autonomously-moving Xenobots. Recently, elegant temporal transcriptomic analysis has shown epidermal cell fate specification during the transformation of Xenopus ectodermal explants (animal caps) to autonomously-moving atypical entities (the basal Xenobots used in this study)64However, in this study we sought to broadly analyze genome-wide transcriptomic changes well beyond epidermal cell fate specification in response to a not previously selected-for embodiment and nascent life history of these autonomously-moving atypical entities - as system that self-assembles without being forced onto an engineered scaffold and exhibits autonomous motion behavior (being functionally closer to a proto-organism than a typical organoid). We see these autonomously-moving atypical entities (basal Xenobots) as an important biorobotics platform because, once their properties are understood, they can be used as a testbed in which to improve our understanding and ability to control growth and form, and ultimately deploy them for many useful purposes. Unlike conventional robotic materials, living cells offer numerous complex behaviors and responses; understanding the plasticity, and molecular-biological responses, of cell collective entities in new contexts is a critical part of morphogenic engineering effortsl9‘31, 32‘65, 66‘67.

[0086] To advance this engineering roadmap, as well as more broadly understand how transcriptomes respond to internal and external environment changes (e.g., for biomedical purposes, as well as basic evolutionary developmental biology), we compared the transcriptome of basal Xenobots to those of age-matched embryo controls. Importantly, we did not focus on geneexpression missing from Xenobots, because it was expected that, being made of just one tissue type, they would lack many transcripts that normal embryos expressed. Instead, we asked what transcripts, other than epidermal markers (expected to be enriched), Xenobots expressed that their age-matched embryos did not. We found at least 537 transcripts to be uniquely upregulated in these Xenobots, representing functional classes such as locomotion apparatus assemblies (cilia and motor proteins), structure formation and multicellularity, stress and immune response, metabolism, and, even sensory perception of sound and mechanical stimuli. Functional experiments confirmed that basal Xenobots, unlike age-matched Xenopus embryos, indeed respond to acoustic vibration stimuli by changing their motion behavior. Phylostratigraphic analysis shows that the majority of these transcriptomic shifts are towards evolutionary ancient transcripts and systems. These changes suggested that alteration of morphology and nascent emergent life history, in wild-type cells, were enough to strongly affect the transcriptome (morphology / behavior driving gene expression). Moreover, we found a significantly greater inter-individual gene variability in these Xenobots compared to their age-matched Xenopus embryos suggesting exploratory adaptation in the transcriptional space to adapt and survive in their new embodiment68-69’70‘71. Another unique aspect that these Xenobots allowed us to investigate was the nature of organismic death and its effects on gene expression. By making a Xenobot out of an embryonic source of cells, the original embryo “dies” - it is no more; but the cells persist and continue on in a new embodiment. Our analysis revealed enrichment of thanatotrascriptomic72, 73, 74, 75, 76genes, seen previously in studies of human post-mortem molecular physiology. Taken together, these data shed light on the feedback of form and function onto cellular genetics, in the context of evolved and engineered living beings transitioning across levels of organization.

[0087] Results:

[0088] RNA sequencing analysis shows no major transcriptional changes between stage 35 / 36 Xenopus embryos raised in 0.1X MMR and 0.75X MMR

[0089] One major difference in the rearing condition of Xenopus embryos and basal Xenobots used in this study is the MMR (Marc’s Modified Ringers) medium concentration: embryos are reared in 0.1X MMR, Xenobots are reared in 0.75X MMR (a higher concentration of salts). Thus, we first asked whether raising embryos in 0.75X MMR induces any significant transcriptionalchanges in comparison to embryos raised in 0.1X MMR. We performed RNA sequencing analysis on stage 35 / 36 Xenopus embryos (stage corresponds to the formation of mature autonomously-moving Xenobots on day 7 at 14°C after explanting ectodermal explants (animal caps) from stage 9 embryos) raised in either 0.1X MMR or raised in 0.75X MMR after stage 9 (stage at which ectodermal explants are removed from embryos and placed in 0.75X MMR to make the basal Xenobots). We collected RNA from three replicate samples (each sample containing ten stage 35 / 36 embryos) for each condition (0.1X and 0.75X MMR) (FIG. 6A). Overall, we observed no separation between groups by principal component analysis (FIG. 6B). However, after surrogate variable analysis and batch-correction, we did observe some separation of the clusters (FIG. 6C). We thus performed differential gene expression analysis and found that in the histograms of significance the / ?- value histograms are relatively flat, with the proportions of the true null hypothesis (non-significant genes) to be 0.996 (FIG. 6D) and all FDRs (False Discovery Rates) to be near one (FIG. 6E). These results indicate minimal changes in the overall transcriptome of 0.75X MMR raised embryos versus 0.1X MMR raised embryos. Differential expression analysis showed that out of -24,500 transcripts only 6 genes were significantly changed (FDR < 0.05) with a log fold change > 2 in the 0.75X MMR raised embryos (FIG. 6F and Table 1). These results show that overall raising embryos in the higher salt medium at 0.75X MMR results in very little transcriptional change - the transcriptome is not highly sensitive to external conditions.

[0090] RNA-sequencing analysis shows major transcriptional changes in the basal Xenobots compared to age-matched stage 35 / 36 Xenopus embryos

[0091] The basal Xenobots are derived by explanting ectodermal explant (animal cap) tissue from stage 9 Xenopus embryos, and reach maturity by day 7: fully differentiated cells, peak cellular health, and peak robust autonomous movement behavior34To test our hypothesis that these Xenobots would have unique and distinct transcriptomic changes compared to normal Xenopus embryos, we compared these Xenobots’ transcriptome to that of age-matched (stage 35 / 36) control Xenopus embryos that were reared in identical conditions (incubation temperature and time) as the Xenobots. We collected RNA from three pooled samples for each condition (Xenobots and age-matched stage 35 / 36 Xenopus embryos) (FIG. 1A) and performed RNA-seq to compare the two transcriptomes. Principal component analysis using surrogate variables and batch-correctionshowed marked separation between the clusters of Xenobot transcriptome and that of age-matched Xenopus embryos (FIG. 7). Differential expression analysis showed that out of 28,009 transcripts, 1,962 transcripts were significantly up-regulated (FDR<0.05) with log fold change >2 in these Xenobots compared to age-matched embryos and 5,053 transcripts were significantly down-regulated (FDR<0.05) with log fold change < -2 in these Xenobots compared to age-matched embryos (FIG. IB). These results show that overall, these Xenobots have major transcriptional differences from age-matched Xenopus embryos.

[0092] The basal Xenobots have greater inter-individual gene count variability than age-matched stage 35 / 36 Xenopus embryos

[0093] In the principal component analysis (FIG. 7), the three Xenobots samples show much wider separation compared to the three age-matched embryo samples. Previous work has shown that cells experiencing novel stressors can randomly change their gene expression profiles until they find configurations that resolve the stress, and that individual cells discover different expression profiles that solve the same problem68-70'71-77-78. We hypothesized that the process of removal from the embryo serves as a novel stressor for the nascent Xenobot tissue, requiring the tissue to adapt to its new embodiment in ways that evolution did not prepare it for. In contrast, while embryos are indeed capable of solving novel problems to achieve their developmental goals79, their developmental trajectory is more stereotyped and likely requires less transcriptional exploration than do these Xenobots.

[0094] We compared the variability of individual transcripts among biological samples using the method in FIG. 2A. Briefly, all 28,009 gene transcripts were ranked based on their mean count values across all samples (3 Xenobot samples and 3 Xenopus embryo samples), combined, and genes for which any sample had a value of 0 were removed. The standard deviations of individual gene transcripts among Xenobot samples and Xenopus embryo samples were estimated and used to calculate the coefficient of variation (CV) for each gene as plotted in FIG. 2B histograms. In Xenobots, genes have significantly higher inter-individual CVs and a broader range of CVs than embryos (Wilcoxon Rank Sum test, p=0) (FIG. 2B). The ranked gene list was split into 100 equalsize bins (percentiles), and the fraction of genes in each bin for which CV of the Xenobots (CVX) was greater than the CV of the Xenopus embryos (CVE) was plotted (FIG. 2C). We found thatalmost all genes have a greater CV for Xenobots than Xenopus embryos (96.06% of all included genes, shown by the red line) (FIG. 2C). Also, the general trend of increasing bin fraction with higher gene count percentile indicated that, the higher the gene expression, the higher is the variability of that gene in Xenobots compared to Xenopus embryos (FIG. 2C). A permutation test (see Methods) revealed that these deviations from the expected (null hypothesis) bin fraction value (0.9606 - the red line in FIG. 2C) across gene count percentiles were statistically significant (dark blue colored bins with p<0.05). Among the top ten most variable genes in these Xenobots (FIG.2D), Oncomodulin gene which encodes a protein with widely distinct functional roles80is most prominent with three oncomodulin genes in the top ten (FIG. 2D). Other interesting genes include A. superbus venom factor 1, salivary glue protein like, neurotrimin, tenomodulin, somatostatin receptor 4, chloride intracellular channel 4 (FIG. 2D). Thus overall, there is significantly greater gene count variability in these Xenobots compared to age-matched Xenopus embryos, possibly indicating exploratory adaptation in the transcriptional space to adapt and survive in their new embodiment.

[0095] Stringent curation of Xenobot transcripts

[0096] The basal Xenobots are derived from ectodermal explants (animal cap explants) which are primarily ectodermal progenitor cells. Most cells and tissues belonging to mesoderm and endoderm are absent in these Xenobots as compared to Xenopus embryos. Hence mesoderm and endoderm transcripts would come out as highly downregulated in these Xenobots compared to age-matched embryos largely due to absence of those cells and tissues in these Xenobots (false positives). Similar to previous studies58, we found no clear path to resolve and differentiate which downregulated transcripts (out of the 5053 downregulated transcripts) are changed due to absence of mesoderm and endoderm (false positives) and which transcripts are truly downregulated in these Xenobots compared to age-matched Xenopus embryos (true positives). Hence, we did not further analyze the downregulated transcripts dataset.

[0097] Conversely, Xenobots are fully derived from ectoderm, but ectoderm only forms a small portion of whole embryo (-10%). Thus, in a head-to-head comparison, ectodermal genes would be artificially enriched in these Xenobots resulting in false positives due to simple proportional enrichment of ectodermal tissues. To adjust for this, we subtracted out the artificially enrichedectodermal / epidermal-specific transcripts from the 1,962 upregulated transcripts to curate a list of transcripts uniquely upregulated in these Xenobots which could not be explained by the increased prevalence of ectodermal cells. To achieve this, we used Klein tools81- a database of single-cell transcriptome of stage 22 entire Xenopus embryo. We subtracted out the entire gene set for embryonic epidermal progenitors in Xenopus embryos, leaving 1742 transcripts that were upregulated in these Xenobots (Table 2). -41% (712 transcripts) of the transcripts were uncharacterized “LOC” genes. We used genome-wide functional annotation tool eggNOG-mapper82to annotate these uncharacterized transcripts using human (most annotated genome) orthologs. We first tested the functional annotation using a small subset of already known Xenopus transcripts and found -80% accuracy with differences mostly being different versions of the same gene. After functional annotation of the uncharacterized transcripts, removing any remaining unannotated transcripts, and removing all duplicate transcripts from short and long chromosomes, we were left with 1078 uniquely upregulated transcripts in these Xenobots compared to age-matched Xenopus embryos (Table 3).

[0098] We found many cilia and motor protein transcripts in the 1078 uniquely upregulated transcripts in Xenobots which suggested the possibility that perhaps even after epidermal gene subtraction there might still be some false positively enriched epidermal transcripts particularly belonging to the epidermal multiciliate cells. To address this issue, we raised our stringency to very high (likely losing some true positives) by again resorting to Klein tools database of singlecell transcriptome of stage 22 entire Xenopus embryos. In addition to the epidermal progenitor gene subtraction already performed, we collected all the genes expressed in the different types of epidermal cells namely, multiciliated cells, alpha and beta ionocytes, and goblet cells. We then subtracted any genes from any of these four cells that were present in our list of 1078 transcripts uniquely upregulated in these Xenobots. We then obtained a highly stringent list of 795 transcripts in which we had high confidence that these transcripts were uniquely upregulated in these Xenobots compared to age-matched embryos. After mapping these 795 transcripts to human orthologs we obtained 537 transcripts which were then used for subsequent comparisons and analysis (Table 4).

[0099] Functional analysis of transcripts uniquely upregulated in these Xenobots compared to age-matched Xenopus embryos

[0100] What kind of genes did the basal Xenobots up-regulate? To understand the biological processes that might be affected by their unique transcripts, we first performed functional enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID)83, 84Using DAVID, we identified enriched biological themes amongst significantly differentially expressed genes and clustered the redundant annotation terms using the Functional Annotation Clustering tool. We identified the most enriched functional clusters and ranked them based on their group enrichment score, which is the geometric mean (in -log scale) of member’s p-values in a corresponding annotation cluster (FIG. 3A and Table 5). Interestingly, despite the very high stringency and removal of genes expressed by epidermal cells, we found enrichment of cilia, ciliopathies, and motor proteins. We also found categories for immune response and signaling, and metabolism / biosynthesis. Interestingly, we found a category of membrane proteins that contained largest number of genes belonging to various functions such as, ion channels and transporters, gap junction and junctional proteins, calcium binding proteins, ATP binding proteins, sound or mechanical stimuli responsive proteins.

[0101] In addition, we also performed a network analysis to identify active functional biological modules85’86. We combined gene expression and interaction data and then applied network embedding, followed by clustering similarly to that performed by87-88. The detected functional modules were then enriched using g:profiler89. This network clustering analysis identified 10 enriched clusters (Table 6). Many of the clusters were similar to the functional enrichment analysis (Tables 5 and 6) such as cilia / cytoskeleton / ciliopathies, immune and stress response, metabolism / biosynthesis (FIG. 3, FIG. 8 and Tables 5 and 6). We also found clusters for extracellular matrix (ECM) / proliferation / multicellular organization, and sensory perception of sound and mechanical stimuli (FIG. 3, FIG. 8, and Table 6).

