Labeled collagen ivα1 protein, labeled collagen ivα2 protein, nucleic acids encoding said proteins, and animals having said nucleic acids

Abstract

The present disclosure relates to: a labeled collagen IVα1 protein; a labeled collagen IVα2 protein; nucleic acids encoding said proteins; and animals having said nucleic acids. The present disclosure also relates to a method for observing the labeled collagen IVα1 protein or the labeled collagen IVα2 protein in the animals.

Description

Labeled collagen IVα1 protein or labeled collagen IVα2 protein, nucleic acid encoding said protein, and animal having said nucleic acid

[0001] The present disclosure relates to a labeled collagen IVα1 protein (labeled COL4A1) or a labeled collagen IVα2 protein (labeled COL4A2), nucleic acids encoding the proteins (labeled Col4a1, labeled Col4a2), and animals having the nucleic acids. The present disclosure also relates to a method for observing the labeled collagen IVα1 protein or labeled collagen IVα2 protein in the animals.

[0002] Precisely controlled remodeling of basement membranes (BMs) is thought to be essential for morphogenesis. However, the molecular and tissue-level dynamics of BMs during morphogenesis and their functional significance remain largely unknown, particularly in mammals, due to limited visualization tools.

[0003] Multicellular organisms are complex composites of cells and extracellular matrix (ECM). The ECM is a complex polymer network composed of proteins and polysaccharides that provides cells with diverse biochemical and biomechanical cues. One of the major components of the ECM is the basement membrane, a thin, dense sheet of ECM that surrounds most tissues in metazoans. １ Basement membranes contain many proteins, including core proteins such as laminin, collagen IV, nidogen, and perlecan, as well as cell- and tissue-specific basement membrane proteins, basement membrane-modifying proteins, and morphogens. １，２ The basement membrane is an evolutionarily ancient ECM, found throughout metazoans and is the earliest ECM structure to appear during development. ３－５ .

[0004] Basement membranes play a crucial role in animal development and homeostasis by functioning as a multifunctional solid-phase cell adhesion substrate and signaling platform. By interacting with cells via integrins and other cell surface receptors, this sheet-like ECM regulates a variety of fundamental cellular behaviors, including cell adhesion, migration, proliferation, differentiation, apoptosis, polarity, and shape. Furthermore, the composition of basement membrane proteins is spatially and temporally specialized during development. ６－８ Therefore, the basement membrane dynamically changes its physicochemical properties and its organizational state, regulating cell fate and behavior. Deletions or mutations in basement membrane genes cause various developmental and homeostatic disorders in multicellular organisms, highlighting the crucial role that basement membranes play in the formation and maintenance of multicellular systems. ９－１１ .

[0005] The basement membrane has long been thought of as a static support structure, like the floor of a building. However, recent studies suggest that the basement membrane is much more dynamic than previously thought, both at the molecular and structural levels. ２，４，１２，１３ For example, molecular turnover of collagen IV has been observed during development in Drosophila and C. elegans and appears to contribute to proper organ development. １４－１６ Furthermore, the assembly of laminin and collagen IV may affect the mechanical properties of the basement membrane and thus the structure of epithelial tissue during tumor progression. １７ Therefore, perfectly balanced basement membrane remodeling and turnover, which can control spatial and temporal changes in the biochemical and biomechanical properties of the basement membrane, are essential for its dynamic function. However, the molecular and tissue-level dynamics of the basement membrane, its underlying regulatory mechanisms, and its physiological functions remain largely unknown.

[0006] A key challenge in basement membrane biology is investigating the dynamics of the diverse, dense, and complex supramolecular matrix across biological hierarchy, particularly in mammals. The primary approach to visualizing basement membrane dynamics is genetic tagging of basement membrane proteins with fluorescent molecules. However, the unique properties of basement membranes often preclude the insertion of large fluorescent proteins without affecting normal basement membrane function. These properties include: 1) a large modular structure; 2) specialized intracellular transport and secretion mechanisms; 3) post-translational processing; 4) assembly of supramolecular complexes; 5) complex intermolecular interactions; and 6) a unique extracellular physicochemical environment, including redox conditions. Some C. elegans and Drosophila organisms have been engineered to express fluorescently tagged endogenous basement membrane proteins and demonstrate normal function. １４，１８ However, functional abnormalities were observed in mice expressing core basement membrane proteins fused with fluorescent tags. １９－２１ Therefore, it remains a major challenge to generate mice that express endogenous core basement membrane proteins fused with fluorescent tags while maintaining normal function.

[0007] The basement membrane consists of two independent self-organizing networks of laminin and collagen IV, interconnected by nidogen and perlecan. ２２ The collagen IV network is crucial in providing the core structure and tensile strength of the basement membrane. ４，２３－２６Collagen IV consists of genetically unique α-chains, designated α1(IV) through α6(IV). Each collagen IV polypeptide is composed of three distinct domains: a cysteine-rich N-terminal 7S domain of approximately 150 amino acids, a long central triple-helical collagen domain of approximately 1300 amino acids, and a globular C-terminal NC1 domain of approximately 230 amino acids. Heterotrimeric collagen IV molecules can interact through the 7S domain to form tetramers, forming crucial inter-collagen disulfide bonds, or through the NC1 domain to form dimers and supramolecular networks. 16,22,27-29 Due to its complex heterotrimeric protein structure and intricate intermolecular interactions, various dysfunctional mutations have been reported throughout the collagen IV gene, causing a wide range of disorders, including embryonic lethality, myopathy, glaucoma, hemorrhagic stroke, nephropathy, and cochlear dysfunction. ２３，３０，３１ Therefore, altering the amino acid sequence of collagen IV or introducing large fluorescent proteins at arbitrary positions may disrupt the synthesis, assembly, deposition, and function of collagen IV molecules, posing challenges to fluorescent tagging. Generating mice expressing endogenously tagged collagen IV with normal function would be a powerful approach to visualize and study basement membrane dynamics in mammalian tissues in health and disease, but this has yet to be realized.

[0008] Recently, a mouse model with fluorescently labeled collagen IVα1 (COL4A1) was developed in Non-Patent Document 1. ６９ However, these mice were embryonic lethal. It is possible that the fluorescently labeled collagen IVα1 does not function normally. ６９ Non-Patent Document 2 is a publication by the inventors on the development of a mouse model with fluorescently labeled collagen IVα2 (COL4A2). ７０ .

[0009] Rebecca A. Jones et al. , 2023, doi: https: / / doi. org / 10.1101 / 2023.09.27.559396Duligengaowa Wuergezhen et al. , 2023, doi: https: / / doi. org / 10.1101 / 2023.10.31.564866

[0010] The present disclosure provides labeled collagen IVα1 protein (labeled COL4A1) or labeled collagen IVα2 protein (labeled COL4A2), nucleic acids encoding the proteins (labeled Col4a1, labeled Col4a2), and cells or extracellular matrices (particularly basement membranes) having the nucleic acids and / or proteins, as well as tissues, organs, organoids, and animals (preferably mammals) comprising the cells or extracellular matrices (particularly basement membranes). Part or preferably all of the genes encoding the collagen IVα1 protein or collagen IVα2 protein have been modified in the cells. The present disclosure also provides methods for observing the labeled collagen IVα1 protein or labeled collagen IVα2 protein, or the extracellular matrices (particularly basement membranes) in the cells, tissues, organs, organoids, and animals.

[0011] We developed a knock-in mouse line in which the endogenous collagen IV gene (Col4a2) was fused to a fluorescent tag. The resulting mice, even homozygotes, grew normally and were fertile. Live imaging of developing hair follicles revealed a spatial gradient in the turnover rate of fluorescently labeled collagen IVα2 protein (labeled COL4A2), which was closely correlated with the rate of hair follicle expansion. Proliferation of epidermal progenitor cells correlated with the rate of expansion of the underlying hair follicle. Epithelial progenitor cells migrated directionally with the expanding basement membrane but not actively on a stable, non-expanding basement membrane. Addition of a matrix metalloproteinase inhibitor delayed COL4A2 turnover, inhibited basement membrane expansion, induced a shift in the division angle of epidermal progenitor cells, and altered hair follicle morphology. Our findings reveal spatially distinct basement membrane dynamics within the contiguous epithelial basement membrane and highlight the importance of basement membranes in orchestrating progenitor cell proliferation, migration, and fate, as well as the macroscopic shape of developing organs.