[0102] In summary, we identified biological processes enriched in these Xenobots compared to age-matched Xenopus embryos. These include cilia and motor proteins, tissue building / multicellular organization, immune and stress response, metabolism shift and perception of sound and mechanical stimuli. Among these the tissue building / multicellular organization andimmune and stress response were unsurprising as these functions are required for building any novel morphology90’91, 92’93, 94. The presence of cilia and motor proteins in spite of our very high stringency was surprising. Lastly, the shift in metabolism compared to embryo and the presence of sensory perception of sound and mechanical stimuli was extremely surprising.

[0103] Transcripts uniquely upregulated in these Xenobots do not include any mesodermal, endodermal, or any axis patterning genes

[0104] To assess whether any mesodermal, endodermal, or axis patterning genes were being up-regulated in the basal Xenobots, we first curated a list of most prominent genes for each of these categories from literature (Table 7). We then compared to see if any of these genes are expressed in our high stringency transcripts upregulated in these Xenobots. None of the critical genes from mesoderm, endoderm, mesendoderm, anterior-posterior patterning, dorso-ventral patterning, or left-right patterning were found to be enriched in these Xenobots. This suggests that it is unlikely that these Xenobots perform trans-differentiation to recover their missing germ layers.

[0105] Xenobots change their motion behavior in response to acoustic vibration stimuli

[0106] Sensory perception of sound and mechanical stimuli was one of the most surprising gene clusters for biological processes identified to be significantly upregulated in Xenobots (FIG. 3). The possibility that basal Xenobots could exhibit novel functional responses to sound is fascinating as it would provide a tractable method to control Xenobot physiology and / or behavior, as well as shedding light on evolutionary developmental biology aspects of sensory capabilities. To functionally test this hypothesis, we exposed Xenobots to acoustic vibration stimulus and documented changes in their motion behavior (FIG. 4). First, we created an apparatus to apply acoustic vibration stimulus to Xenobots (FIG. 4A). A preliminary frequency survey showed that Xenobots respond to vibration stimulus by changing their motion behavior within the frequency range of 50Hz to 300Hz with the strongest response at 300Hz. Hence, we chose a frequency of 300Hz for our experiments. The amplitude was reduced to the point where there was no visible movement of liquid in the dish and then the frequency and amplitude were kept constant throughout all experiments. We recorded time-lapse videos of Xenobot motion for 10 min before the vibration stimulus, 10 min during the vibration stimulus, and for 10 min after the vibrationstimulus (total 30 minutes of observation for each Xenobot). We first performed two essential controls.

[0107] To test whether subtle movement of liquid medium in response to vibration may be physically moving the Xenobot we used day 1 Xenobots as controls, because at that time point cellular differentiation has not yet occurred and there are no multiciliate cells: they are non-motile (FIG. 4B and Supplemental Movie 1 (not shown)), and thus would reveal any passive movement induced externally. Day 1 Xenobots are especially good controls as they have roughly the same size and mass as the day 7 autonomously motile Xenobots (FIG. 4F). Day 1 Xenobots showed no movement within the intervals before, during, and after vibration stimulus (N=9, One-Way ANOVA with Tukey’s multiple comparisons test, p>0.05) (FIG. 4C, H, and Supplemental Movies 1 & 2 (not shown)) suggesting that whatever movements we observe cannot be due to a passive displacement of inactive material by simple physical vibration.

[0108] Next, to test whether vibration stimulus causes a generic change in structure and / or function of cilia leading to change in motion behavior, we tested the effect of vibration stimulus on age-matched stage 35 embryos (FIG. 4D). In addition to cilia-based gliding, stage 35 embryos also have muscle-based movement. Hence, we treated them with 0.05% tricaine (MS-222)95which blocks sensory-motor responses and thus inhibits muscle-based movement while leaving the cilia-based movement intact. Age-matched stage 35 embryos showed the expected cilia-based motion (FIG. 4E and Supplemental Movies 3 & 4 (not shown)) but did not show any significant difference in motion behavior between the time intervals of before, during, and after vibration stimulus (N=5, One-Way ANOVA with Tukey’s multiple comparisons test, p>0.05) (FIG. 4E, H, and Supplemental Movies 3 & 4 (not shown)). This result suggests that vibration stimulus is not causing a generic change in structure and / or function of cilia and shows no significant effect on cilia-based motion in age-matched stage 35 embryos. Would the Xenobots respond differently than embryos, as suggested by the RNAseq data?

[0109] We then tested the effect of vibration stimulus on day 7 autonomously motile Xenobots (FIG. 4F). Day 7 autonomously motile Xenobots show characteristic rotational motion behavior34before exposure to vibration stimulus (FIG. 4G and Supplemental Movie 5 & 6 (not shown)). However, upon exposure to vibration stimulus, day 7 Xenobots show marked change in motionbehavior with a more linear / arcing motion behavior and a significant increase in peak velocity (N=7, One-Way ANOVA with Tukey’s multiple comparisons test, p<0.01) (FIG. 4G, H, and Supplemental Movie 5 & 6 (not shown)). After the vibration stimulus they return to rotational motion behavior similar to pre-stimulus levels (FIG. 4G and Supplemental Movie 5 & 6 (not shown)). These results reveal that day 7 freely-behaving Xenobots specifically respond (in form of changed motion behavior) to vibration stimulus in ways that inactive (passive) Xenobots and age-matched normal embryos do not.

[0110] Xenobot transcripts show enrichment of thanatotranscriptomic genes.

[0111] We next turned our attention to additional aspects of the RNAseq results. Recent studies have shown the surprising fact that cells actively turn on specific genes after an organism’s death - the so called thanatotranscriptome73, 75‘96, 97‘98, 99. This is fascinating as it provides a way to dissociate death of an organism from that of its cells, thus shedding light on deep issues of multi cellularity and emergent levels of organization in biology. We hypothesized that the data from human thanatotranscriptome studies indicated the cells’ attempt to survive in another form after the demise of the organism. In the case of mammals, living in dry air, this would be futile, but in the case of amphibians, this could work and indeed, these Xenobots represent just that scenario - the original body is destroyed, but some of the cells live on in another form. Would these Xenobots express thanatotranscriptome genes?

[0112] As control we used all the genes expressed in Xenopus embryonic epidermal progenitor cells + multiciliated cells + alpha and beta ionocytes + goblet cells (obtained with Klein tools database) a total of 2635 genes (without LOC genes and mapped to human orthologs - Table 8). From the literature we curated the thanatotranscriptomic genes (336 genes) (Table 9). Then we determined what percentage of transcripts uniquely upregulated in these Xenobots (537 genes mapped to human orthologs - Table 4) are in the thanatotranscriptome set in comparison to the controls (total list of genes from all epidermal cell types - 2635 genes - Table 8). We found that the Xenobot-upregulated genes had more than double (3.5%) of thanatotranscriptome genes compared to control genes (1.6%) (Table 10). Enrichment analysis of these Xenobot genes overlapping with the thanatotranscriptome suggests they mostly belong to immune activation, stress response, and insulin signaling. This suggests that the self-assembly process of theseXenobots following the harvesting of their source cells from an embryo may have similarities to the death process in other metazoan including humans.

[0113] Evolutionary age of enriched Xenobot transcripts

[0114] To test the hypothesis that the basal Xenobots were acquiring more ancient transcriptomic patterns as a result of their morphogenesis and nascent emergent life history, we performed a phylostratigraphic analysis100'10L 102'103, l04‘105. For this analysis we did not need to remove LOC genes, and we did not need any mapping to human orthologs so the fullest extent of the gene lists were used. We used PhylostratR that applied BLAST to compare the whole Xenopus genome to at maximum 5 different proteomes of representative organisms of different ages in each evolutionary tree strata. Subsets of all genes in Xenopus genome associated with each stratum were shortlisted. Xenobot transcripts and control transcripts were then compared against these shortlisted genes. To determine the evolutionary age of transcripts from these Xenobots, we used both the unique Xenobot transcripts (1450 genes including LOC genes without mapping to human ortholog) and all the Xenobot up-regulated transcripts (1812 genes after removing short and long gene duplicates) and compared them to the controls (all the genes expressed in epidermal progenitor cells + multiciliated cells + alpha and beta ionocytes + goblet cells - 3374 genes including LOC genes without mapping to human orthologs). Going as far back as Vertebrata, there was not much difference between overlap of controls vs Xenobot transcripts. However, going further back in evolutionary time showed major enrichment of Xenobot transcripts overlap in comparison to controls for example, Bilateria, Eumetazoa, and Metazoa showed much more overlap with Xenobot transcripts than controls (FIG. 5). In addition, in these (Bilateria, Eumetazoa, and Metazoa) phyla the unique Xenobot genes showed more overlap than all Xenobot up-regulated genes (FIG. 5) indicating that the unique Xenobot genes were enriched for older phyla even in comparison to its own overexpressed genes suggesting specific enrichment of evolutionary older genes. Overall, this indicates that the transcripts upregulated in these Xenobots are particularly evolutionarily ancient genes.

[0115] Discussion:

[0116] In this study, we try to make inroads into the relationships between genomes, transcriptomes, and overall morphology2. This is a fundamental question in biology with manyknowledge gaps concerning the range and plasticity of the latent space around a genome with respect to transcriptome and morphology, and lack of predictive power for the properties of novel constructs20, 106, 107and chimeras25, 108. Tremendous progress in the fields of synthetic biology, synthetic morphology, and biorobotics has made available biological model systems that are unencumbered by a history of environmental selection forces and serve as great model systems in which to understand the emergence of novel molecular-genetic, physiological, and morphological phenotypes.

[0117] In this study we ask the question, how does the transcriptome change in response to cellular collectives acquiring non-evolved embodiments and behaviors in absence of any genomic editing, drug treatments, or heterologous interventions - to understand the top-down influence of morphology over transcriptome. Our goal was to utilize a synthetic living system to identify unique transcriptomic changes in it, in comparison to its native (wild-type) context. For this we exploited the Xenobots platform which includes multiple different kinds of autonomously-moving biobots all derived from Xenopus embryonic cells33’34, 35, 36, 39, 109. We analyzed the basal Xenobots which are autonomously-motile, self-assembling multicellular entities derived from Xenopus ectodermal explants (animal caps)34The Xenopus ectodermal explants have a rich history of serving as a tractable model system for investigating epidermal cell fate specification and cell fate plasticity in response to various chemical stimuli51’52, 53‘54, 53’56‘57. In addition, mucociliary organoids derived from them have helped understand mucociliary epidermis dynamics58’59’60‘61’62’63.

[0118] Recent transcriptomic analysis has looked at epidermal cell fate specification during transformation of these ectodermal explants to the autonomously-moving entities (basal Xenobots)58-64. However, in this study we investigate the transcriptome of these Xenobots in comparison to their native wild-type embryonic tissue using a different transcriptomic analysis beyond epidermal cell fate specification, to understand how these Xenobots’ morphology and behavior affects their transcriptomic changes. We found significantly greater inter-individual gene variability in these Xenobots suggesting adaptive exploration of the transcriptional space to adapt to their novel embodiment and associated stressors. Gene ontology and network analysis of unique (non-epidermal marker) transcriptomic changes showed both, expected shifts in multicellular organization and immune / stress response along with surprising changes in metabolism and sensoryperception of sound and mechanical stimuli. Indeed, these autonomously motile basal Xenobots respond to acoustic stimulation by changing their motion behavior while motile age-matched embryos and passive non-motile Xenobots do not. Physlostratigraphic analysis showed transcriptional changes reflecting evolutionary ancient genes. We also found significant enrichment of thanatotrascriptomic genes suggesting signatures of organismal death in them. Overall, all these transcriptomic changes in these Xenobots suggest that alteration of morphology and nascent emergent life history were enough to strongly affect the transcriptome (morphology / behavior driving gene expression).

[0119] Since these Xenobots are raised in different media concentration (0.75X MMR) as opposed to embryos (0.1X MMR) we first wanted to see if there are any major transcriptome changes in response to the different media concentration. Since the ectodermal explants were excised from embryos at stage 9 and incubated in 0.75X MMR for making these Xenobots, we incubated embryos in 0.75X MMR from stage 9 onwards until age-matched stage to mature day 7 Xenobots. Raising embryos in 0.75X MMR after stage 9 remarkably showed normal embryonic development with no developmental morphology defects and embryos looking similar to their sibling raised in 0.1X MMR. Comparing transcriptome of embryos raised in 0.1X MMR to 0.75X MMR showed no major transcriptome differences (only 6 transcripts out of -24,500 transcripts showed significant differences) (FIG. 6). This was very surprising and suggests that the embryonic transcriptional regulation is very well shielded from external environmental changes. This might be due to the presence of tight junctions in the outmost ectodermal layer which compartmentalizes the embryos from their external environment. If embryos are raised in 0.75X MMR from pre-stage 9 then they do not undergo normal gastrulation and do show developmental defects, suggesting as previously documented that the pre-gastrulation embryos are much more susceptible to environmental differences than a post-gastrulation embryo.

[0120] To deal with false positives we used scRNAseq data base of Klein tools81. However, the caveat is that we had to use scRNA data from stage 22 embryos whereas our age-matched embryos were stage 35. This stage 22 scRNA dataset is the best accessible results we have as there are no stage 35 scRNAseq datasets which we could use to remove epidermal progenitor transcripts. However, by stage 22 epidermal cell differentiation has already occurred64, 81. In addition, toeliminate false positives, we took an extremely stringent approach by removing all genes expressed in epidermal progenitor cells, multiciliated cells, alpha and beta ionocytes, and goblet cells from our Xenobot dataset. In fact, this stringent curation may have resulted in the loss of some true positives but it was worth it to give us good confidence in our results. Overall, our highly stringent list of Xenobot transcripts is a good starting point to look into unique transcriptional aspects of these Xenobots and to begin understanding how genome / transcriptome relates to novel morphologies. In future, single cell RNA sequencing of Xenobots might allow us to look at regional and cell type specific expression patterns in Xenobots and how ectodermal progenitor cells distribution and profile diverge between Xenobots and embryos.

[0121] We compared and analyzed the transcriptome of these Xenobots with their age-matched Xenopus embryos to determine effects of altered morphology and nascent emergent life history on transcriptome. Our results give an indication of the kinds of transcriptional changes induced by change of morphology and behavior (no genomic editing or transgenes). We found at least 537 transcripts uniquely upregulated in these Xenobots (Supplementary Dataset 4). These transcripts indicated enriched functions such as locomotion apparatus assemblies (cilia-based motion and motor proteins), structure formation and multicellularity, stress and immune response, shift in metabolism, and sensory perception of sound and mechanical stimuli (FIG. 3, FIG. 8, Tables 5 and 6). The majority of these shifts were towards evolutionary older systems which likely get suppressed when they are part of the organism. Phylostratigraphic analysis shows the transcripts uniquely upregulated in these Xenobots compared to the wild-type tissue are enriched for evolutionarily ancient genes and systems (FIG. 5). This is fascinating and suggests that cellular functional plasticity can roll backwards along the “ontogeny recapitulates phylogeny” axis, utilizing gene ensembles and systems from their distant evolutionary past that might be relevant in their new configuration and environment. Future work will examine possible links to similar phenomena (a shift toward ancient, unicellular transcripts) in cancer110and potential implications for the relationship between cancer and multicellularityni-112.

[0122] The transcripts uniquely upregulated in these Xenobots compared to the wild-type tissue also showed enrichment for thanatotrascriptomic genes113which are transcripts awakened in cells and organs after the overall organism dies. These transcripts mainly includedimmune / stress response and insulin signals. What this suggests is that these Xenobots have some sort of memory / information of them not being part of “living organism”. One possibility is that this might be evidence that the cells experienced the Xenobot construction event as the death of their parent organism; it is also likely that the thanatotranscriptome is limited by the fact they retained multicellularity; perhaps dissociating the cells and having them reboot multicellularity (as occurs in a different type of Xenobot114) might show a much stronger thanatotrascriptomic response. One challenge in this field has been that it was not possible to test whether these death-induced genes were functionally active. Indeed, what kind of function could even be assayed at the end of life? The Xenobots platform, as a whole, offers a path forward, because in future work, the thanatotranscriptome could be down-regulated via RNAi or morpholinos, to examine whether this would induce any negative effects on their continued survival or Xenobot functionality. All in all, the refactoring of cells after the death of a body provides a fascinating way to engineer an extension of the normal cycle of development, adulthood, and death toward new possible life histories that could cycle between unicellular and multicellular forms11?’116.

[0123] When cells are challenged and need to make quick changes to their gene expression to adapt to novel conditions, they exhibit exploratory adaptation by changing many different genes simultaneously and different cells change different sets of genes to overcome the same challenge 68, 69, 70, 71 When comparing the overall transcriptome of the basal Xenobots and age-matched Xenopus embryos using PCA, the three Xenobots samples showed much wider separation compared to the three age-matched Xenopus embryo samples (FIG. 7). Further, our analysis of gene count variability showed significantly higher inter-individual gene variability in these Xenobots compared to age-matched Xenopus embryos (FIG. 2). This increase in inter-individual gene variability in these Xenobots suggests that these Xenobots might be exploring the transcriptomic space (of possible gene expression profiles) to discover transcriptomes that allow them to improvise and adapt to their new embodiment. This is supported by the top ten most variable transcripts in these Xenobots which show prominent presence (3 genes out of ten) of oncomodulin that encodes a protein usually found only during early embryonic cells and tumor cells and performs widely distinct functions80including maintaining sensory perception, tissue regeneration, immune response, and strong antioxidant properties80. Other genes in the top tenmost variable transcripts in these Xenobots include: tenomodulin which is involved in tissue maintenance by supporting stem cell renewal and preventing senescence and aging117-118-119, adhesion proteins neurotrimin120and salivary glue protein-3 like protein121, A. superbus venom factor 1 an immune regulator122, somatostatin receptor 4 a regulator of hormone and secretory protein secretion123, chloride intracellular channel 4 that belongs to an extremely conserved (across prokaryotes and eukaryotes) family that can perform different and independent functions at the membrane and the cytoplasm including fundamental cellular processes such as regulating mitochondrial function, exosome communication, membrane trafficking, and pH maintenance124,125, and tyrosine aminotransferase which is involved in gluconeogenesis - an alternate metabolic pathway126.

[0124] All of these functions are likely to be useful in the capacity to adjust to novel embodiments and challenges. Xenobots are an intriguing model in which to characterize problem-solving during traversal of transcriptional, physiological, and other unconventional spaces90'127Individuals may explore the option space for many possible paths to adaptations to new ways of existence. Alternatively, the greater transcriptomic differences among these Xenobots may be a reflection of heterogeneity introduced to the these Xenobot population by variability in their manual production process, either simply due to variability in cell numbers between individuals, or as a transcriptional memory of the unique “lived experiences” of each Xenobot128, 129, 130. Finally, it has been observed that embryos in groups assist the morphogenesis of others and develop more uniformly with fewer defects131. While the embryo groups in the present study were small (10 individuals), they may still have some capacity to synchronize their transcriptomes that these Xenobots lack. In future studies, a spatial transcriptomic analysis of individual Xenobots would provide a better understating of this adaptive exploration process.

[0125] We saw not a single transcript overlap between the uniquely upregulated transcripts in these Xenobots in comparison to wild-type tissue and genes representing the mesoderm, endoderm, and axis patterning clusters (Table 7), suggesting that there was no “contamination” of the explanted ectodermal tissue from which these Xenobots were derived with mesoderm, endoderm or axis patterning genes. Likewise, it did not support transdifferentiation of ectoderm cells into other lineages.

[0126] Overall, both functional enrichment analysis and the network clustering analysis showed largely overlapping functional clusters (FIG. 3, FIG. 8, Tables 5 and 6). Even with the stringent subtraction conditions including subtraction of all multiciliate cell genes, we still saw cilia assembly and motor proteins as major enrichment categories in both gene ontology analysis and the network clustering analysis. This suggests that there might be significant upregulation of generation / making of multiciliate cells in these Xenobots compared to embryos. The multiciliate cells in embryos may be much more stable and long-lasting compared to these Xenobots which might be making them at a higher rate due to rapid turnover. This is partly supported by the observation of both proliferation and cell death categories being upregulated in these Xenobots suggesting a higher turnover of cells. Also, previously it has been reported that the density of multiciliate cells is significantly higher in these Xenobots compared to embryos34, this could be another reason why we see upregulation of cilia assembly and motor proteins. It is also possible that there are some unique and subtle differences in cilia assembly and adjoining motor proteins and their functions in these Xenobots’ morphology configuration. Overall, there is definitely a major shift in cilia assembly and motor proteins and perhaps multiciliate cell dynamics in these Xenobots; this remains an area of active investigation.

[0127] Subcategories involved in tissue building, repair, and multicellular organization were ATP binding, calcium binding,, and subcategories in stress and immune response signals mainly included cytokines, heat-shock proteins, and MMPs. It has been postulated that stress / immune response is important indicator of deviation from homeostasis and target morphology states and involved in moving the system by repair and tissue building processes to reach its final state90-91’ 92, 93.94wasinteresting t0see V-ATPase among these genes, as it is not only deeply conserved (evolutionarily ancient) but has also been postulated to be one of the key elements required for generation of multicellularity132- 133, 134, morphogenesis135‘136‘137, 138, and cancer139, 140, 141. These genes represent candidates for control knobs that will be tested for their ability to enable guidance of the self-organization process to produce other types of Xenobots of different shapes and morphologies.

[0128] It was surprising to see sensory perception of sound and mechanical stimuli as an upregulated category in these Xenobots. Initially we thought it might be erroneous or crossassignment of cilia related transcripts but transcripts in this category were not cilia related. They indeed contained transcripts known to be involved in sound and mechanical stimuli perception such as: GJB2 which is a gap junction protein involved in sensory perception (among other functions) and mutational loss of this protein leads to hearing loss in humans and mice142, 143, 144stereocilin143- the mechanoreceptor for sound waves / vibrations, proteins involved in inner ear membrane formation, inner ear ion channels, and many other stereocilia (inner ear hair cells) specific proteins. Importantly, the expression data were experimentally confirmed in a functional experiment. We tested the ability of these basal Xenobots to respond to acoustic vibration stimulus (FIG. 4 and Supplementary Movies 1-6 (not shown)). Observable changes in their motion behavior (FIG. 4 and Supplementary Movies 5 & 6 (not shown)) confirm that they do indeed respond to acoustic stimulus. This change in motion behavior is not due to physical movement of medium or simple mechanical effect of vibration on cilia function (FIG. 4 and Supplementary Movies 1-4 (not shown)), as indicated by controls (immotile bots and ciliated age-matched normal Xenopus embryos, neither of which responded to the stimulus).

[0129] Future work will detail the specific mechanisms leading from stimulus to change in behavior and characterize additional motility patterns that may be inducible by diverse stimuli. Importantly, the field of morphological computing reveal how the physical properties of bodies serve as an integral part of their cognition146Morphology (including material structure) can exploit system-environment interaction to achieve useful behaviors147’148. Advances in this field dissolve simplistic distinctions between physics and active behavior: cognition, especially of non-neural agents, is beginning to be understood as a network of inseparable physical systemenvironment interactions which implement behaviors via analog computation and not explicit symbolic computations that require a conventional central controller149‘150‘151. The Xenobots’ rich mix of ciliary action (itself a highly active multiscale system), internal bioelectric and calcium physiology, and fluid dynamics make Xenobots an exciting model system in which to unravel morphological computing and behavioral responses in novel morphologies with wild-type genomes152, 153'154-155>156>157

[0130] Xenobots behavioral response to acoustic stimulus suggests several points for consideration. First, these basal Xenobots express high levels of acoustic-related genes, and aresensitive to such stimuli, whereas age-matched Xenopus embryos are not. The induction of whole cluster of transcriptomic machinery sufficient for a functional behavioral response, in a wild-type genome (in the absence of synthetic circuits or added induction by novel cell populations), may shed light on evolutionary developmental biology consequences of changes of morphology and nascent emergent life history. This in turn suggests a future research program to attempt to identify novel sensory-behavioral capabilities in other minimal, organoid, and bioengineered living constructs which may heretofore have gone un-noticed. Second, from the perspective of biorobotics and the use of this model system as useful synthetic living machines, the ability to reversibly modulate their behavior via acoustic stimuli opens fascinating possibilities for control. Work is on-going to map the frequency, waveform, and amplitude domains and link them to predictable changes in behavior (both via motility and via gene expression and physiological state). Real-time modulation of the hydroacoustic parameters in closed-loop controllers is a possible path forward for guided functionality in bio-robotics in Xenobots and beyond.

[0131] What would the role of such sensory perception of sound and mechanical stimuli be in such an aneural system like these Xenobots? And how does that regulate its morphology and movement behavior? A role of acoustics in aneural systems is not unprecedented. Recent studies have shown that coral larvae which are aneural mass of cells with cilia-based movement similar to these Xenobots, perceive reef sounds and distinguish acoustics of healthy reef vs unhealthy reefs and preferentially migrate towards healthy reefs158-159‘160‘161. In addition, they also distinguish acoustics of mother reef vs other healthy reefs and preferentially migrate towards mother reefs. It is not known how (mechanism of action) they are able to achieve this acoustic perception and it remains an area of investigation. Thus, it is possible that such acoustics-based sensing and movement is one of the evolutionarily ancient mechanisms of perceiving the environment and reacting to it via motion before nervous system evolved leading to coordinated movement behavior. Given the presence of machinery for perception of sound and mechanical stimuli in these Xenobots, acoustic perception and response similar to coral larvae should be examined in Xenobots.

[0132] Although it is well known that yolk platelets provide the energy source and essential nutrients for Xenopus during embryonic development162, l63‘164, little is known about themetabolism happening within the embryos. Some evidence suggests that during early Xenopus embryonic development (pre-gastrulation) the pentose cycle and glutamate-aspartate cycle are predominant165, 166, 167The Embden-Meyerhof pathway and Krebs cycle although functional, contribute little during pre-gastrulation stages but their relative contribution shows major increase after gastrulation165, 166, 167. Interestingly, in the basal Xenobots there seem to be upregulation of different metabolic processes such as gluconeogenesis (which has been shown not to occur in embryos166) and ketone metabolism and metabolic process breaking down cholesterol and steroids. These were associated with presence of many liver-related transcripts involved in metabolism, although these Xenobots have no liver. It is interesting to see upregulation of gluconeogenesis and ketone metabolism in these Xenobots which are highly conserved metabolic processes of generating glucose from non-carbohydrate carbon substrates and is found in microorganisms, bacteria, fungi, plants, and animals168‘169‘170‘171, 172‘173‘174, suggesting that metabolically these Xenobots might be shifting to an ancient mechanism to meet their energy requirements. This perhaps is due to the majority of yolk (energy source of embryos) being on the vegetal side of the embryo and since these Xenobots are derived from the animal side, they may have disproportionately lower yolk, thus needing to manage energy use given the limited amount of energy source. Interestingly, V-ATPase, the deeply conserved proton pump known to be involved in many functions (generating multicellularity, morphogenesis and cancer133, 134, 135, 136-137, 138, 139, 140, 141, 175^ js ajso|<nown t0 reulate mitochondrial metabolism176, 177and was found to be upregulated in these Xenobots as part of metabolism cluster, thus suggesting an overall shift in the metabolism in these Xenobots. Further detailed and deep investigation first into the metabolism and energy usage of embryos and then of these Xenobots will help reveal the shifts in metabolism. Understanding this will allow us to manage the energy requirements and control the overall nascent emergent life history of these Xenobots. Along these lines it has been shown that external supply of glucose can drastically increase the life-span of these synthetic biobots34.