[0012] The present disclosure provides, for example, the following inventions: (1) A nucleic acid encoding an engineered collagen IVα2 protein, wherein the nucleic acid has an insertion of a nucleic acid encoding another protein (which may be a labeled protein, for example; the same applies hereinafter) directly or via a linker into the 7S domain (preferably N-terminal to the 7S domain, more preferably after the C-terminus of the signal sequence of exon 3, e.g., at the C-terminus), wherein the nucleic acid encoding the engineered collagen IVα2 protein is capable of producing a collagen IVα2 protein fused with the other protein, wherein the engineered collagen IVα2 protein is capable of forming a trimer with two collagen IVα1 proteins, and wherein a non-human animal (preferably a mammal, the same applies hereinafter) having the insertion in all of its endogenous collagen IVα2-encoding genes can survive and reproduce to adulthood. (2) The nucleic acid according to (1) above, wherein the other protein comprises a fluorescent protein. (3) A non-human animal having the nucleic acid according to (1) or (2) above. (4) The non-human animal according to (3) above, wherein at least one of the nucleic acids encoding endogenous collagen IVα2 in the genome has the insertion and expresses the fusion protein. (5) The non-human animal according to (3) or (4) above, wherein all of the nucleic acids encoding endogenous collagen IVα2 in the genome have the insertion, express the fusion protein, and are capable of surviving and reproducing to adulthood. (6) The nucleic acid according to (1) or (2) above, which is mRNA. (7) An engineered collagen IVα2 protein that is the translation product of the nucleic acid according to (6). (8) A heterotrimeric complex comprising the engineered collagen IVα2 protein according to (7) above and two collagen IVα1s. (9) An isolated animal cell, wherein at least one or all of the nucleic acids encoding collagen IVα2 contained in the cell are the nucleic acid according to claim 1 or 2. (10) An isolated organoid, tissue, or organ comprising the animal cell according to (9) above.(11) A method for obtaining the non-human animal described in (4) or (5) above, comprising obtaining a next generation of the non-human animal described in (4) or (5) above from sperm of a male of the non-human animal described in (4) or (5) above and an egg of a female of the non-human animal described in (4) or (5) above.

[0013] (21) A nucleic acid encoding a modified collagen IVα2 protein, the nucleic acid having an insertion of a nucleic acid encoding another protein (which may be, for example, a labeled protein, the same applies below) {e.g., a fluorescent protein} via a linker at the N-terminal side of the 7S domain of exon 3 and after the C-terminal end of the signal sequence, the nucleic acid encoding the modified collagen IVα2 protein can result in the production of a collagen IVα2 protein fused with the other protein, the modified collagen IVα2 protein can form a trimer with two collagen IVα1 proteins, and a non-human animal having the insertion in all of its endogenous collagen IVα2-encoding genes can survive to adulthood and reproduce. (22) A nucleic acid encoding a linker (e.g., a nucleic acid encoding a GS linker, particularly (GGS)) at both ends of the nucleic acid encoding the other protein. ｎ or (GGGS) ｎwherein n is a natural number between 2 and 5. (23) A non-human animal having the nucleic acid of (21) or (22) above. (24) The non-human animal of (23) above, wherein at least one of the nucleic acids encoding endogenous collagen IVα2 in its genome has the insertion and expresses the fusion protein. (25) The non-human animal of (23) or (24) above, wherein all of the nucleic acids encoding endogenous collagen IVα2 in its genome have the insertion, express the fusion protein, and survive and reproduce to adulthood. (26) The nucleic acid of (21) or (22) above, which is mRNA. (27) An engineered collagen IVα2 protein that is a translation product of the nucleic acid of (26). (28) A heterotrimeric complex comprising the engineered collagen IVα2 protein of (27) above and two collagen IVα1. (29) A method for obtaining the non-human animal described in (24) or (25) above, comprising obtaining a next generation of the non-human animal described in (24) or (25) above from sperm of a male of the non-human animal described in (24) or (25) above and an egg of a female of the non-human animal described in (24) or (25) above.

[0014] (41) The non-human animal according to (4), (5), (24), or (25) above, which has been born. (42) The non-human animal according to (4), (5), (24), or (25) above, which has grown to a reproductive stage or is an adult. (43) The non-human animal according to (41) or (42) above, in which the entire gene encoding endogenous collagen IVα2 protein has been replaced with a nucleic acid encoding the modified collagen IVα2 protein. (44) The non-human animal according to (41) or (42) above, in which a portion of the gene encoding endogenous collagen IVα2 protein has been replaced with a nucleic acid encoding the modified collagen IVα2 protein.

[0015] (51) A cell having the nucleic acid according to (1), (2), (21) or (22) above. (52) The cell according to (51) above, wherein at least one of the nucleic acids encoding endogenous collagen IVα2 on the genome has the insertion and expresses the fusion protein. (53) The cell according to (51) above, wherein all of the nucleic acids encoding endogenous collagen IVα2 on the genome have the insertion and express the fusion protein. (54) The cell according to any one of (51) to (53) above, which is a cell selected from the group consisting of pluripotent cells, somatic cells, tissue stem cells, tissue progenitor cells, and germline cells. (55) An isolated tissue or organ comprising the cell of (54) above. (56) An isolated organoid comprising the cell of (54) above.

[0016] (61) A nucleic acid encoding a modified collagen IVα1 protein, wherein the nucleic acid has an insertion of a nucleic acid encoding another protein (which may be, for example, a labeled protein; the same applies below) directly or via a linker in a region encoding the 7S domain (preferably N-terminal to the 7S domain, more preferably after the C-terminus of the signal sequence of exon 3, e.g., at the C-terminus), wherein the nucleic acid encoding the modified collagen IVα1 protein is capable of producing a collagen IVα1 protein fused with the other protein, wherein the modified collagen IVα1 protein can form a trimer with one collagen IVα1 protein and one collagen IVα2 protein, or wherein two modified collagen IVα1 proteins can form a trimer with one collagen IVα2 protein, and wherein a non-human mammal having the insertions in all of the collagen IVα1 proteins can survive and reproduce to adulthood. (62) The nucleic acid according to (61) above, wherein the other protein comprises a fluorescent protein. (63) A non-human animal having the nucleic acid according to (61) or (62) above. (64) The non-human animal according to (63) above, wherein at least one of the nucleic acids encoding endogenous collagen IVα1 in its genome has the insertion and expresses the fusion protein. (65) The non-human animal according to (63) or (64) above, wherein all of the nucleic acids encoding endogenous collagen IVα1 in its genome have the insertion, express the fusion protein, and are able to survive and reproduce to adulthood. (66) The nucleic acid according to (61) or (62) above, which is mRNA. (67) An engineered collagen IVα1 protein that is the translation product of the nucleic acid according to (66) above. (68) A heterotrimeric complex comprising two of the engineered collagen IVα1 proteins according to (67) above and one collagen IVα2. (69) An isolated animal cell, wherein at least one or all of the nucleic acids encoding collagen IVα1 contained in the cell are the nucleic acids according to (61) or (62) above. (70) An isolated organoid, tissue, or organ comprising the animal cell according to (69) above.

[0017] Figures 1A-1J show the development and results of a 4D imaging method for visualization of the extracellular matrix. Figure 1A shows a schematic diagram of the molecular structure of the basement membrane and the fluorescent protein insertion site in the collagen IV α2 chain. Figure 1B shows the genotypic distribution of offspring from a cross between Col4a2-eGFP heterozygous and Col4a2-eGFP heterozygous mice. Sample sizes are shown in Table 2. Figure 1C shows the appearance of 8-week-old wild-type and Col4a2-eGFP homozygous (homo) mice. Figure 1D shows a representative immunofluorescence image of an E15.5 embryo of a Col4a2-eGFP mouse stained with eGFP. Scale bar: 3 mm. Figures 1E-1H show representative immunofluorescence images of adult Col4a2-eGFP mouse tissues (scale bar: 50 μm). Figure 1E shows that in P56 adult dorsal skin tissue from heterozygous mice, eGFP (green) colocalized with collagen IVα2 (COL4A2) (red) in the epidermal basement membrane (closed white arrowhead), hair follicle epithelium (closed yellow arrowhead), and blood vessel-like tissue (open arrowhead). In P56 adult dorsal skin tissue from homozygous mice, eGFP (green) was distributed similarly to heterozygous mice, colocalizing with collagen IV (red) in the epidermal basement membrane (closed white arrowhead), hair follicle epithelium (closed yellow arrowhead), blood vessel-like tissue (open arrowhead), and arrector pili muscle (arrow). In adult kidneys, eGFP (green) was detected in Bowman's capsule (closed arrowhead), mesangial matrix (arrow), and collecting duct (open arrowhead). In the adult brain, eGFP (green) is localized to capillaries (closed arrowheads) and fracton-like structures (open arrowheads). DAPI (blue) was used as a counterstain for nuclei. Figure 1I shows a representative fluorescent stereomicroscope image of cultured dorsal skin from an E12.5 embryo of a Col4a2-eGFP mouse. A magnified image of the dotted rectangular area is shown. Closed arrowheads indicate primary hair follicles, and open arrowheads indicate secondary hair follicles. Scale bar: 200 μm. Figure 1J shows a representative snapshot image of a 3D maximum projection of a cultured skin excision from an E12.5 embryo of a Col4a2-eGFP mouse. The closed arrowhead indicates a ring-shaped accumulation of COL4A2-eGFP signal in the neck region of the hair follicle. The hair follicle indicated by the closed arrowhead in the left panel grows perpendicular to the observation plane.The dashed rectangle indicates a hair follicle growing horizontally from the curled surface of the epithelium. Snapshot images of a 3D time-lapse maximum projection of this hair follicle are shown in the right panel. Open arrowheads indicate blood vessel-like structures. Scale bar: 50 μm. Figure 1K shows the eGFP insertion site in Col4a2-eGFP. Figure 1L shows the eGFP insertion site in Col4a1-eGFP. Figures 2A-2I show that the rate of basement membrane expansion varies spatially and is synchronized with the direction of cell migration. Figure 2A is a schematic diagram of the definition of regions within the developing mouse hair follicle. Tip: The bending interface region between the prematrix and the dermal condensate. Lower stalk: The lower half of the hair follicle, extending to the tip region. Upper stalk: The upper half extending to the junction. Junction: The bending neck where the follicular epithelium and interfollicular epidermis meet. The molecular and organizational dynamics of the basement membrane were measured within these distinct basement membrane regions. Figure 2B shows representative confocal images of hair follicles in excised dorsal skin from Col4a2-eGFP mice used to measure changes in hair follicle length. The dotted rectangle indicates the area selected for photobleaching. Changes in basement membrane length were measured at the tip (blue line), lower shaft (orange line), and upper shaft (gray line) of the hair follicle during a 7-hour culture period. Scale bar: 20 μm. Figure 2C shows quantification of changes in basement membrane length during a 7-hour culture period (n = 9 hair follicles, from four independent explants / experiment). Values ​​are shown as mean ± SD. A two-tailed unpaired t-test was used. Figure 2D shows fluorescence images for measurements of BM thickness in the upper, lower, and upper shaft hair follicles. Figure 2E shows the measured BM thicknesses per site (n = 13). Figure 2F shows representative immunofluorescence images of developing hair follicles in fetal skin of Col4a2-eGFP mice stained with eGFP (green) and Ki67 (magenta). Ki67 was used as a proliferating cell marker, and DAPI (blue) was used as a nuclear counterstain (upper panel). We also attempted to detect cell proliferation based on EdU incorporation, and Hoechst was used as a nuclear counterstain. Scale bar: 20 μm.Figure 2G shows quantification of the percentage of Ki67-positive nuclei in the tip, substalk, and suprastalk regions (left panel) (n = 6 hair follicles from 4 explants / experiments) and the percentage of EdU-positive cells (right panel) (n = 18 hair follicles from 7 explants / experiments). Values ​​are shown as mean ± SD. A two-tailed unpaired t-test was used. Figure 2H shows representative snapshots from a 3D time-lapse movie of hair follicles developed from explants of Col4a2-eGFP;mem-tdTomato mouse embryonic skin. The relative positions of attached epithelial basal progenitor cells (indicated by white arrowheads) from the basement membrane (lower photobleached edge) and a reference point on the basement membrane (upper photobleached edge, indicated by yellow arrowheads) were measured at 0 and 9 hours and 30 minutes in both the lower stalk region (orange line) and the upper stalk region (gray line). Scale bar: 20 μm. Figure 2I shows bar graphs of the normalized length change (ratio to initial length) of the distance of basal progenitor cells from a reference point on the basement membrane (cell displacement) and the distance of the photobleached edge of the basement membrane from a reference point on the basement membrane (basement membrane expansion) (n = 7 cells, from independent in vitro cultures / independent hair follicles from independent experiments). Values ​​are shown as mean ± SD. Figure 2J shows bar graphs of the normalized length change (ratio to cell displacement length) of basement membrane expansion and calculated cell-autonomous migration. Data are based on Figure 2I. Values ​​are shown as mean ± SD. Figure 2K is a schematic diagram of the combined movement of cells and the basement membrane. Figure 2K shows that the movement directions of the basement membrane and cells are aligned, and that cells spontaneously move further on the basement membrane. Figures 3A-3C show spatially distinct collagen IV turnover. Figure 3A shows representative time-lapse images of a COL4A2-eGFP fluorescence recovery after photobleaching (FRAP) experiment. Confocal single-color images (top) and pseudocolor images (bottom) show the basement membrane of an excised hair follicle photobleached at the tip (blue arrowhead), lower stalk (orange arrowhead), and follicle-interfollicle junction (gray arrowhead) regions before and after photobleaching at selected recovery times. Scale bar: 20 μm.Figure 3B is a line graph of the normalized fluorescence recovery of COL4A2-eGFP over 4 hours (n = 6 hair follicles, each from an independent explant / experiment). Values ​​are shown as mean ± SD. A two-tailed unpaired t-test was used. Figure 3C shows the normalized mean intensity of COL4A2-eGFP in the extinction region of the FRAP experiment in Figure 2B at 3 hours and 30 minutes. Figure 3D shows representative maximum projection images of COL4A2-mKikGR in developing hair follicles at the indicated times after photoconversion in Col4a2-mKikGR mouse embryonic skin explants. Scale bar: 20 μm. Figure 4A outlines the experimental procedure for MMP inhibition. Figures 4B-4K show that matrix metalloproteinases are required for COL4A2 uptake, basement membrane expansion, and hair follicle morphogenesis. Figure 4B shows representative snapshot images of a COL4A2-eGFP FRAP experiment using developing hair follicles in Col4a2-eGFP mouse embryonic skin explants (with or without MMP inhibitors). The images show the differential fluorescence recovery rates of COL4A2-eGFP (green) after photobleaching the tip (blue arrowhead), apex (orange arrowhead), and junction (gray arrowhead) regions of control and 3 μM batimastat-treated (MMP inhibitor) hair follicles. Scale bar: 20 μm. Figure 4C shows quantification of the normalized mean intensity values ​​of COL4A2-eGFP fluorescence in the photobleached regions of the FRAP experiment in Figure 4B (n = 6 hair follicles for the control and n = 6 hair follicles for the inhibitor-treated conditions, from one and four independent explants per experiment, respectively). MMPi indicates MMP inhibitor treatment. Values ​​are shown as mean ± SD. A two-tailed unpaired t-test was used. Control data from this experiment are also shown in Figure 3B. Figure 4D shows the quantitative data of the mean intensity at 3 hours and 30 minutes from Figure 4C. Figure 4E shows the change in BM length in the early stage (0 hours to 7 hours) under control and MMP-inhibited conditions. n = 8 for the control experiment, n = 12 hair follicles for the inhibitor-treated condition, each from 4 independent ex vivo cultures / experiment.Figure 4F shows the changes in BM length at the late stage (16-23 hours) under control and MMP-inhibited conditions (n ​​= 9 hair follicles from six independent explant cultures / experiments for the control and n = 5 hair follicles from three independent explant cultures / experiments for the inhibitor-treated condition). Values ​​are shown as mean ± SD. A two-tailed unpaired t-test was used. Control data from this experiment are also shown in Figure 2C. Figures 4G-4O show the morphological changes of developing hair follicles cultured under control and MMP-inhibited conditions during the early MMP inhibitor treatment period (0-16 hours). Figure 4G shows representative confocal images (for Figures 4G-4O, n = 10 hair follicles from five independent explant cultures / experiments for the control and n = 14 hair follicles from four independent explant cultures / experiments for the inhibitor-treated condition). Figure 4H shows the changes in the shape factor S of hair follicles. The shape factor S was calculated by dividing the length of the hair follicle (LHF) by the width of the bulb (WHF). Figure 4I shows the change in hair follicle length after 0 hours and 20 hours under control and MMP-inhibited (MMPi) conditions. Figure 4J shows the change in normalized length of hair follicles after 0 hours and 16 hours under control and MMP-inhibited (MMPi) conditions. Figure 4K shows the change in hair follicle width after 0 hours and 20 hours under control and MMP-inhibited (MMPi) conditions. Figure 4L shows the change in normalized width after 0 hours and 16 hours under control and