[0133] From the gene ontology and network analysis categories it seemed that the basal Xenobots had activated evolutionarily-ancient functions. To test the hypothesis that these Xenobots were acquiring more ancient transcriptomes as a result of their morphogenesis and nascent emergent life history, we performed a phylostratigraphic analysis. Here we compared the control transcripts(all transcripts from epidermal progenitor cells + alpha and beta ionocytes+ multiciliated cells+goblet cells) with transcripts uniquely upregulated in these Xenobots. The controls and Xenobot transcripts were equally enriched until Vertebrata strata. However, although the controls have a much larger transcript dataset (3374), the Xenobot transcripts (1450 uniquely upregulated transcripts) were much more enriched than controls in the older strata Bilateria, Eumetazoa, and Metazoa. This again suggests that these Xenobots have upregulated transcripts and programs that are evolutionarily ancient. Along those lines, the cellular physiology of these Xenobots appears to be closer to evolutionarily ancient organisms with a single layer of differentiated cells with no nervous system and vasculature and cilia-based movements. The phylostratigraphic analysis also shows that these Xenobot genes are evolutionarily basal and found in organisms with simpler body plan. This fits in line with the observation of shifts in metabolism, sensory perception process, and multicellular organization towards evolutionarily ancient processes. This indicates that when cells and tissues are liberated from their organisms, they can tap into their evolutionary history to kickstart ancient processes and adjust their energy requirements to reach novel morphology goals.

[0134] Taken together, the results of this study reveal a control arc that is complementary to the conventional control of morphology by gene expression: to some extent, the genome is being used as a “resource book” by the cellular collectives to kick start various processes as per the conditions and needs of atypical multicellular configurations. Plasticity in form, function, and molecular physiology is gaining interest, as it impacts on basic evolutionary biology, biomedical applications, and synthetic bioengineering91, 178> 179, 180, 181, 182, 183, 184, in way that augments current bottom-up approaches of DNA engineering and synthetic biology circuits. We see this work as part of a future roadmap in which a wide variety of synthetic systems is examined to see ways in which living organisms adapt to new environments on-the-fly and solve evolutionarily-novel problems in transcriptional, morphological, and physiological spaces. Discovering how groups of cells manage their molecular-biological and metabolic resources to achieve adaptive ends in novel scenarios will facilitate the ability to control and generate different morphologies from the same genome by reshaping the morphological landscape to create new attractor states, control their behavior and responses and manage their nascent life histories. Such knowledge will serve biomedical purposes (controlling multicellularity during regeneration, birth defects, and cancer), advance bioroboticsand bio- Al (design systems with morphology and behavior to solve specific problems) and explore the space of possibilities of life-as-it-can-be.

[0135] Materials and Methods:

[0136] Animal Husbandry:

[0137] All experiments were approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) under the protocol number M2023-18. Xenopus Laevis embryos were fertilized in vitro according to standard protocols185and reared in either 0.1X Marc’s Modified Ringer’s solution or 0.75X Marc’s Modified Ringer’s solution (only post stage 9). Xenopus embryos were housed at 14 °C and staged according to Nieuwkoop and Faber186.

[0138] Basal Xenobot construction:

[0139] The basal Xenobots used in this study were derived from Xenopus embryonic ectodermal explants, also known as “animal caps”, as the starting material51, 52, 53, 54The basal Xenobots are the most basic version of Xenobots (no sculpting or engineering or mixing of different tissues)33. These Xenobots were constructed manually from Xenopus Laevis embryos as described previously34Briefly, in vitro fertilized embryos were reared at 14°C in 0. IX Marc’s Modified Ringer’s solution (MMR) until Nieuwkoop and Faber stage 9186. At stage 9 embryos were transferred into a petri dish coated with 1% agarose made in 0.75X MMR and containing 0.75X MMR solution. Using surgical forceps, the vitelline membrane was removed from the embryos and animal cap epidermal progenitor cells were cut out of the embryos as per previously described protocols51-52,53, 54The ectodermal explants were placed on the agarose with inside surface facing up. Over a period of 2 hours the ectodermal explants round up into a spherical shape. These are then incubated at 14°C on 1% agarose in 0.75X MMR for 7 days with daily cleaning. By day, 7 the tissue differentiates and transforms into an autonomously moving synthetic epidermal entity (basal Xenobot) used in this study. These Xenobots was not further modified, and were used as mature at day 7.

[0140] RNA extraction:

[0141] Total RNA was extracted from either stage 35 / 36 Xenopus Laevis embryos or from autonomous moving fully mature Xenobots (7 days post extraction of ectodermal explants fromembryos). For stage 35 / 36 embryos we had 3 replicates for each experimental sample and each sample contained 10 pooled embryos. For Xenobots we had 3 replicates for each experimental sample and each sample contained 50 pooled Xenobots - necessary to gather enough RNA from markedly small mass of Xenobots (FIG. 2A). Total RNA was extracted using Tri-reagent (MRC, Inc) as per the manufacturer’s protocol. Total RNA quality and quantity was measured using NanoDrop spectrophotometer (Thermo Fisher Scientific).

[0142] rRNA depletion and RNA-sequencing:

[0143] Total RNA was sent to the Tufts Genomic Core. RNA quality was measured with bioanalyzer, and high-quality RNA was used for library preparation with the Illunima Stranded Total RNA with Ribo-Zero Plus. Libraries were multiplexed and single-end 75-nucleotide sequencing was performed with 30 million reads per sample on Illumina NextSeq 550 High output. Raw read files were used for initial analysis at Bioinformatics and Biostatistics Core at Joslin Diabetes Center.

[0144] Transcriptomic analysis:

[0145] The reads were trimmed for adapter “CTGTCTCTTATACACATCTCCGAGCCCACGAGAC (SEQ ID NO: 1)” and polyX tails, then filtered by sequencing Phred quality (>= QI 5) using fastp187Xenopus laevis genome sequences and gene annotation were downloaded from the NCBI genome database, version 10.1. GenomeGenerate module of the STAR aligner188was used to generate the genome indexes. STAR aligner option was set to sjdbOverhang = 75 for 76-bp reads, as was ideal. Adapter-trimmed reads were aligned to the genome using STAR aligner with the two-pass option. Reads were mapped across the genome to identify novel splice junctions in the first-pass. These new annotations were then incorporated into the reference indexes and reads were re-aligned with this new reference in the second pass. Gene expression was estimated from the gene alignments using RSEM tool for accurate quantification of gene and isoform expression from RNA-Seq data189. Low expressing genes were filtered out by only keeping genes that had counts per million (CPM) more than 0.6 in at least 3 samples. There were 28,009 genes after filtering. Counts were normalized by weighted trimmed mean of M-values (TMM)190Voom transformation191was performed to transformcounts into logCPM where logCPM=log2(106*count / (library size*normalization factor)). PC A analysis was performed to provide an overall view of the data. Differential gene expression analysis between groups was performed using limma192.

[0146] Inter-individual gene count variability analysis:

[0147] A schematic of the method is shown in FIG. 2A. The analysis was performed on gene count data from the 3 Xenobot pools and 3 age-matched Xenopus embryo pools in MATLAB. The 28,009 genes were ranked by the values of the means of their counts across all 6 pools of Xenobots and Xenopus embryos. Genes with a count of 0 in any of the 6 pools were removed, leaving 25,276 gene transcripts. Because the count value of a given gene in a given Xenobot pool represents the mean value of all the individual Xenobots in that pool, the standard deviation of the 3 Xenobot pools gives the standard error of the means (SE) of the Xenobots, and likewise for the age-matched Xenopus embryos. The SE is related to the number of individuals per pool (n) and the standard deviation of the individuals within the pools (o) with the equation SE = o / sqrt(n). By multiplying the SE by sqrt(n) (where n = 50 for Xenobots, n = 10 for age-matched Xenopus embryos), G can be calculated. Dividing o by the mean count value of the 3 pools gives the coefficient of variation (CV) for Xenobots (CVX) and for age-matched Xenopus embryos (CVE). The CVs for all genes in the Xenobots and age-matched embryos were plotted as histograms with the area under the curves normalized to 1 (FIG. 2B) and the two distributions compared with a Wilcoxon Rank Sum test in MATLAB, returning p=0, indicating highly significant difference between the distributions. The ranked gene list was then split into equal-size bins (percentiles) and the fraction of genes in each bin for which CVX > CVE was calculated and plotted in FIG. 2C, with black line indicating fraction of 0.5. The fraction of all genes for which CVX> CVE was found to be 0.9606 and was plotted as the red line. To assess whether any bin values were outside the range of values expected by chance, a permutation test was used to determine the typical distribution of bin values around the red line. First, the order of CVX - CVE gene pairs list were shuffled, and the bin analysis was performed on the shuffled list to determine a set of bin fractions. This was repeated 1000 times for different shuffles to produce a distribution of bin fraction values for each bin. To determine statistical significance of the true bin values’ differences from the red line, each true bin fraction value was compared to the distribution. The p value was defined as the proportion of thedistribution that was further (in absolute value) from the distribution mean (red line) than the true bin fraction. Bins with p values of p<0.05 were deemed statistically significant and were colored dark blue; otherwise, bins were colored light blue, revealing significant variation of bin fractions across gene count percentiles. To generate the list of top 10 most variable Xenobot genes CVX values were used. Genes for which any transcriptome in the group of 3 replicates had a count value of 0, and genes for which all count values in the 3 replicates were <10, were excluded.

[0148] Functional Enrichment Analysis:

[0149] To understand the biological processes and pathways enriched in RNA-seq comparisons, we performed functional enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID)83‘84Gene lists of interest (significantly differentially expressed genes with false positive genes subtracted) were uploaded to the DAVID Analysis Wizard. Functional Annotation Clustering was performed to identify significantly enriched functional annotations and these annotations were grouped into related clusters based on shared genes. Stringency classification was set to the default recommendation of “Medium”. Each individual annotation was assigned a p-value and each cluster of annotations received an enrichment score, which is the geometric mean (in -log scale) of member's p-values in a corresponding annotation cluster. Enriched functional clusters were ranked based on their group enrichment score.

[0150] Xenobot network clustering Analysis:

[0151] We performed a network analysis for identifying active functional biological modules85’86. We combined gene expression and interaction data, we extracted the xenobot protein-protein interaction (PPI) and we applied network embedding followed by clustering similarly to87, 88. We used the MNMF network embedding and clustering algorithm193and imposed a number of clusters equal to 15. To construct the synthetic proto-organism network, we filtered the genes of interests, found the human orthologs in the different conditions using HCOP194Then, we extracted the corresponding network using the STRING database195. The detected functional modules after network embedding and clustering are enriched using g:profiler89

[0152] Phylostratigraphic analysis:

[0153] We used phylostratR to perform the phylostratographic analysis of xenobot transcripts196. The focal species was set to ‘8355’ for Xenopus Laevis. This package automates several key processes in evolutionary analysis (1) it constructs a clade tree from species listed in UniProt, aligned with the latest NCBI tree of life; (2) it prunes the clade tree such that it retains a phylogenetically diverse selection of representatives for each phylostratum; (3) it compiles a database of protein sequences from hundreds of species from the constructed clade tree and sourced from the UniProt Proteome database (in total 329 species in our study, including the yeast and human proteomes that we added manually) (4) it applies similarity search by conducting pairwise BLAST between the proteins encoded in the focal species (here the Xenopus Laevis) and all proteins from all target species within the clade tree; (5) it determines the 'best hits' and infers homology for each gene of the focal species against each target species; (6) it assigns each gene to a phylostratum correlating with the oldest clade for which there is an inferred homolog. Genes found exclusively in the focal species are identified as orphan genes and are categorized under the phylostratum ‘Xenopus Laevis'. The phylostratas we selected are: All living organisms, (Eubacteria, bacteria and their descendants), Eukaryota, Opisthokonta, Metazoa, Eumetazoa, Bilateria, Deuterostomia, Chordata, Vertebrata, Gnathostomata, Euteleostomi, Sarcopterygii, Tetrapoda, Anura, Xenopus, Xenopus Laevis. We then mapped the age of genes with our xenobot overexpressed genes in the different conditions and the control.

[0154] Mapping to human orthologs:

[0155] For the network analysis and comparisons with the thanatotranscritome, we mapped Xenopus genes to their human orthologs using HCOP197.

[0156] Acoustic Vibration stimulus setup:

[0157] For controlling and providing acoustic vibration stimulus, we used a setup (FIG. 4A) containing a speaker (RECOIL RW8D2 Echo Series 2-ohms, 400Watts) connected to a digital audio amplifier (Kinter K3118 Texas Instruments, 20Watts output) operated using a laptop computer. An online tone generator (szynalski.com / tone-generator / ) was used to set a sine type wave with frequency of 300Hz. The computer volume and online tone generator volumes were set to 100%. A P60 petri dish coated at the bottom with 2-3mm layer of 1% agarose made in 0.75X MMR was placed on top of the speaker. 14mls of 0.75X MMR was added into the petri dish. Thevolume of amplifier was set to 3.5 at which point no physical movement of media in the dish was observed in response to acoustic vibration stimulus. These settings were kept unchanged throughout all acoustic vibration stimulus experiments. An iPod camera mounted on a Stemi SV6 dissection microscope eyepiece was used to capture time-lapse imaging. Subjects (day 1 Xenobots, day 7 Xenobots, or age-matched stage 35 embryos) were placed in the center of petri dish. Timelapse recording was conducted for 10 mins before initiation of vibration stimulus, followed by 10 mins of vibration stimulus, and lastly 10 mins after vibration stimulus was tuned off.