MMP-inhibited (MMPi) conditions. Figure 4M shows the change in shape factor S after 0 hours and 20 hours under control and MMP-inhibited (MMPi) conditions. Figure 4M shows the shape factor S after 0 hours under control and MMP-inhibited (MMPi) conditions. Figure 4O shows the shape factor S at 16 hours under control and MMP-inhibited (MMPi) conditions. Figure 4P shows the changes in hair follicle morphology due to MMP inhibitor treatment. Data are from the follicles used for FRAP analysis. Scale bar: 20 μm. Figures 4Q-4T show quantitative analysis of the shape of developing hair follicles cultured under control or MMP-inhibited conditions. Figure 4Q is a representative image of the measurement of shape factor S. L. ＨＦ is the length of the hair follicle, W ＨＦ indicates the width of the hair follicle. The shape factor S is L ＨＦ WＨＦThe S was calculated by dividing by . The graphs show the changes in hair follicle length (Figure 4R), hair follicle width (Figure 4S), and shape index (Figure 4T) over a 20-hour culture period. Ibid. Ibid. Ibid. Figure 4U shows a graph of the rate of change in S over a 20-hour culture period (n = 8 hair follicles obtained from 7 independent explant cultures / experiments for the control and n = 8 hair follicles obtained from 5 independent explant cultures / experiments for the inhibitor-treated conditions). Values ​​are shown as mean ± SD. A two-tailed unpaired t-test was used. Figures 5A-5F show that matrix metalloproteinase inhibition alters the daughter cell allocation angle of dividing cells. Figure 5A shows representative snapshot images and cell division tracking data of developing hair follicles in Col4a2-eGFP; mem-tdTomato mouse embryonic skin explant cultures. The basement membrane and plasma membrane were visualized with COL4A2-eGFP (green) and mem-tdTomato (magenta), respectively. Scale bar: 20 μm. Figure 5B shows quantification of cell division of epithelial basal progenitor cells in the experiment shown in Figure 5A (n = 6 hair follicles, each from an independent explant / experiment). Figure 5C shows representative results of an EdU incorporation assay. Dividing cells were labeled with EdU (magenta). All cells were counterstained with DAPI (blue). Scale bar: 20 μm. Figure 5D shows quantification of the percentage of EdU-positive basal epithelial cells in the tip, lower stalk, and upper stalk regions (n ​​= 7 hair follicles from three independent explants / experiment for control and n = 5 hair follicles from three independent explants / experiment for inhibitor-treated conditions). Values ​​are shown as mean ± SD. Two-tailed unpaired t-tests were used. Figure 5E shows the daughter cell allocation angle of proliferating basal epithelial progenitor cells relative to the basement membrane zone in the experiment in Figure 5A. Representative images of vertical and horizontal divisions are shown. Scale bar: 5 μm. Figure 5F is a pie chart showing the daughter cell arrangement angle relative to the BM under control and MMP inhibitor-treated conditions. Figure 5G is a plot showing the daughter cell allocation angle relative to the basement membrane under control and MMP inhibitor-treated conditions (n ​​= 48 cells from 11 hair follicles obtained from 7 independent explants / experiments for control; n = 36 cells from 8 hair follicles obtained from 8 independent explants / experiments for inhibitor-treated conditions).Figure 6 shows a model illustrating the role of basement membrane dynamics in hair follicle morphogenesis. This model summarizes the spatially distinct molecular and tissue-level dynamics of the basement membrane and explains its crucial role in controlling cell and tissue dynamics during hair follicle morphogenesis. The green line represents the basement membrane. The magenta cells and basement membrane zone represent tracer epithelial basal cells and basement membrane zones, respectively. Blue cells represent epithelial progenitor cells that divide horizontally relative to the basement membrane, and red cells represent epithelial progenitor cells that divide perpendicularly relative to the basement membrane. The following key features are observed during normal hair follicle development: (1) the rates of COL4A2 turnover and basement membrane expansion show spatial gradients; (2) epithelial progenitor cells divide both horizontally and vertically; (3) cells passively migrate on a directional expanding basement membrane rather than actively migrate on a stable, static basement membrane; and (4) epithelial duct structures elongate in a directional manner. On the other hand, in hair follicles treated with MMP inhibitors, (1) COL4A2 turnover and basement membrane expansion are suppressed. (2) Progenitor cells change the daughter cell allocation angle from horizontal to vertical. (3) Laterally extending epithelial tubular structures are formed. Figure 7 shows the generation of Col4a2-eGFP knock-in mice. A. Targeting strategy for generating Col4a2-eGFP knock-in mice. The eGFP protein was inserted into the N-terminal region (between A28 and Q29) of the 7S domain of the COL4A2 protein. To minimize structural interference of the inserted fluorescent protein with the structure and function of the COL4A2 protein, a flexible linker rich in glycine and serine, GGSGGSGGSGGS (SEQ ID NO: 3), was added to both ends of the eGFP protein. L, linker; LF, left forward primer; LR, left reverse primer; RF, right forward primer; RR, right reverse primer. B. Representative image of PCR screening to distinguish wild-type, heterozygous, and homozygous Col4a2-eGFP mice. Fabpi-200: Internal control primer pair for detecting the Fabpi gene. 50 bp DNA ladder RTU (GeneDirect) was used. C. Genomic sequence of the Col4a2 gene and linker boundary.D. Representative immunofluorescence images of eGFP-stained tissues from Col4a2-eGFP mouse embryos at E13.5, E15.5, and E17.5. Scale bar: 3 mm. E-G. Representative immunofluorescence images of tissues from Col4a2-eGFP mouse embryos. (E) At E15.5 (left panel), eGFP (green) signals are detected in the epidermal basement membrane (closed white arrowhead) beneath the keratin 14-positive (red) basal epidermis, the basement membrane of the hair follicle (closed yellow arrowhead), dermal vascular structures (open arrowhead), and the panniculus carnosus muscularis (arrow). At E17.5 (middle and right panels), eGFP (green) is detected in the perlecan-positive (red) epidermis and vascular basement membrane. F) In the E15.5 kidney, eGFP (green) appears around tubular epithelial structures (closed arrowheads). (G) In the E15.5 brain, eGFP (green) appears in the cerebellar basement membrane zone (closed arrowheads) and vascular capillaries (arrows). DAPI (blue) was used as a nuclear counterstain. Scale bars: (E, F) 20 μm, (G) 200 μm. H-K. Representative immunofluorescence images of adult Col4a2-eGFP mouse tissues. (H) In the dorsal skin tissue of P56 adult heterozygous mice, eGFP (green) colocalizes with collagen IV (red) and perlecan (red) in the basement membrane of the epidermis (closed white arrowheads), hair follicle epithelium (closed yellow arrowheads), arrector pili muscle (arrows), and blood vessel-like tissue (open arrowheads). (I) In the dorsal skin tissue of P56 homozygous mice, eGFP (green) is distributed similarly to heterozygous mice and colocalizes with perlecan (red). (J) In the adult kidney, eGFP (green) is detected in Bowman's capsule (closed arrowhead), mesangial matrix (arrow), and collecting duct (open arrowhead). (K) In the subventricular zone, eGFP (green) colocalizes with perlecan (red) in capillaries (closed arrowhead) and fracton-like structures (open arrowhead) surrounding GFAP+ (red) astrocytes. DAPI (blue) was used as a nuclear counterstain. Scale bar: 50 μm. Figure 8A shows the design sites of junction PCR primers for the unaltered and altered Col4a1 alleles. Figure 8B shows the genotypes of offspring obtained by mating Col4a1-eGFP heterozygous mice. The genotype can be determined based on which band in Figure 8A is amplified.Wild-type (WT) refers to a sample lacking the modified allele, heterozygotes (heterozygotes) refer to a sample in which one of the two alleles is the modified allele, and homozygotes (homozygotes) refer to a sample in which both alleles are modified. Samples in which the 1522 bp band or no band is detected with the LF-RR primer set for detecting the WT allele, and the 608 bp or 478 bp band is detected with the LF1-LR and LF2-RR primer sets for detecting the modified allele, are homozygotes. In other words, F1, M1, and M3 in the top panel are homozygotes. Figure 9A shows the staining of basement membrane in adult dorsal skin tissue sections from Col4a1-eGFP homozygous mice. COL4A1-eGFP protein (green) colocalizes with collagen IV (red) and perlecan (red), which are basement membrane components. Figure 9B shows the results of basement membrane staining in adult dorsal skin tissue sections from Col4a1-eGFP heterozygous mice. COL4A1-eGFP protein (green) colocalizes with collagen IV (red) and perlecan (red), which are basement membrane components. Figure 10 shows the re-photobleaching procedure for measuring BM expansion. Panel A shows representative images of the re-photobleaching procedure. Panel B shows representative images of changes in BM length. Panel B shows the procedure used to quantify the changes in BM length quantified in Panel A. Panel C shows a maximum projection image of Col4a2-eGFP in a hair follicle. Figure 11 shows the results of 3D BM structure analysis. Panel A shows a representative confocal image of a hair follicle surrounded by linear BM structures. Panel B shows representative confocal z-stack maximum projection and single z-plane images of a cultured skin explant from an E12.5 embryonic Col4a2-eGFP mouse showing a pore at the bottom (arrowhead). Panel C shows representative confocal z-stack maximum projection and single z-plane images of a hair follicle in a cultured skin explant from an E12.5 embryonic Col4a2-eGFP mouse treated with an MMP inhibitor (batimastat). Figure 12 shows snapshot images from a 3D time-lapse video of developing hair follicles from a Col4a2-eGFP; mem-tdTomato mouse.Panels A-B show representative snapshot single z-plane images of cells and BM at the lower stalk (A) and upper stalk (B) during tracking at 30-minute intervals (from 0 hours to 9 hours 30 minutes). Figure 13 shows the results of replicate imaging of COL4A2-mKikGR in developing hair follicles. Z-stack maximum projection snapshot images of COL4A2-mKikGR in embryonic skin explants from COL4A2-mKikGR mice are shown at the indicated times after photoconversion. Figure 14 shows Col4a2 mRNA expression in developing hair follicles. Panel A shows representative confocal images of Col4a2 mRNA expression in control and MMP inhibitor-treated developing hair follicles acquired using RNAscope. Panel B shows quantitative analysis of Col4a2 mRNA expression in control and MMP inhibitor-treated developing hair follicles in different tissue regions.

[0018] <Definition of Terms> As used herein, a "cell" may be an animal, plant, bacterial, or fungal cell. Animals include vertebrates, which include mammals, fish, and birds. As used herein, "mammals" include, for example, rodents such as mice and rats, livestock animals such as pigs and cows, pet animals such as rabbits, dogs, and cats, and primates such as monkeys. Fish include, for example, medaka and zebrafish. Birds include chickens. As used herein, an animal may be a non-human animal, a non-human mammal, or a non-human primate. A vertebrate is an animal that can be genetically modified.

[0019] As used herein, "nucleic acid" may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids may or may not contain introns. Nucleic acids may be isolated or in a form present in a cell nucleus. When modifying an endogenous gene of a cell or animal, DNA encoding a marker protein is introduced into the natural gene region, and in this case, the modified nucleic acid may contain introns. Alternatively, when a nucleic acid is knocked into a cell as a foreign gene, the foreign gene may not contain introns (e.g., have a cDNA sequence).

[0020] As used herein, "modification" means manipulating a gene sequence artificially so that the resulting sequence differs from that which occurs in nature.

[0021] As used herein, "collagen" refers to a component of the extracellular matrix, a structural protein that provides strength, flexibility, and structure to connective tissues such as skin, bone, cartilage, and tendons. The extracellular matrix is ​​a complex polymer network containing proteins and polysaccharides, the primary component of which is basement membrane. Basement membranes contain core proteins such as laminin, collagen IV, nidogen, and perlecan. Two independent self-organizing networks of laminin and collagen IV form a central structure, interconnected by nidogen and perlecan. It has been suggested that basement membranes are much more dynamic than previously thought, both at the molecular and structural levels. Collagen IV includes α1(IV), α2(IV), α3(IV), α4(IV), α5(IV), and α6(IV). Each collagen IV contains three distinct domains: a cysteine-rich 7S domain approximately 150 amino acids long located at the N-terminus, a triple-helical collagen domain approximately 1,300 amino acids long located in the center, and a globular NC1 domain approximately 230 amino acids long located at the C-terminus. Examples of database accession numbers for the amino acid sequences of collagens IVα1 and IVα2 from various animals are listed in Table 1. Those skilled in the art will be able to appropriately obtain the amino acid sequences, and, if necessary, the nucleotide sequences, of collagens IVα1 and IVα2 for animal species other than those listed in Table 1.

[0022]

[0023] Collagen IVα2 contains an approximately 150 amino acid cysteine-rich 7S domain at the N-terminus, a approximately 1300 amino acid triple-helical collagen domain in the center, and a approximately 230 amino acid globular NC1 domain at the C-terminus. Heterotrimeric collagen IV forms a tetramer via the 7S domain, which can be stabilized by disulfide bonds between collagen molecules, but it has also been reported that it can form a dimer via the NC1 domain to form a supramolecular meshwork. Collagen IVα2 is known to form a heterotrimer with two molecules of collagen IVα1. The gene to be modified can be collagen IVα1 or collagen IVα2.

[0024] As used herein, the term "another protein" refers to a protein or a fragment thereof that is different from the protein to be linked. The "another protein" may be a labeling protein that labels collagen IVα1 or collagen IVα2 without changing the physiological function of these collagens, or may be a functional protein that confers physiological functionality to these collagens. The other protein may have a molecular weight of, but is not particularly limited to, about 5 kDa to 50 kDa (e.g., 10 kDa to 30 kDa).

[0025] As used herein, a "labeled protein" refers to a protein that is linked to and labeled with another protein. Examples of labeled proteins include, but are not limited to, peptide tags (including tags for affinity purification), fluorescent proteins, and fluorescently labeled tags. Functional proteins also include other proteins with physiological functions (e.g., endogenous proteins) and sensor proteins (e.g., pH sensors or force sensors).

[0026] As used herein, a "fusion protein" refers to a protein containing two or more different proteins linked together. In a fusion protein, the two or more different proteins are linked directly or via a peptide sequence such as a linker. A linker is a peptide inserted between two proteins or tags when binding them, and is usually designed to have appropriate flexibility and length so as not to interfere with the structure or function of the proteins. The linker may be, for example, a flexible and inactive linker such as a GS linker. A fusion protein is preferably encoded by a single messenger RNA (mRNA). In the mRNA, regions encoding two or more different proteins are preferably linked in-frame with or without a sequence encoding a peptide sequence. "Protein A is fused to protein B" means that protein A forms the fusion protein together with protein B. When a fusion protein is referred to as "protein A-protein B," it includes proteins in which protein A is fused to the N-terminus of protein B, proteins in which protein A is fused to the C-terminus of protein B, and proteins in which the other protein is inserted into either protein A or protein B. Similarly, when a gene encoding a fusion protein is expressed as "gene X-gene Y," gene X and gene Y are linked in-frame, but this includes cases in which gene X is linked upstream of gene Y, cases in which gene X is linked downstream of gene Y, and cases in which either gene X or gene Y is inserted inside the other.

[0027] As used herein, "N-terminal side" refers to a region closer to the N-terminus than the center of a defined region, and "C-terminal side" refers to a region closer to the C-terminus than the center of a defined region. The "N-terminal side" and "C-terminal side" respectively include 1 / n (where n is a real number from 2 to 10) on the N-terminal side, for example, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, and 1 / 10 on the N-terminal side, and 1 / n (where n is a real number from 2 to 10) on the C-terminal side, for example, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, and 1 / 10. N-terminal side 1 / n means the region closest to the N-terminus when the region closer to the N-terminus than the center of the defined region is divided into n equal parts, and C-terminal side 1 / n means the region closest to the C-terminus when the region closer to the C-terminus than the center of the defined region is divided into n equal parts.