[0158] Behavior imaging and analysis:

[0159] Movement behavior was recorded using an iPod camera mounted on a Stemi SV6 dissection microscope eyepiece. ProShot app was used to capture time-lapse imaging of one frame every 2 seconds. Time-lapse recordings were compressed to 30 frames per second movie which results in 30 second movies of 30 mins of motion tracking (10 mins before stimulation, 10 mins during stimulation, and 10 mins after stimulation). Thus 1 second of movie = 1 min of actual time. Motion tracking of behavior captured in time-lapse videos was done using Ethovision XT v.15 (Noldus Information Technology). A scaled background image was used to set scale for distance. Motion tracking was completed and recorded. Any tracking errors were manually corrected in the track editor. All X-Y coordinates of each subject for every frame across the time-lapse video were exported for data analysis.

[0160] Data availability:

[0161] RNA-sequencing data generated during the study are available in the NCBI GEO public repository with accession numbers (GSE275807 and GSE277182).

[0162] Table 1 - List of genes significantly changed in stage 35 / 36 embryos raised in 0.75X MMR in comparison to those raised in 0.1X MMRSignificant P value with FDRUP inO.75X MMRGene Gene biotype Gene product nameLOC121401044 IncRNA uncharacterized LGC121401044 LOC121398999 protein_coding interferon-induced very large GTPase 1-like LOC108703876 protein_coding A-kinase anchor protein 9, transcript variant X4

[0163] Table 2 is shown in FIG. 11: List of upregulated genes after klein tools epidermal sub

[0164] Table 3 - List of upregulated genes after annotation and duplicate subtraction

[0165] Table 4 - List of genes after klein tool all individual cells sub.A4GNT ATP8B3 CDKL4 DLX3 AVPR2 CDKN2B DLX4 ABCA12 B3GALT5 CDS1 DNAAF11 ABCB5 B4GALT1 CFAP144 DNAAF6 ABO BARX2 CFAP206 DNAH14 ACOX1 BBOF1 CFAP221 DNAI1 ACP3 BCL2L12 CFAP251 DNAI3 ADA2 CFAP299 DNAI4 ADAD2 BCL3 CFAP46 DNAI7 ADH7 BDH1 CFAP52 DRCC1 ADPRM BICDL2 DSG1 ALOX12B BMP2K CFAP58 DTX3 AMDHD1 BOK CFAP68 DYNAP AMY2A BPI CFAP73 DYNC2I1 ANKK1 BSND CFAP74 DYNLT5 ANKRD35 C10orf67CFAP97D2ANKRD53 C20orf85 EDN2 ANKRD65 C22orfl5 CH25H EFCAB6 ANKRD66 C22orf23 CHDC2 EGR1 ANO1 C4orf45 CIB3 ELF 5 AOX1 C6orfll8 CIBAR2 ELOVL3 AP1G2 CAPS2 CLDN23 ENKUR AREG CAPSL CLEC2B EPPK1 GASP 10 CL1C3 ERAP1 ARHGAP25 CBLB CNBD2 ERICH3CBLC COL6A5 ERICH6B ARHGAP9 CBLN4 COX15 ESYT3 CCDC107 CRACR2A FAM115C ARHGEF38 CCDC108 CREB3L4 FAM162B ARHGEF4 CCDC13 CROCC2 FAM166C ARMC2 CCDC135 CRYBG2FAM167A A RM H l CCDC148 CSDC2ARMH4 CCDC178 FAM81B CSGALNACT1ARSD CCDC180 FAM83C ASAHI CCDC24 CST3 FAM83E ATF3 CCDC78 CYP27A1 FAM83F ATG16L2 CCDC89 CYP4F22 FAM83H ATP13A2 CCL19 DCST1 FAS ATP6V1F CCNO DDIT3 FCGR1A CD4 DENND3 FCN2 ATP6V1G3 CDH26 DHX35 FHIP1A ATP8A1 CDHR4 DLEC1 FL ACC 1FN1 GSG1 KIF16BMGC81152 FOSB KIF27GTF2IRD2BFRMPD2 KLF15MGC82715 FSIP1 HELZ KLF3FUCA1 HES2 KLHL26MGC84752 FUT2 HEXD KRT19FUT6 LDHB MGST2 HIST1H1EFXYD3 LDLRAD1 MINDY4B GAB3 LEKR1 MLKL HLA-DMALEMD1 MMP1 GADD45ALENG9 MMP10 HSD17B11GALE2 LEP MMP11 GALK2 HSF3 LEXM MMS19 GALNT3 HSPA1B LHB MOCOS GANG HTR3A LIF MORN1 GAS2L1 HTR7 MPZ LKAAEAR1GAS2L2 IER3 MPZL1 GCM2 IFITM3 LMBRD2 MR1 GFUS IFNAR2 LNX1MSANTD1 GGT1 IFNGR2 L0NRF1IFNLR1 LPAR3 MT1A GGT6 IGF2 LRFN4 MUC17 GJB2 IGFBP2 LRRC56 MYH7 GJB4 IGSF3 LRRC69 MYO15A GJB7 IKBKE LRRC74B NAT 16 GLB1L2 IKZF2 LRRC9 NECTIN4 GLIPR2 LRRIQ4 NEK10 GLO1 IL17C NFAT5 GMDS IL17RA LY86 NFATC2 GNA15 IL17RE LYPD2 NFKBIZ GNE IL17REL MACC1 NIP AL 1 GNG10 IL1B MAP3K14 NIPAL2 GP2 IL22RA1 MAPK15 NKX2-3 GPR116 INHA NMUR2 MARCHF10GPR156 INPP5E NODI GPR174 INSL5 MESP1 NOS2 GPR37 IRF3 MGAT4DGPR37L1 IRX4 NOXI MGC131348GPRC5C ITPRTD2 NOXO1 GPRC6A KCNA3 NPL MGC52622KCNE1B NUP160 GRAMD1CKCNQ1 NXPE2 MGC53199KDM7A NXPE4 GRAMD2BKIAA1161 ODAD2 MGC68910GREB1L KIF13A ODAD3ODAD4 REPS2 SLC66A1 TIRAP OTOG RGL1 SLC7A11 TLR1 OTOP2 RGS2 SLC8B1 TMC2 OXCT1 RGS22 SLITRK6 TMC7 PADI1 RHCG SLPITMEM125 PANK1 RH0BTB1 SMPD3PAQR8 RII D1 SMPDL3ATMEM130 PARP4 RIPK3 SOAT2PATJ RITA1 SOCS3TMEM212 PCDH20 RNF222 SOWAHAPCK1 RNF223 SP6TMEM213 PCSK4 RSPH1 SPACA9PDE9A RSPH14 SPAG17TMEM232 PEX11G SAMHD1 SPATS 1PHF19 SCNN1G SPDYCTMPRSS11D PHTF2 SERIN C2 SPRYD3PIH1D2 SERPINB4 SRMSTMPRSS13 PIK3API SESN1 STAB1PKC1 SGMS2 STAT3 TNF PKD1L2 SH3KBP1 STEAP1 TNFAIP2 PKDREJ SH3RF2 STK33 TNFRSF9 PKN1 SIGLEC1 STK38L TNFSF10 PLAC8 SKAP2 STKLD1 TNIP2 PNPLA1 SLC10A2 STMND1 TNIP3 POLR2L SLC11A2 STPG2 TNK1 POU2AF2 SLC14A2 STRCTOGARAM2 POU2F3 STRTP2SLC16A12PPARG SUCLG2 TP73 PR0M2 SLC18A1 SWAP70 TPMT PROSER2 SYCP2 TPRG1L SLC25A22PRRG2 SYCP2L TPTE PRSS21 SYT15 TPX2SLC25A37PRSS27 SYT8 TRAF1 PRXL2C TANKSLC25A48 TRAPPC3L PSME1 TBATAPTGER4 SLC26A2 TCAF2 TRERF1 SLC26A4 TCHP TRIM 16 PTPN18 TCP 10 TRIM69 SLC30A10PTPRJ TDRD5 TRPM2 SLC35G1 TEX26 TRPM5 RAB11FIP1SLC37A1 TFAP2C TRPV6 RAB7B SLC41A3 TFE3 TSPAN15 RAD50 SLC45A3 TGM3 TSTA3TTC12 RASGEF1ASLC47A2 TGM5 TTC21ATTC6XB5809687 XB5993457 TTLL10TTLL8XB5810926 XB980013 TTLL9TXNRD1 YPEL2XB5811292UAP1L1 ZC3H12A UBE2U ZFYVE28 XB5812047ZP4 UPK1AXB5815362USP42USP43XB5832479UTS2BVHL XB5857145VPS13CVSIG8XB5863530WDR27WDR49XB5872520WEE2WNK3XB5880541WNT10AWNT16XB5897453WNT6XB1001290 XB5899881XB13580952 XB5922676XB 22061511 XB5932119XB22063314 XB5949052XB22064002 XB5949738XB22164552 XB5952314XB22164556 XB5953580XB22169588 XB5957062XB5717875XB5965586XB5740125XB5969033XB5799088XB5993342

[0166] Table 5 - Functional enrichment of categories for upregulated transcripts after all cell type sub.Category P valueCilia / cytoskeleton 2.90E-09Ciliopathies 3.42E-12Immune response 7.52E-06Motor proteins 5.26E-04Membrane proteins 5.86E-05Immune signaling 0,003837227Metabolism / biosynthesis 0.003817847

[0167] Table 6 - Network clustering analysis clusters after all cell type sub.CategoryCilia / cytoskeleton / ciliopathiesKetone metabolism, EGF / EGFR signalingSensory perception of sound and mechanical stimuliInterleukins / serotonin signaling / GPCRsImmune signaling / death processesNucleotide sugar metabolismECM / proliferation / multicellular organizationMetabolims / biosynthesisimmune response / Stress responseMetabolism - cholestrol and steroids

[0168] Table 7 is shown in FIG. 12: List of Mesodermal -Endodermal and Axis patterning genes.