[0028] As used herein, the term "corresponding" in the context of amino acids and positions in amino acid sequences means that the amino acids align at the same positions when two or more amino acid sequences are aligned. Amino acids at corresponding positions typically confer equivalent functional characteristics to proteins across multiple amino acid sequences.

[0029] <Fusion Protein and Nucleic Acid of the Present Disclosure> The present disclosure provides a fusion protein containing a labeled protein and a collagen IVα1 or IVα2 protein, and a nucleic acid encoding the fusion protein. Specifically, the fusion protein and the nucleic acid may have the following configuration: The nucleic acid encodes the fusion protein, and the nucleotide sequences constituting the nucleic acid are linked in-frame. In the following description, the labeled protein is an example, and it is clear that the labeled protein may be a protein other than collagen IVα1 or collagen IVα2.

[0030] The present disclosure provides an engineered collagen IVα2 protein having a labeled protein inserted into its 7S domain-encoding region (particularly the N-terminal side of the 7S domain), for example, at a position corresponding to any of the 25th to 35th, preferably the 26th to 33rd amino acids (preferably a position corresponding to between the 28th and 29th amino acids) of the amino acid sequence of mouse collagen IVα2 protein (e.g., the amino acid sequence registered under Accession: AAA50293.1), directly or via a linker; the engineered collagen IVα2 protein can form a trimer with two collagen IVα1 proteins; and a non-human animal having all of the collagen IVα2 proteins modified can survive and reproduce to adulthood. The present disclosure also provides a nucleic acid encoding the engineered collagen IVα2 protein. In a preferred embodiment, the labeled protein is inserted via a linker, preferably via linkers on both the N- and C-terminal sides of the labeled protein. In a preferred embodiment, the linker is a flexible linker, for example, a GS linker consisting of glycine and serine. The linker consists of a flexible G and an S for increasing water solubility, and has a structure in which S is interposed between consecutive Gs, typically with Ss interposed at equal intervals between consecutive Gs. Examples of GS linkers include (GGS) ｎ , (GGGS) ｎ , or (GGGGS) ｎ{where n can be an integer from 2 to 6, for example, an integer from 3 to 5, for example, 4}. GGGS is set forth in SEQ ID NO: 4, and GGGGS is set forth in SEQ ID NO: 5. The putative signal sequence is the region from positions 1 to 25. In one aspect, the non-human animal survives normally to adulthood. "Normal" means that no abnormalities are observed (e.g., satisfying one or more or all of the following selected from the group consisting of: appearance indistinguishable from a wild-type animal; modified collagen is deposited in the basement membrane without ectopic deposition or deposition defects; no obvious abnormalities are observed in the tissue structure of organs; and offspring obtained by mating animals heterozygous for the modified collagen gene exhibit Mendelian inheritance and survive normally even when both alleles of the endogenous collagen gene are replaced with the modified collagen gene).

[0031] According to the present disclosure, there is provided an engineered collagen IVα1 protein, which has an insertion of a labeled protein directly or via a linker into the 7S domain (preferably N-terminal to the 7S domain, more preferably C-terminal to the signal sequence of exon 3), for example, an insertion of a labeled protein directly or via a linker into a position corresponding to any of the 27th to 37th, preferably the 28th to 35th, and more preferably the 29th to 33rd amino acid sequences (preferably between the 30th and 31st amino acid sequences) of the amino acid sequence of mouse collagen IVα1 protein (for example, the amino acid sequence registered under Accession: AAA50292.1), wherein one engineered collagen IVα1 protein can form a trimer with one collagen IVα1 and one IVα2 protein, or two engineered collagen IVα1 proteins can form a trimer with one collagen IVα2 protein, and wherein a non-human mammal in which all of the collagen IVα1 proteins have the above-mentioned modifications can survive and reproduce to adulthood. The present disclosure also provides a nucleic acid encoding the modified collagen IVα1 protein. In a preferred embodiment, the insertion of the labeled protein is via a linker, preferably via linkers on both the N-terminal and C-terminal sides of the labeled protein. In a preferred embodiment, the linker is a flexible linker, for example, a GS linker consisting of glycine and serine, for example, (GGS) ｎ , (GGGS) ｎ , or (GGGGS) ｎ {where n can be an integer from 2 to 6, for example, an integer from 3 to 5, for example, 4}. The putative signal sequence is in the region from amino acids 1 to 27. In one aspect, the insertion position is not between amino acids 28 and 29 of the amino acid sequence of collagen IVα1 protein. In one aspect, the non-human animal survives normally to adulthood.

[0032] The modified collagen IVα1 protein can form a heterotrimer with collagen IVα2 protein and be incorporated into the extracellular matrix (particularly basement membrane). The modified collagen IVα2 protein can form a heterotrimer with collagen IVα1 protein and be incorporated into the extracellular matrix (particularly basement membrane).

[0033] According to the present disclosure, there is provided a nucleic acid encoding a modified collagen IVα2 protein, wherein the modified collagen IVα2 protein has an insertion of a nucleic acid encoding a labeled protein either directly or via a linker into a region encoding the 7S domain (preferably N-terminal to the 7S domain, more preferably C-terminal to the signal sequence of exon 3), for example, an insertion of a nucleic acid encoding a labeled protein either directly or via a linker into a position corresponding to any of the 25th to 35th, preferably the 26th to 33rd amino acids {preferably a position corresponding to between the 28th and 29th amino acids} of the amino acid sequence of mouse collagen IVα2 protein (e.g., the amino acid sequence registered under Accession: AAA50293.1), wherein the nucleic acid encoding the modified collagen IVα2 protein is capable of producing a collagen IVα2 protein fused with the labeled protein, and the modified collagen IVα2 protein is capable of forming a trimer with two collagen IVα1 proteins, and wherein a non-human mammal having the insertions in all of its collagen IVα2 sequences is capable of surviving and reproducing to adulthood. In one embodiment, the non-human animal survives normally to adulthood.

[0034] According to the present disclosure, a nucleic acid encoding an engineered collagen IVα1 protein has an insertion of a nucleic acid encoding a labeled protein directly or via a linker into a region encoding the 7S domain (preferably N-terminal to the 7S domain, more preferably C-terminal to the signal sequence of exon 3), for example, an insertion of a nucleic acid encoding a labeled protein directly or via a linker into a position corresponding to any of the 27th to 37th, preferably the 28th to 35th, and more preferably the 29th to 33rd amino acids {preferably a position corresponding to between the 30th and 31st amino acids} of the amino acid sequence of mouse collagen IVα1 protein (for example, the amino acid sequence registered under Accession: AAA50292.1), the nucleic acid encoding the engineered collagen IVα1 protein can result in the production of a collagen IVα1 protein fused with the labeled protein, the engineered collagen IVα1 protein can form a trimer with one collagen IVα1 protein and one collagen IVα2 protein, or two of the engineered collagen IVα1 proteins can form a trimer with one collagen IVα2 protein, and A nucleic acid is provided in which a non-human mammal having the insertion in all of collagen IVα1 survives and reproduces to adulthood. In one aspect, the non-human animal survives normally to adulthood.

[0035] According to the present disclosure, it is preferable that a labeling protein is introduced into either the collagen IVα2 protein or the collagen IVα1 protein as described above, but this does not exclude the introduction of a labeling protein into both of them.

[0036] The present disclosure also provides a fusion protein that is a translation product of the nucleic acid.The present disclosure also provides an extracellular matrix (preferably a basement membrane) containing the fusion protein.

[0037] According to the present disclosure, the other protein includes one or more selected from the group consisting of a labeled protein such as a peptide tag, a fluorescent protein, and a fluorescently labeled tag, and a functional protein. In a preferred embodiment of the present disclosure, the labeled protein includes or is a fluorescent protein.

[0038] Examples of peptide tags include, but are not limited to, His tags, FLAG tags, Myc tags, HA tags, GST tags, MBP tags, V5 tags, T7 tags, S tags, AVI tags, Strep tags, and SUMO tags, which can be suitably used for protein purification and protein detection.

[0039] Examples of fluorescent proteins include modified GFP fluorescent proteins (more specifically, for example, GFP and fluorescent proteins having a GFP-like β-barrel structure (i.e., GFP-like proteins), point mutants thereof, and circular permutations thereof). GFP-like fluorescent proteins such as (sfGFP), EGFP, Citrine, Venus, mVenus, YFP, mApple, mOrange, mCherry, BFP, TagBFP, mTurquoise, and Cerulean, mHoneydew, mBanana, tdTomato, mTangerine, mStrawberry, mPlum, mScarlet, mNeonGreen, mNeptune, and NirFP, as well as circular permutants of these variant fluorescent proteins (for example, fluorescent proteins that are circular permutants having a helical structure at the C-terminus; adding the notation "cp-" as a prefix to the protein name indicates that the protein is a circular permutant). Many known GFP variant fluorescent proteins include those with substitutions in which the order of the β-sheets constituting the β-barrel is swapped, and those with a β-barrel structure in which the number of β-sheets is reduced, and these can be used as the fluorescent protein of the present invention. A circularly permuted mTurquiose (cpmTurquoise) can be used as the fluorescent protein. Since mTurquiose has a helical structure at its N-terminus, a circularly permuted cpmTurquiose with a helical structure at its C-terminus can be used. Known examples of mTurquiose include mTurquiose-DR, mTurquiose-GL, mTurquiose-GV, mTurquiose-RA, mTurquiose2, mTurquiose2-G, mTurquiose-146G, and mTurquiose-146S, as well as modified fluorescent proteins thereof. One or more selected from the group consisting of these can be used as is or after being converted into a circularly permuted mutant.

[0040] Fluorescent proteins also include photoconvertible fluorescent proteins such as KiKG from Favia favus, a type of stony coral, and its variants (e.g., KikGR1 and mKikGR). Photoconvertible fluorescent proteins change their fluorescent properties upon irradiation with light of a specific wavelength. For example, photoconvertible fluorescent proteins can change their fluorescence from green to red upon irradiation with light (e.g., laser light). For example, mKikGR normally has an excitation maximum at 505 nm and a fluorescence maximum at 520 nm, emitting green fluorescence upon excitation. However, upon irradiation with ultraviolet (UV) light, it changes to have excitation maxima at 360 nm and 580 nm, a fluorescence maximum at 590 nm, and emitting red fluorescence upon excitation. When a photoconvertible fluorescent protein is expressed in an animal and photoconverted, the localization and disappearance of the photoconverted fluorescent protein can be observed, as well as the production and localization of newly synthesized photoconvertible fluorescent proteins.

[0041] Fluorescent proteins also include phytochrome-based near-infrared fluorescent proteins, including iRFP and miRFP. iRFP contains the PAS domain and GAF ​​domain (PAS-GAF) of the bacteriophytochrome RpBphP2 of Rhodopseudomonas palustris, emits near-infrared fluorescence upon incorporating biliverdin IXa, lacks the C-terminal α-helix necessary for dimerization, and has the following mutations: S13L, A92T, V104I, V114I, E161K, Y193K, F198Y, D202T, I203V, Y258F, A283V, K288T, and N290Y (see Filonov et al., Nat. Biotechnol., 29(8): 757-761, 2011). miRFP contains the PAS and GAF ​​domains (PAS-GAF) of the bacteriophytochrome RpBphP1 of Rhodopseudomonas palustris, emits near-infrared fluorescence upon incorporating biliverdin IXa, lacks the C-terminal α-helix required for dimerization, and has various mutations (see Shcherbakova DM et al., Nature Communication, 7, Article number: 12405, 2016). Examples of miRFPs include, but are not limited to, miRFP670 (excitation / emission at 642 / 670 nm), miRFP709 (excitation / emission at 683 / 709 nm), and miRFP703 (excitation / emission at 673 / 703 nm), as well as miRFPs derived from these miRFPs. These phytochrome-based near-infrared fluorescent proteins have a helical structure at the C-terminus of the GAF domain and can be used to create fusion proteins with Affimers. In addition, miRFPs derived from the GAF domain of the cyanobacteriochrome NpR3784 from Nostoc punctiforme, such as miRFP670nano, can also be used to create the fusion proteins of the present invention.As miRFP670nano, 18 mutations, V7M, F25C, M26V, Y27F, P31E, S41A, A48S, N51K, Q55R, T57R, I72Y, G82N, H87Y, N99I, N117H, C119L, L136Q, and Q139V, have been introduced into the GAF domain of NpR3784. miRFP670nano can be used as a fluorescent protein in the present invention.

[0042] Examples of fluorescently labeled tag proteins include modified proteins derived from the dehalogenation domain of haloalkane dehalogenase from bacteria of the genus Rhodococcus. The dehalogenation domain of haloalkane dehalogenase has a pocket near Asp106 in the active center into which a ligand is inserted. The -NH-CH group of the ligand is inserted into this pocket. ２ CH ２ -O-CH ２ CH ２ -O-(CH ２ ) ６ The ligand is a fluorescent substance, and the group -NH-CH ２ CH ２ -O-CH ２ CH ２ -O-(CH ２ ) ６By introducing -Cl, a fluorescent substance can be bound to the tagged protein. Examples of dehalogenation domains of haloalkane dehalogenases that can be used for this purpose include those that have lost their dehalogenase activity, such as an active mutant (H272F) in which the histidine at the active center has been converted to phenylalanine. The binding of a ligand to the dehalogenation domain is suitable for polarized light observation. The present invention provides the use of the dehalogenation domain of haloalkane dehalogenase and its active mutants in fluorescence observation. Fluorescent substances that can be used for the ligand include coumarin, Oregon Green, diAcFAM, tetramethylrhodamine (TMR), STELLA Fluor 650, STELLA Fluor 700, STELLA Fluor 720, indocyanine green, Alexa Fluor 488, and Alexa Fluor 660, each of which is commercially available as a Halotag ligand.

[0043] Examples of pH sensors include, but are not limited to, fluorescent proteins that can visualize pH changes, such as pHluorin, SNARF-1, and functional variants thereof. Examples of force sensors include, but are not limited to, fluorescence resonance energy transfer (FRET)-based tension sensor modules, which can detect mechanical deformation based on the fluorescence intensity that changes in response to changes in the distance between two proteins.

[0044] According to the present disclosure, there is provided a cell having a nucleic acid encoding the fusion protein. The cell may have the modification in at least one or all of the nucleic acids encoding endogenous collagen IVα2 protein in its genome. The cell may have the modification in at least one or all of the nucleic acids encoding endogenous collagen IVα1 protein in its genome. The cell may also have an exogenous nucleic acid encoding the fusion protein.

[0045] According to the present disclosure, there is provided an animal, preferably a mammal, having a nucleic acid encoding the fusion protein. The animal, preferably a mammal, may have the modification in at least one or all of the nucleic acids encoding endogenous collagen IVα2 protein in its genome. The animal, preferably a mammal, may have the modification in at least one or all of the nucleic acids encoding endogenous collagen IVα1 protein in its genome. The animal, preferably a mammal, may have an exogenous nucleic acid encoding the fusion protein.

[0046] The nucleic acid may be DNA or mRNA, or may be DNA and mRNA. The mRNA may be spliced ​​mRNA. The mRNA may produce a modified fusion protein.

[0047] According to the present disclosure, there are provided a nucleic acid encoding the fusion protein, and an animal, preferably a mammal, having the fusion protein. In a preferred embodiment, the animal, preferably the mammal, has the modification in at least one or all of the nucleic acids encoding endogenous collagen IVα2 proteins in its genome. As a result, all collagen IVα2 proteins expressed by the animal, preferably the mammal, are the modified collagen IVα2 proteins.

[0048] In a preferred embodiment, the animal, preferably the mammal, has the modification in at least one or all of the nucleic acids encoding endogenous collagen IVα1 protein in its genome, such that all collagen IVα1 proteins expressed by the animal, preferably the mammal, are the modified collagen IVα1 protein.

[0049] In one preferred embodiment, the animal, preferably the mammal, can be a disease model.

[0050] In a preferred embodiment, the animal, preferably the mammal, is a fetus. In a preferred embodiment, the animal, preferably the mammal, is born. The animal, preferably the mammal, is reproductively developing or is an adult.