[0169] Table 8 - List of control genes - epidermal proj+all cells.VGLL1 ATP11C UQCRC2 COLGALT1ADRB2 WDR34TTPA012I10 KIF13B ACYP1TEGG023J21FGF14 CCKAR CISD2 C3ORF14 PAXX PRR15LALFM2 TTLL13P VEGFD TNEU136G08SMC6 CCDC129 CHST4 REEP3 HPGD FBN1TM4SF1 HSH2D AGRP TMEM238SDF2 UNC45A OMP1 ELOC CFAP65 TRIM44 C17ORF97MAP9 TTGB4 PEX2 MRPL43 C2CD2 DNAH3 MGC84061LMNTD1 FAT2 ODF3 ENCI GA D I MGC69416 HSP90AA1MPP7 GDC 14- NT5M COPA NIP ALA ALPHA CCDC83 MIDI SLC43A2 ESRP1 STAP2 VCAN CEP41 SMPD1 RUVBL1 SEC23A BTN2A1TMEM218 HOXB4 ENTPD6FAM 160 AlRNF128 SIRT3 ATP 1 Al A GAN PPP1R9A AK7 HSP90B1 ITSN2 PPM1F ATP6V0E1 KREMEN2TRY10 TMEM38BPLXNA2 CCR5 ATP2A3 SPATA48 HRASLS PPP1R32 ARRDC3 TFCP2 LUZP1 CYP4B1 BOD1 ATP5A PNP WASL MGC69473COL28A1 MRPL57 SAMD12 CCDC30 ATAD3 STX19 LIMD2 KLP4 SLC38A2 GPHA2 DQB1 AP3B1 UCMA MAP4K5 SCEL APX ZDHHC6 LRRC61 TNFRSF21SOCS5 FUZ CACNA2D3SAMD15 CA5B GPD1L SLC41A2 C4ORF45 TMBLM4 SLC2A12 LRBA OPTN KIAA0513 SLC44A2 CHKA UNC119B APPL2 ASAP3 TADA1 POSTN MGC64589DZANK1 XSIISLC25A46 RASL11B CCDC153 SP7FABP7 PM20D1 EP3 SECISBP2LKTN1 RETREG1 NBEAL2 PLEKHA1 AS3MT PRAM1 TRAF3LP2 SMPX DGKATMEM115 RAB27A COPB1 RINICOL26A1 COXI NUP50 POLE4 GABRA6 CLRN2 PRIMA1MGLL RBMS2 LAMT0R1 ZMYND12ABCB6 GDPD5 MY05B CC2D2A RHBDL2 SYTL2 CMPK1 TCTN2 EFCAB2 KRT76 ACSBG2 STAM2 KCNJ15 MGC82342GOT! MR0H1 FHL2 CRIPT EEF1AO GABRD PACRGL DOCK5 CRACR2B C15ORF65DNAJB13 AIRE RELT TIMM9 CNOTIO MMP3 C19ORF12GSTP1 CCDC146 TBC1D9 TCTN3 TBRG1 YIFIB RUVBL2 FOXO3 BTC HBA CETN3 SVIP COX7A2L CHURC1C10ORF143LGALSL NCBP2MGC147188 CC2D1A BRK1MGC132021SPAG6 ARHGAP32MA AT SI AAED1 DSCR1 C4ORF19 VAMP3 MGC81785 SLC25A34KIF1C CFAP97 TAF1B LAMB4 TGFBIMGC115231 CAT2 SRPRAMGC68680RAB28 MEIG1 PDIA6 USP20 ESRRA EVI 5 CIDEA CROCC C4ORF3 PTPA B3GALT4 CTTN PC CFAP54 GALNT4 PPP2R5C HACL1 IFT20 TTC13 HMGCS1TMEM127 MGC82022 ETS1 CSRP1H0MER2 LRRC18 GABARAPL2 TMEM63CARC1 R3HDM4 BLNK STK10 TRAF3IP3PRRC1 SFRS15 CROT MGC82636MLX TPD52 TMSB4 ZDHHC12 TECR CCDC34 AP1S1 GALC RNF152 FLJOO38O IFT74DNAJB1 PARP14 GCH1 LAMTOR2ABI1 ANKAR HEATR4 TRAF3IP1 MAML3 ERLEC1 CAMKV 7-Sep RASAL1 KIAA0205TMEM39APHLDB3 AK9 ATF5 NCCRP1 SH3BP4 NTPCRFAM 169 A INPP5F PITPNM2 ARG2TTC5 RIBC2PXDC1 SPECC1L ABCC3 MGC 147226PARD3B GNG4 ARL2BP INTA6 KCT2 SLC25A3 S0X7 ALAS1 NIT2MARVELD2 LRRC72 SOCS4 BAHCC1PGM3 TBC1D16 TAD A3 COL6A1 PDLIM7 MT ACAT1 LAPTM4A PPIB DNAH1 HPDL SLC35B1 STK39 SSTR5 LARP4 C6ORF132LRRK1 FPR2 MAP2K1 TEX33ARHGAP6 MGC131121HDDC3 RNF32 FCGBP KCNQ4 ARHGEF26 C20ORF196TACR1 SMAP1 GFPT1 PRKD1 PLSCR2 BBS9 TG0LN2 TMBIMI RAPGEF3 PRSS1 CFAP36 PDIA3 LY6G6C PPP3R1 POL OVOL2 ACSF2 HEXDC GENE PHB DNAAF4 CDCP1 SPTLC3 HYDIN MGC131037 MGC 53705BAHD1 PARVB GRHL3 ZNF414 CLPTM1 GK ZNF827 STAT1 ENDOU CBWD2 ABCB9 MBTPS2 KCNIP1 TNEU113N12SLCO2A1 TAGLN2 RAB23 M0B2 LRRC43 TMEM43 GPR65 CCDC87 TEKT1 ATP 1 OB TMEM154RBM11 EMX2 AP1B1 LUM CHMP4C VTCN1 CYB5D1 HTATIP2 TM9SF3 CDC42SE1 MGC82073IL17D COROIC LGI1 CLDN1 TNPARHGEF28 TMEM2 DHRS13 TSPAN6SNAP29 TUBA POLQ MGC80358DNAJC18 MGC 84053 NAALADL2ERO1A NEK1 RIN3 CLTC AFAP1 DNAH12 TMBIM6 COL5A2 NAGA C10ORF67ATP1B4 NCOA2 ARHGAP40ELF3 IST1CYB561D1 AOPEP IQCK ECM1 TEKT2 FBXW9 GLRX APOE AMIGO2 ABCB7 BLOC1S2 TRAM2 ALDH1L2 FZ3 CCDC160ZNF474 WIPF3 F. ME6 MGC115716HADHB CHMP2B END0D1GUCY2F NF2 MUC4 FURIN RAB3D MOCS2 IFT81 C2CD3 EPB41L5 ISOC2 RNF11 TPRG1 DSP KI AA 1109 CERS2 RCN1 ANKRD50 SLC30A8 LRP2BP TYR03 XHOXC8TGAS104B22FLn PLEKHG3 IFT88 SFXN1 MFSD6L TMC6TGAS115G12VWA3B REEP5 FAM189A1MRPL46 TSTD1 PAFAH1B3AFTPH MY018AGORASP1 METTL24 CIB1C22ORF15ALCAM GMPR2MGC75785 MYOF ARMC9 GRK6FA 129B GOLGB1 PIP4K2C DYNC1I1 FAM83A YWHAQ TNS3 HPCAL1 SPARC EPB41L1 CALM1 TTC39A F13B DLGAP4 SLC25A5 KIF9 TAF1C FREM3 TLR8 HERPUD1 CD69 MSLN MOB3B IN PP I CD40LG CHMP2A MARS2 MRPS33 RREB1 P3H1 RUFY4 CLTA WDR5B STK31 INF2 DNAJC19 CCDC166 PNPLA2 CEP89 REC8 CN2 C0X5A PTPRN2 CPPED1 HMX2 ADGRF5 KIF6IVNS1ABP PYP FAM83B ATP5DKCNK5 GRASPTEGG036F07CCDC105 MKRN1 ACSL1 STXBP2 MALL C2ORF50 PLPP3 PLEC GNA14 MSX2 SH3RF1 BAG4 TRPV4 CPNE1 VSIG10L ZNF185 SLC12A4 RAPH1MGC76224PTK6 MKI. I HKI LIG4 VPS29 ATP5J2 DGKQ ALDH1L1 ITPR3 BAMBI CDC42EP3ACTB LRRIQ1 ITGB1 USP34 TEKT4 LRRC63 PPARGC1ANANOS3 PLS3 TNFRSF6 FBXL7 MRPL51 DCDC2 KCNK6 TP53I3 LPD 0T0P3 EPS8L1 FLRT3 PDCD6IP NDUFV1 CCDC103 RNF121 CD151 RSU1 FOXB1 IL4R TLK1 AADACL4 PLCL2 ARCN1 SLIRP PSCA C9ORF16 NCKAP1 CFAP44 LITAF KLF5 ARMC3 CCDC112 FBX015 RS ADI SYNE1 CCL21 USP32 SDF4H1KESHI YDJC RAP2B ADAMTS15MCM10 EFC AB 7TGAS081I01 GOLPH3L AARSD1 SGK3FN3K RSPH9 LIMA!SHANK2 PIGO RIPK4A TNEU058C09IDH3B NPHP1TMEM263 PP2D1 XIRF KAZNTMEM72C11ORF49 GADD45B MGC82408EFC AB 10NGB IL17RC MGC78865 EPB41L4ATMIGD1 ZPLD1 ATPBS XMT CCDC170 NT5DC2 NEURL1 DHPS XTP11 NEDD9FKBP7 DYNC2H1 MGC68592ERICH2 UGCG GENEB LRRC6 COG4 GOPC OVOL1 ADAM9 FAM234B TPD52L1TEGG038A23 USP33 GOLM1 NDFIP2PDCL3 SLC2A10 PDE7B C7ORF31 DOCK1 UBC ATP6V1B2PPP2CA UPK3B NDUFS5 TEX264 CELSR1 MAPK1 FBXO36 TXNDC9 PIEZO2 MCF2L TFAP2ANLRC4 RASSF3 SFT2D2 MTFP1 G0LGA3 PARP3 CAPN7 TNFSF1OL CYTH1 PTPRN TMOD3 AKAP13 DCBLD1 GOLGA2 MORN5FAM214AKIAA1217 ZC2HC1A FAM177B N0M01 SGK1RABGAP1LMUC16 TMEM8 XMAD4 MRPS10 ACSL4 ARL10 MYO IE MAGI2 SPAG8TNEU006N05 COL1A1 HADH2 RAD9ACYB5A SNX31 MON1B KIAA1024LSAT1 ACOT13 NIM1K CCDC181 TBC1D32 FRMD4B OSCP1GALE RINT1MGC108213 FUCOLECTIN FADS2ERRFI1 FAM47E ASAH2 AQP3 TGFB1 MOK KCNS2 XPO6 CFAP99 SPTAN1STAMBPL1 XGALECTIN RAB2A RSPH6ADAGFL5 FAM210B SEMA3D C1ORF116HBP1 SYNE2 PKM RPS6KA5 TR1M32MGC80748ATP6AP1 IQCA1 KCNRG FAHD1 NDUFB4 CASKIN2 PABPC1 MNS1 ABCA3 PMFBP1 ATP5L LAMA4 MCOLN2 ARF4 POC5 FAIM RTN3 GORASP2 DZIP1L PXDN C4ORF47 WDR93 MRPS24 FA 166B FGFR1OP TM2D2 PHLPP2 LMAN2L DOCK8 SEC61B TMEM216SLIT2 COTL1C10ORF27MYCBP PDCD10 C2ORF24 GNG13 LRRC27 PDXDC1 CAV CHMP5 CANT1 TRPV5EG243628LY75 CCDC151 PDCD6 DNAJA4 NRAS ZNF232 H2AFY VASN PSPH KRT5 NDUFS3 SESN3 ATF6 C8ORF37 LRRC49 B3GNT2 PKHD1L1 HYOU1 BTBD11 GCGRP MSRA ZNRF1 HMCN1 RBM38 RD3 KATNB1GNPNAT1 MAP3K2 LRRC10 TOLLIPDMRT2 ADPRHL2 MCUB TUBZ1 NAXE ATP11B LSR UPK2KIF26B WWC1 STARD3ADAMTS17 TM9SF1 MGST3TMEM30BSTYK1 COX7B NDUFS8 SYNRG ARI.6 SMAD6 NAALAD2 MAP4K1 PIRTMEM268 ARHGAP39 JUP C8ORF34LDLR2 SGPP1TMEM145 MGC64475WDR35 SLC35B3GDF2 CTBS SDSL MGC78963BBS2 DAP MAP3K19 RNASEK AK KIF21A VW A3 A GLUL SIGMAR1 SRP68 MYL12B SYTL1 EDNRB TNFAIP8 KIZ POLR3E ZBBX PLEKHM1 KRT18 FGF7 PPP1R36 KMT2C C8ORF58 TRIM45 PPP2R5A VGRBP60TBC1D10BHSPA1A FAM89A XCAD SNAI3 CXCL2 IFFO1 CCL28 CXADR TTC38 ARAP1 NRG1GNG5 TOR4A MGC 154789 MGC68744DDT ARPC1B PTAFR NDUFA6 LDB2 STMN1 ASB10 FDX1 PRICKLE3 GATA2 PPIL6 DNAH8 DYNLT1 GGH IQCG PICK1 ALDH1 TEGG047A04IFT46 CAPN5 TUBA1A FBXL20 PPP1R42 TEGG009A16CD84 BBS1 SLC39A10TWF1 LRRC74A TMED1 SLC7A2 LAD1 SIX6OS1MGC 107884CHSY1 EDIL3 NR1I2 XPO5 MKNK1 ATP6V1G1MY06 CAVIN 1 APMAP TTC23L NUBP2 TUBE1 TMPRSS2 MYZAP VSTM5 CISDI MYO9A PISD RHOF POSTB4GALNT1ACTN4 ZBTB22 EVPL P4HA2 PKN2 LRRD1 GCNT4 PDPK1 WDR19 IRAK2 UBE2J1 HOXA7 ATP5H ATRN TOBI PRR29 GRB7 WDR38 BCKDHB PPP2R3C PHLDA2 SDHD HHIP CCDC61 SLC9A7 TENM3 LARP6 TBL2ODF3L2 THF. G NDUFS2SLC22A23 CCDC17 NR1D2 DNMBPCASP3 TTC39C CAV1A RAB11A RHOA TFCP2L1 EIG121L PDHA1 H0XD8 NUCB MDH1B TNEU093E11MUC5B SOD3 ANKMY1 DNAH6 CPTP ACTR2 DNAI2 G0LIM4TMEM114 MGC86295ZCCHC14TEGG001L15LARGE2 EFHD1 MGC82833SSR3 MAP1LC3A TMEM63ADIP2B MGC147194FNDC7 PRKG1 DENND2DMGAT4C MIGA2 EIF2AK4 CCDC39 DEGS1 MGST1 MRTFB POU4F3 IFT122 NDUFA10 TUSC5 UBR3 SRI EFCAB12CFAP69 HEL SMC04 FAM126AB9D2 F8A1 CALU DYNLRB1 TRPM4 CYB561 TTC9 A KN ADI CNIH4 MOCS1GAS8 DEGS3 MRPL32 ZAN RBM47 CFAP126 MKS1 HIC2 WDR1 STT3A BCAR3 ARL13B TTC36 EMP2 MYOID F0X01 CGN SPEF2 EMX1MGC83163 TRIM39 KIFAP3 EZH1DNAH9 CPEB4 ATP2C2MGC132047 CISH CAPN2 CYP2D6CLDN4 PCDH9 VPS13D ARHGEF5 SOD2 PERP USMG5 GRHPR LCTL SLC33A1 FAM161B SH3BGRL2COX5B TJP2 TGAS021D03TMEM18 SLC35B4MGC83164DYNC1H1 VMA21TMEM131L ATP13A1 NR6A1 STX121PMK REL3 LMAN1MGC81419 AGBL5 KIRREL3 JPH3CFAP157 KRT222 MCRIP2 MGC84118COL 18 Al KANK1 ATP6V1C2NT5C1A ACER3 DHCR7 ANKRD22C1GALT1C1 ATP6V0A4 MGC81120COG7CLDN8 CD163L1 CADM4 CCDC157EGFL6 TBCEL DIP2CMGC83511 ECHDC1 MAPK13 SGPP2SPATA6L STK24 NAB1NC0A1 SCNN1A MYO5C ATP6V0D2C3 CD 164 BPGM ADGB CCDC15 PTGS2C1ORF194PENDRIN GCLC PLEKHB2 RNF149 RPN2 HIP1R ULK4 EDF1 ATP13A CPM RIN2 NT5C3 WDR60 NDUFB2 SIAE CASD1 CARD9 KCTD9 MPC2 ZBTB43 TDE2MGC79146 MGC83130CCDC176 PIGR TMX2 LIPH TRIM37 SMARCA2IGFBP5 TTC24 XIRG RETSAT MRPL20 XALDB1 GNAZ PRKCQ PCDH ODF2 PTPRH SLC7A4 LRRFIP1 UPK3ATGAS014P14 SPEF1 CAPN8 TP63SLC35G2 MSRB2 ATP9A MGC79725JMJD1C NADK MGC81165ATF4 SAXO2 NSF UBAP1 ERBB3 MDM1 PTPRU PROM RALA S1OOA11 MGC76064SLC3A2 ACAA2 ENTPD4 PIGQ HGF RPAP3CDC42EP2KRTAP4 SNX22 GMPPA SZT2 LIMD1 PKP21MPGI C6ORF154 MGC78972 TMEM140KATNAL1 MSR1 APBB2 SLC10A3 TGAS076M01HUNK TFG ARMET BMP7 RAB1A PTGR1 COQIOB ZNF395 LRRC45 TMTC4 MGC75872CYFIP1 CXORF58 SLC4A1 AHU XKR9 KSR1 LRGUK PLCB3 RTN4IP1 GRIN1 ASCC2 TM6SF2 BCAR1 MGC78979SHOX2 DTD2TGAS071004IQUB TTC25 CMAH TMED10 LRRC71 YWHAE TLE4 TMTOPS BTG3 TANCI KRT12 DYNLRB2 VIMP SCN3B DAPL1 FRK SEC14L2 EHFBCR TTLL5 CXCL12 POLK CCDC81 CEP83 ATP6V0B YIPF5 M0RN3 ZNF238 IRF6 RHOBTB2 GPX8 CRP DHFR MGC85375MAK FBXL13 ABCD1 STK26 PITPNB MGC 64293NME7 PACRG CCNG1 SDS UBXN11 CFAP43 FH0D1 CLBA1 DPP8SLC26A11 SARS DHX32 KIAA0232TJP1 BMP LYN TBCB COL4A6 TMEM26 WU RAP1B ATP5G3 SLCO1C1 LRRC31TMEM164 XRRA1 AD ADICETN4 BIXI TES SGSM3 CSTA SLC41A1 PPP1R3C CA2 OGDH ECU MYCBPAP TGAS141P18 CCDC68 GNG7SPNS1 BSG TUBB4TNEU137P08AGBL3 ARRDC4GPR18 DYM GLIS2 ZDHHC1 LAMB1 PPP4R1 TRPM8B FAM49B AK8 PTPN21 EFNB1 HSPB11 CEBPG SCARB1B4GALNT3 MGC81264ARID3A SMKR1YIPF1 ESYT1 SDHBFAM241A DPY30 PCK2TTPA012C15TLR5 2-SepMGC80993 PI4K2A RBBP8NL URADFAAH2 VDAC2 MAE PITPNC1 DAB2IP FGFR2 CIDEC MARVEL DI FAM227B CLDN6 LZTFL1 EPAS1 TWF2 LRAT DAPK2 ODF2L F0XI1E LMLN SLC16A3 VWA5A ATP12A PPP1R13L SPTLC2 MRPL54 TMC5 CDC25D AGBL2 FUCA BCL6 PTPRK VCL DRC7 UFSP2 CLDN7 CCDC63 MFAP3L STXBP1 AFF1 ARMC4 CYC1 KDELR3 GPX7 DNAAF1 ANKRD9 ANAPC4 AGRN SAMD11 ETFB DMXL1 GRPEL1 SLC25A32RDD2 CHEF KIAA1549LMGAT4A SEC31B ZBTB20D0PEY2 FTH1 RABEPK MGC 108497ATP1B1 MED27KIAA0319L KLHDC9 MKKSMGC 108338TM SPATA4 CTNND1 NEDD4L PNMA2 GTF3A FOPNL KCTD14 TMEM74 COX7A2 TIMM10 1FT22 GCHFR KCNH3 MGC81583IDH3A COL6A3TCTEX1D2ESG1 ASPH IL20RA OXNAD1 DCTN2 CFAP57 TMEM41BCOX6A1 HECTD3ANKDD1B EFC AB 11 TMEM51ARHGEF16PTK2B CYCT WNT2 FGL1 RTP2 ATP6AP2 ITK BMP3 USP2 MAPK12 TTC30A H0XA1 UXS1 SIMC1 SPATA45 ARG1 ARL11 ARFGEF2 T2R54 SPATAI 8 MBNL2 SOX21 TPPP3 PLA2G4A MTUS1 MYH10 CAT ID4 B3GALT2 ST6GAL2ARHGAP45NECTIN1 LLGL2 CD38 LSP1 RNF225 BIN3 BMP5 DHCR24 FAM26B TTC39B ADORA2AC9ORF43 FAM3B PRKCI AGR3 SLC12A2 INPP5A FOCAD KUR APIP ARF6 LPCAT3 EFC AB 1 IFT57 SLC25A29DISP1 ITGA2 ITM2ABETASCRN2 UFL1 PFKM DSCR6 BCAP29 RAB14 DCST2 EHD4 TTC22 MAP7D2 HAGH T0MM5 DAW1 CRYBG3 CFAP47 ANKRD26 PAK2 CHMP1A QKI GPR1 COX 17 PRKCB GRAMD4 MISP3 PTBP3 SYNGR2 BAIAP2 TIMM8A UQCRB HDAC6 SCLT1 AKAP14 C5ORF42 CEP 126 GDI2 SPG 11 TLL2 RBL2 KRT8 DRCIMGC98907 PTGER2 CLUHKREMEN1MDK GRHL1 CNDP1 UBE2W MIC 13 LAMA5SGK2 NWD1 NDUFB10 COX6B1 CEP 19 ASCC3 ZCWPW1PCDH7 CPT1ATNFRSF1A MTHFD2L IDH2 LRWD1DNAL4 TPBG CRYBG1 COL14A1 GLRA1 OAT CPAMD8 PHYHD1 DUSP1 CCDC57 MYD88 SUGT1 CCNI ACADL ROPN1L WDR66 ACTG1 FBXO31 SHISA2 COL7A1 CLINT 1 AVL9 C16ORF46SEC 13 ATP6V0D1UQCRQ RASEF MGC 115323GAL3ST4 NDUFA8 MDH1 SLC37A4 SNTB2 TRPC2 NUS1 ITFG1 XRIP1 TEX9 ST6GALNAC2DOPEY 1MGC108118COL3A1 KALRN TOMI UQCR10 FH CCDC175 CNFN T2R37 CLIC1 TFPI2 CMBL CPEB2 WNT3A ARL1ATP6V1E1 RANBP3L TEKT3 WDR78ARAP2 CRYBA2 DLL4 HSD17B12DRC3 HYAL4 SEC23B RDH7 1TGA5 TTC9B SERPINH1SYT1 CEP290 SPTLC1 RILPL2 TTP AL PAPSS2 FAM46C PANK3 MYO 10 ACY3 SLC39A7 RNFT1 CRTAP KATNAL2 ADGRG4 LMCD1 CCM2 CEP 162 VIT MRPL30 GLT8D2 CFAP61 WDR44 SEC24D CHMP1B COL6A2 SUCLG1 PIBF1 CCDC189 KIAA1671 ACACB SFRPX FKBP14 SLC35D2 SPATA6 IFNAR1 TTC1 CRISP3 ARPC4 RMDN2 ANOS1 NDRG2 THTPA STPG1 ITCH MAN1A1METTL21C TBC1D14 SLC12A6MGC84172SV2A STIM1 COL4A5 ELF1 CAPN9 SMDT1MGC83153 SSNA1 TIMM22MGC82549SPINT1 NXPE1 SEC31A CCNA1 ZNF750 SFT2D1 C17ORF98MRPS11 NDUFA7FAM161ALRRN4 PF20 CLDN18 COQ4 SLC22A5 CTDSP2 ENO4 ATP8B1 ATP2B4 SLC27A6 PCNX4PIH1D1 ENDOG NEIL2 FBXO43 MAN1B1 UBE2H FOS EVC2 LBR ELM03 PKC CAPN13 SULT1E1 NVD COMT GMPPB RNPEP CLCN3 AP2A2 LRRC59 COL11A1 RAI14 GPD1 LOXL3 DSG2 PLEKHS1 PNKDTEGG127O10 GNS ERICH6 ATP6V1ARAB39B MVP AHNAK TREM2 SLC13A1 TRPA1 GOLT1B PPARGC1BTLDC2 B3GNT9 NDUFAB1SPSB3 MEI1 HK2 PFKFB1 HACD3 C9ORF84 FKBP9 AUTS2 ABCG2 CCDC77 JUNB SEC61G CALCRL SYDE2 ANXA11 CALP2 EPGN TEX36ZMYND10PIP5K1B M0RN2 CCDC126 ITGB3 GALNT10 PRKCZ SSH CLCN1 EPN3C4H3ORF84 ERBB2 UGT8 MINDY4CYP1D1 TRRAP MGC81930 TMEM45BDNAAF5 E1F1 DYNLL2 PTPRF SEMA3B ISL2 LRRC23 TBC1D23 CDR2 HIGD1A SPA17 KIAA1468 SLC25A4 CANX ZNF703 CCDC96 MGC108307CEP68 SYNJ2 DEPDC7 COPG1 RAB36 TSPO FSTL3 COPE PLCH2 P2RY1 ASAP2 DIAPH1 GGPS1DCUN1D2 NBR1 TACC3 HRBSLC6A15 SLC6A12 BBOX1 ILVBL WDR92 SMTN HMX3LSS PCYT1A RFLNB FNDC3B VM01 C4ORF22 EFHC2 ARHGAP1UAP1 BBIP1 APOL3 OTOGL XCEN DNASE 1L3 C1ORF189ABCC5 DNAH5 HTT TCTE1 T2R11 LGALS3 PHACTR4 SCFD1 LPCAT4 COL1A2 COPZ1 XFRP DNAAF3 DADI FXYD2 BCDIN3D TMED5 PGLYRP4 HGC6 COBLL1 GBX2 TACSTD2 PLXNB2 GCMI CL RRM2BANK1SLC22A31 TMEM255A CYGBB IQCE LRRC57 RGL2 ZFP36 PDE3A NQO1 OLFM4RABGGTA IRAK IBP 1MGC78960 NFE2L2 ANXA2STX3 FSTL5MGC80982ATP13A4TEGG049G06 RAB5A GLUD1AXDND1 VAPA RHPN2 ATM CCDC27 SH2D4A ABCC1 MAP3K10FREM1 TNK2MGC68897 STH LRTOMTDPCD NDUFA1 MGC 147326EHBP1L1 QPCT NDUFAF1 DCTN4 SLC25A20 TMEM131F2RL2 ZSWIM7 TTMP SLC4A9TEGG014M13 CDC42EP1CFL1C20ORF85USAG1 DNAJB14 ATP8A2 PCBD2 BCKDHA UQCRFS1 C22ORF23SMC IB TM4SF18 FREM2 LRRC46 SERP1 ANKRD42 ADAMTS5GPAA1 SH3YL1 CAMK2G TGFA RUSC2 FAM227A N0XRED1NF MGC81183 SPTSSB CHAC1 LMX1BMAP3K9 EFHB CCDC125 MGC76024REV3L G0LGA4 NBEAL1 CAM AGPAT4 MGC 146293 TNEU022002SATB2 MFSD6 TEX49 NHSL2 TSPAN13 RSPH3 FER1L4 IFT140 ATPIF1 CAI 2 FAM96A MAGT1 KIF3B B9D1 CEP78 HS6ST1 MIA3 CLUAP1 HHIPL1 ASPRV1 GATA3 SDPR ZZEF1 ETHE1 NLRX1 RAB9B CCDC42 CCDC33 SH3BGRL3IMMT CCDC171 CAMP1 SON RTDR1SELENBP1 C11ORF16 ESRRG CUZD1HOXC9 MID1IP1 OCA2PFN4 CHST10 FAM213A XCASPASELAMP1 TSPAN36 LBH LRRC1 METTL9 SMAD8B ARVCF SCN5ACABCOCO 1 AD AMTS 1 LPAR2 EDEMIMYO9B CSTB BRD3TNEU001N23 APLP2 CEP170BMGC80171TLCD2 RAD52FHST1H2AMXGLY4 BAIAP2L1MGC68493DNAH11 TBC1D30 DAAM1 ITGA3 SURF1 CCDC47 GPX1 RGS14 LEF RFLNA SLC25A24TRPV1 EPHA5 KCNMB3 DYSF GJB3 RER1 TMEM123SAPCD2 PIG37 P2RY11 PTPRB FAM83G ITPK1MARCKSL1WDR88 CPT1B EML3 SQSTM1 PTPN1 PIGW MBOAT2 CYSLTR1 NDST1FAM221A DUSP22 SURF4 FESIFT43 CCDC155TMEM255B MGC81105 C1AP1N1 MTMR12CAPZA1 SLC6A6 IGF2BP3 MGC83501DDOST YPEL1 MAST3 CPN1 LRRIQ3C20ORF27 TMEM161B DNHDI TJP3MAP3K15 CLEC SH3GLB2 MGAT5 ED AR BLVRB EZR ALG5 STOX2 ARL3 KIF1B SLC10A7 PRSS29 HEBP1 ACTR3 UQCRH TTC16 OCIAD1 PARM1 MAP7 FAM69C DLL3 RANBP9 CFAP161 PLEKHA8 CLDN34 FAM26D NXN OR51E1 CFAP100 XESP PRSS8 FAM174BCOL4A3BP KI AA 1324 SLC23A2C9ORF116NDUFA11 FLVCR2 ZNF438 TRPM7 TESCCDC42BPA C6ORF183 GFOD1 RNF19BSCAMP2 YWHAZ MSMB MEM01 CFAP45 VPS37A RG PPP4R4 MPC1 DNAH10 COX7C DBT APOCI NPC2 NDUFA5SNX27 SH3RF3 ITLN1SPTSSA SLC24A5 1A11MGC 131054 STARD10 XIQGAP1 EYA2IDHI MGC68472 MGC68615TMEM17MGC83272SBF2 DNAAF2FAM 184 A CAP IB TTC7A ACOT2LRRC75B US01 GIPC GNTI KRT AP3S1 MP68TMEM167ACCDC169 RPS6KATMEM248 FAM107B CEP55TRAPPC6BP4HB ELMOD1 CDC34 GPR160 HRH2 CATIP KCNE5 UQCC1 CFAP70 DNALI1 SACM1L ZNF362 FLNA FBN2 TMED2 PRR15 ATP5J MGC85216GBF1 DTHD1 OSTC SH3TC2 RASSF6 DMBT1 MGC81191FBXW10MGC75752NXPH3 PTH AIDA PIK3CB LEPROT STON2RAB11FIP2 SQOR EP400 CFAP53NT5C2 MY ADM TEX43 ENOSF1 SM1M15 SZL MYB RNF25 GTPBP1 TFB2M TPRN PAWR C2ORF70 EPB42 FAM228B CYSTM1 NUDT6 FBP1 NOGO AKNA TMPRSS4 P3H2 EFHC1 LRRC15 IRAKI SLC13A5 ZCCHC4MGC 107969PYCR2 RYR3 ANKS1B SLC7A7 NDUFA12 ANO9 CAB39L TBCA HDLBP HADV36S1 C12ORF63 PGK1 LPIN2PDGFB CAMK2D SSBP2 CHMP4B LCA5 SCUBE1 IKB1P SULF2 NDUFV3 ARF1 NUPR1 TMEM246ATP6A1 KIAA0319FAM183A FAM57A EPS8L2 STAG3SDCBP DNAJC3 HAL GJC1 SPATA5 ADAT1 NPNT CASIMO1 DSE GAS6 ERMP1ADAMTSL4 CCDC92 LAMC1MGC 84197PPARD PCM1 AGTPBP1 LRRC36 PPP3CA SNRNP25 SPHK1 RAB35 SEC61A1 AK5STAMBP AKAP9 PDE4DIP MGC131013SEC22BMGC83403INHBA XB FAAH CLHC1 LNPK PDLIM1FAM 162 ASPTBN2 TMEM184C C14ORF37NIPAL3 MUC15 PGA5 STK4 SERINC3 MAP 10 BMAL1 LPIN3 TMEM205 ATP6V0A1TIE! PMM2 CYTH2 SMAD3 NME5 MGC115316SYAP1 GLT1D1 PFKP P2RX1 CMC1 ILI0R. BC10ORF53 ISYNA1 PTPN12 HOXA9ARC3 GDPD2 SPAG16 CDCREL BBS5 TNIK SCAMPIMGC82377 HOPX CEP 164 FUT10DUSP22A TSPAN1 AMD1 TNEU085H23TTC29 UGDH FAM131ATMCC3 MCEE GNB3 CARMIL 1 PCYT2 PLXDC2 MGC145311ECM2 FAM 174 SEMA6A NDF1P1 FRRS1 PLBD2 LINC00116TBX3 GRHL2 TMEM138CNNM3 C14ORF80 MAPK1IP1LNRXN3 VDAC3 PLSCR1 ACER1 RPS27 MGC80649RAB19 ATP6V1C1 TMEM258ATP6V1H PREPL EXTL2 ID2 TEX47 NME9 NEK 5 UPS3 LRCH3 CHCHD7SPATAI NEK11 NCMAP DBNL B4GALT6 NFKBIA MGC 80268PRDM11 MAP3K1 COQ8B SLC35A3 CASP6 FAN KI FLNB SEMA3F LMAN2 VAMP8FAM19A2 MNX1 BHLHE41 ATP5BFRAS1 STOM MTUS2 RALBP1 PLCB4 F10 P16XIC2 FOXJ1 INSRR NLRP12 SYT2 PDHE1BETAHIVEP2 SL CCDC65 DHRS7 XEXT1 NDRG4 FRMD8 TEPP BORCS5 OSGIN2 AGBL4 A DIRT ASB9 LIN28A MAGI3 PEX16 KIT

[0170] Table 9 - Thanatotranscriptome genes.DIABLO ADAM28 SIK1 MS4A15 AHNAK ARG2 CLDN3 PFKP FOSB ABTB2 GNAI1 RGS4 ACER3 ABHD3 OSBPL6 GPR31 ITGA6 KPN Bl SMIM19 LEP SLC26A4 SVEP1 ADO NPB CA4 RHOT2 EGR2 PLEK2 UBQLN4 HSPA1A DDIT3 SPATA6L XDH PPP2R5D KLHL12 CRHR1 CHD3 TMED10 ELF3 ASB15 FOS JUN NFKBIA STX10 ZMYND8 CBX7 RPS6KA3 CAPN11 PRDX2 HIF1A RAPGEF1 MIDN RTBDN EPAS1 GRM7 KCNH2 MKNK2 HIF3A ROR1 KLF9 RASGRP4 ATF3 STARD13 MPDU1 RASSF6 CLDN4 TOX2 EGR1 RGL1 CLDN6 SLC14A2 TNP01 CYBA CLDN9 ZFAND4 LIMD2 PTGIS CLDN17 DBX2 WIFI KCNB1 CLDN8 GLTP TTC28 MYO3A BATF3 PCSK1 BIK OGFR LRRC59 ZNF281 TRPM5 EPHA2 RMI2 PDE4B EPHA7 PHLDB3CEACAM1NFIL3 IFNAR2 CRIPT SOCS3 IFNGR1 CASP3 TOE1 DA AMI IL22RA2 CYP2W1 RBM19DUSP2 IL1B TNS1 GADD45ARPF2 MAP3K2 TNFRSF19MCM5 SBK1 ELL HM0X1 LTBR LTA KLF14 USP18 GRWD1 IL17C SIM2 MMP14 PNP IL22 BCL2L11BCL6 COX 17 PDE4C HTR1B PR. Fl EIF1AX CHST15 C4B HSPE1 EIF3J RPS12 TUBB2A IGSF9 LUC7L2 MY01G KATNAL1 PTGFR KTN1 COL 19 Al TTLL10 IRF2BPL CEND1 IGHV2-26 TPR ARFTP1 YEATS2 FYTTD1 GSE1 GEMIN6 SLC5A10 ZNF107 ATP6V0C BANP SLC38A4 PLEKHG3 RAB3IP SFT2D1 PARG FGD5 CDC42AADACL3 LONRF2 DCLRE1C KISSI RBL1 UNG ABCC5 GOLT1A ZNF724 SEMA4C ABCA12 GRAMD1AEPHB3 TUB A3 C RNASEH2BDEFBI 35 ZNF101 CALR KCNK5 C7 RALGPS1 SOX30 GRK4 ORC4 SEZ6 RBAK RPS18 TSPY26P SNTG1 PTPRD MECOM PPHLN1 SPRY1TMEM161B SRRMI FBXO17 SIL1RPS23 UCHL3 POLR2B SERP1NC1 MMS22L MPDZ EEA1 TMEM182USP15 TMT1A C4orf33 MAN2B1 IL15RA HERPUD2 COMMDIO FBXOIO LARP7 ZNF652 ZNF780A PRSS23 E2F4 SLFN13 GDPD1 CHST9 UBXN4 CEP170B CYP8B1 KLK1 PPP ICC ADRA2B CAI NUDT18 UBTF NLGN1 MDGA2 OGFOD1 RIPPLY3 WDFY4 TNFRSF9 LM04 CREB5 ZNF611 LDLRAD3 CYP2R1 ARID 1 A ZNF175 CSNK2A1 TGIF1 PAK4 PPARGC1AHLA-DOB HCN1 HECW1 LYAR PPM1E CCR4 SGSH SEMA6D NAV2 CBX3 LASPI RASA1 ZFP36L1 CACNA1B ZNF14 RPL26 GCGR DPYSL5DENND1ARBM45 PHB2 WDR19 SALL1 PPP1R3B TIMM8A SERPINB8 SP3 RTL1 BEX4 ZNF564 CPSF7 YTHDF2 ZDHHC14 POTI CPSF6 HSF2BP IL5 MEF2AABCA3 SCN8A PROK2TNFRSF14 MCAM SKAP2MS4A4A ZNF273 PPP2R1A RAB2A SP110 FRS3 PGF TTGB6 ITSN1 JMJD1C TRIO ARHGAP17CD22 ATP11C CNOT6L GXYLT2 FRYL SEMA6C IFTTM1 SPAG7 ZNRF2 LCP2 CBX6 FBXW12 KLF15 KCNV2CCDC138 DEGS2 REV3LDYTN MS YIP FAM219ANDUFC2 CRYBG3

[0171] Table 10 - List of thanatotrascriptome genes upregulated in xenobots ABCA12 LEPATF3 RGL1DDIT3 SKAP2EGR1 SLC14A2FOSB SLC26A4GADD45A SOCS3IFNAR2 TNFRSF9IL17C TRPM51LIB TTLL10KLF15

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[0173] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0174] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0175] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

1. CLAIMS2.We Claim:

1. A method of modifying motility of an engineered multicellular organism, the method comprising (a) exposing the engineered multicellular organism to an acoustic stimulation.

2. The method of claim 1, wherein the engineered multicellular organism comprises ciliated cells.

3. The method of claim 2, wherein the ciliated cells are ciliated epithelial cells.

4. The method of claim 1, wherein the engineered multicellular organism comprises vertebrate cells.

5. The method of claim 1, wherein the engineered multicellular organism comprises amphibian cells.

6. The method of claim 1, wherein the engineered multicellular organism comprises Xenopus cells.

7. The method of claim 1, wherein the acoustic stimulation comprises sound at a frequency of about 5 Hz to about 1000 Hz.

8. The method of claim 1, wherein the acoustic stimulation comprises sound at a frequency of about 50 Hz to about 500 Hz.

9. The method of claim 1, wherein the acoustic stimulation comprises sound at a frequency of about 50 Hz to about 300 Hz.

10. The method of claim 1, wherein the acoustic stimulation comprises sound at a frequency of about 300 Hz.

11. The method of claim 1, wherein the engineered multicellular organism is exposed to the acoustic stimulation for at least about 1 second, at least about 10 seconds, at least about 30 seconds,at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours.

12. The method of claim 1, wherein the method further comprises (b) ceasing the acoustic stimulation.

13. The method of claim 12, wherein the acoustic stimulation is ceased for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours.

14. The method of claim 12 or 13, wherein the method further comprises (c) exposing the engineered multicellular organisms to an acoustic stimulation.

15. The method of claim 14, wherein the engineered multicellular organism is exposed to the acoustic stimulation for at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, or at least about 10 hours.

16. The method of claim 14 or 15, wherein the acoustic stimulation of (c) is the same as the acoustic stimulation of (a).

17. The method of claim 14 or 15, wherein the acoustic stimulation of (c) is different from the acoustic stimulation of (a).

18. The method of claim 1, wherein the method comprises modifying the motility of the engineered multicellular organisms in an aquatic environment.

19. The method of claim 1, wherein the engineered multicellular organisms are genetically modified.

20. The method of claim 1, wherein the engineered multicellular organisms comprise an antisense oligo or a morpholino.

21. The method of claim 1, wherein the engineered multicellular organisms comprise a heterologous polynucleotide.

22. The method of claim 21, wherein the heterologous polynucleotide comprises a sequence encoding a transcript.

23. The method of claim 21 or 22, wherein the heterologous polynucleotide further comprises a regulatory sequence operably linked to the sequence encoding a transcript.

24. The method of claim 22 or 23, wherein the transcript encodes a polypeptide, optionally, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.

25. A system for modifying motility of an engineered multicellular organism, the system comprising (a) an acoustic stimulator; and (b) an engineered multicellular organism.

26. The system of claim 25, wherein the acoustic stimulator comprises a sound component.

27. The system of claim 26, wherein the sound component is a mechanical sound component or an electronic sound component.

28. The system of claim 25, wherein the acoustic stimulator is a speaker.

29. The system of claim 25, wherein the acoustic stimulator is configured to emit a frequency of about 5 Hz to about 1000 Hz.

30. The system of claim 25, wherein the acoustic stimulator is configured to emit a frequency of about 50 Hz to about 500 Hz.

31. The system of claim 25, wherein the acoustic stimulator is configured to emit a frequency of about 50 Hz to about 300 Hz.

32. The system of claim 25, wherein the acoustic stimulator is configured to emit a frequency of about 300 Hz.

33. The system of claim 25, wherein the engineered multicellular organism comprises ciliated cells.

34. The system of claim 33, wherein the ciliated cells are ciliated epithelial cells.

35. The system of claim 25, wherein the engineered multicellular organism comprises vertebrate cells.

36. The system of claim 25, wherein the engineered multicellular organism comprises amphibian cells.

37. The system of claim 25, wherein the engineered multicellular organism comprises Xenopus cells.

38. The system of claim 25, wherein the engineered multicellular organism is in a liquid medium in a vessel.

39. The system of claim 25, wherein the engineered multicellular organisms are genetically modified.

40. The system of claim 25, wherein the engineered multicellular organisms comprise an antisense oligo or a morpholino.

41. The system of claim 25, wherein the engineered multicellular organisms comprise a heterologous polynucleotide.

42. The system of claim 41, wherein the heterologous polynucleotide comprises a sequence encoding a transcript.

43. The system of claim 42, wherein the heterologous polynucleotide further comprises a regulatory sequence operably linked to the sequence encoding a transcript.

44. The system of claim 42 or 43, wherein the transcript encodes a polypeptide, optionally, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.

45. The system of claim 38, wherein the vessel is physically connected to the acoustic stimulator.

46. The system of claim 38, wherein the vessel is about 1 meter or less from the acoustic stimulator.

47. The system of claim 38, wherein the vessel is about 1 centimeter or less from the acoustic stimulator.

48. The system of claim 25, wherein the system further comprises (c) one or more controller, processor, or memory.

49. A method comprising emitting sound from the acoustic stimulator of the system of claim 25.

50. The method of claim 49, wherein the method modifies the motility of the engineered multicellular organism.

51. A method of conferring sound sensitivity to an engineered multicellular organism, the method comprising introducing a heterologous polynucleotide to the cells of the engineered multicellular organism, wherein the heterologous polynucleotide comprises a sequence encoding a polypeptide, wherein the polypeptide comprises a sound responsive protein or a mechanical stimulus responsive protein.

52. The method of claim 51, wherein the heterologous polynucleotide further comprises a regulatory element operably linked to the sequence encoding the polypeptide.

53. The method of claim 51 or 52, wherein the cells of the engineered multicellular organism express the polypeptide.

54. The method of claim 51, wherein the engineered multicellular organisms comprise or consist of ciliated cells.

55. The method of claim 51, wherein the engineered multicellular organism comprises vertebrate cells.

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