[0051] In a particularly preferred embodiment, even if part of the gene encoding collagen IVα1 protein or collagen IVα2 protein on the genome is replaced with a nucleic acid having the above-mentioned modification, the animal, preferably the mammal, is not lethal, and even if all of the genes encoding collagen IVα1 protein or collagen IVα2 protein on the genome are replaced with a nucleic acid having the above-mentioned modification, or even if the animal, preferably the mammal, does not have any other nucleic acids encoding collagen IVα1 protein or collagen IVα2 protein that have normal functions.

[0052] In some aspects of the present disclosure, the labeled protein is EGFP. In some aspects of the present disclosure, the labeled protein is mKikGR.

[0053] <Cells, Extracellular Matrices, Tissues, Organs, and Organoids of the Present Disclosure> According to the present disclosure, animal cells, preferably mammalian cells, having a modified genome are provided. The cells may be cells selected from the group consisting of pluripotent cells (e.g., pluripotent stem cells such as ES cells and induced pluripotent stem cells (iPS) cells), somatic cells, tissue stem cells, tissue progenitor cells, and germline cells.

[0054] In accordance with the present disclosure, there are provided germline cells, germ cells, and gametes, particularly sperm and eggs, of animals, preferably mammals, having a modified genome. In accordance with the present disclosure, there are provided pluripotent cells (e.g., pluripotent stem cells, such as ES cells and induced pluripotent stem (iPS) cells) of animals, preferably mammals, having a modified genome.

[0055] The cells may be derived from any tissue, including, but not limited to, the stomach, small intestine, large intestine, lung, pancreas, kidney, liver, thymus, spleen, prostate, ovary, uterus, bone marrow, skin, muscle, and peripheral blood. In some embodiments, the cells are non-blood cells. In some embodiments, the cells may be derived from a specific cell type in a tissue (e.g., epithelial cells, endothelial cells, epidermal cells, stromal cells, fibroblasts, adipose tissue, mammary cells, mesangial cells, pancreatic beta cells, neurons, glial cells, exocrine epithelial cells, endocrine cells, skeletal muscle cells, smooth muscle cells, cardiac myocytes, osteoblasts, embryonic cells, immune cells, etc.). In some embodiments, the cells may be normal cells or cancer cells. The cells may be isolated cells and may be mammalian cells, such as human cells.

[0056] The present disclosure also provides an extracellular matrix comprising the modified collagen IVα1 or modified collagen IVα2 of the present invention. The extracellular matrix can include, for example, a basement membrane.

[0057] The present disclosure also provides isolated organs or tissues containing the cells of the present invention. The isolated organs or tissues may contain the extracellular matrix (particularly the basement membrane) of the present disclosure. These organs or tissues may be obtained from animals, preferably mammals, of the present disclosure.

[0058] The present disclosure also provides cultured organoids comprising the cells of the present invention. Organoids are artificial tissues that contain multiple cells, preferably containing extracellular matrix (particularly basement membrane), and have a three-dimensional structure that can be cultured in a culture medium. Organoids include, but are not limited to, brain organoids, intestinal organoids, liver organoids, cardiac organoids, kidney organoids, lung organoids, pancreatic organoids, stomach organoids, skin organoids, retinal organoids, breast organoids, prostate organoids, and ovarian organoids, and are widely used as disease models, drug discovery research tools, or developmental research tools. Organoids comprising the modified collagen of the present disclosure can provide additional value to these applications.

[0059] In some aspects of the present disclosure, the labeling protein is a fluorescent protein. In some aspects, the labeling protein is EGFP. In some aspects of the present disclosure, the labeling protein is mKikGR. In some aspects, the labeling protein is a fluorescent protein, and the cells, extracellular matrices, tissues, organs, and organoids of the present disclosure are suitable for fluorescence observation.

[0060] <Method for observing fusion proteins of the present disclosure and cells or extracellular matrices (particularly basement membranes), tissues, organs, organoids, and animals (preferably mammals) containing the fusion proteins> In the fusion proteins of the present disclosure, the labeled protein may include one or more selected from the group consisting of peptide tags, fluorescent proteins, and fluorescently labeled tags. Peptide tags can be detected using other proteins (e.g., antibodies) that recognize the tags. For example, peptide tags may be detected using labeled antibodies, or the peptide tags may be complexed with a primary antibody and then detected using a labeled secondary antibody that recognizes the primary antibody. Fluorescent proteins can be detected by fluorescence emitted by irradiation with excitation light. Fluorescently labeled tags can be detected by adding a fluorescent label and then irradiating it with excitation light, resulting in fluorescence.

[0061] In a preferred aspect, an animal, preferably a mammal, expresses a fusion protein of the present disclosure. More preferably, the animal, preferably a mammal, has been genetically modified such that some or preferably all of the nucleic acids encoding endogenous collagen IVα2 proteins have the modification, and some or preferably all of the expressed collagen IVα2 proteins have the modification. In a preferred aspect, in the animal, preferably a mammal, the modified collagen IVα2 protein is incorporated into the extracellular matrix, more preferably the basement membrane, as collagen IVα2 protein.

[0062] In a preferred embodiment, an animal, preferably a mammal, expresses the fusion protein of the present disclosure. More preferably, the animal, preferably a mammal, is genetically modified such that some or preferably all of the nucleic acids encoding endogenous collagen IVα1 protein have the modification, and some or preferably all of the expressed collagen IVα1 proteins have the modification. In a preferred embodiment, in the animal, preferably a mammal, the modified collagen IVα1 protein is incorporated into the extracellular matrix, more preferably the basement membrane, as collagen IVα1 protein.

[0063] According to the present disclosure, a method for observing these animals, preferably mammals, is provided. The observation target site may be one or more sites selected from the group consisting of the epidermis, oral cavity, esophagus, stomach, duodenum, small intestine, large intestine, tissue, organ, and the surface thereof. Because collagen IVα1 protein and collagen IVα2 protein can be incorporated into any basement membrane in the body, any site in the body can be observed.

[0064] When the labeled protein is a peptide tag, the observation can be performed on a lysate of the excised sample containing the surface or on a section of the sample containing the surface.When the labeled protein is a fluorescent protein or a fluorescently labeled tag, the observation can be performed by irradiating the observation site with excitation light.

[0065] Observation can be performed at multiple time points. Observation can detect changes in the distribution of fluorescent proteins. Observation can, for example, estimate the activity of matrix metalloproteinases. Observation can, for example, estimate changes in matrix metalloproteinase activity. Observation in the presence and absence of a test compound can estimate the activity of the test compound on matrix metalloproteinase activity. For example, if matrix metalloproteinase activity is improved in the presence of the test compound, the test compound can be identified as an activator of matrix metalloproteinases. Furthermore, for example, if matrix metalloproteinase activity is reduced in the presence of the test compound, the test compound can be identified as an inhibitor of matrix metalloproteinases.

[0066] In one embodiment, the animal, preferably a mammal, can be a disease model. In this embodiment, the observation method of the present disclosure can observe collagen IVα1 protein or collagen IVα2 protein in the disease model and estimate the function or role of collagen IVα1 protein and collagen IVα2 protein in the disease. Furthermore, in the presence of a test compound, the function or role of collagen IVα1 protein and collagen IVα2 protein can be estimated in the disease model, and whether the test compound has a therapeutic or preventive effect on the disease in the disease model can be evaluated.

[0067] The fusion protein of the present disclosure and the cell or extracellular matrix (particularly basement membrane), tissue, organ and organoid that have said fusion protein can be observed under fluorescent microscope when labeling protein is fluorescent protein.The fusion protein of the present disclosure and the cell or extracellular matrix (particularly basement membrane), tissue, organ and organoid that have said fusion protein can be observed under fluorescent microscope when labeling protein is tagged protein, if necessary, the sample that comprises cell or extracellular matrix (particularly basement membrane), tissue, organ and organoid can be sectioned, fixed, and stained by immunocytochemical staining or immunohistochemical staining method, and can be observed under fluorescent microscope.

[0068] When the subject is alive, it may be useful to observe changes over time. Therefore, the observation method of the present disclosure may include time-lapse observation. Time-lapse observation may involve quenching (photobleaching) of a fluorescent protein and / or photoconversion of a photoconvertible fluorescent protein. This allows for observation of changes in the localization and / or distribution of a newly produced collagen IVα1 protein or collagen IVα2 protein in the subject. Furthermore, when photoconversion of a photoconvertible fluorescent protein is involved, it may be possible to observe either or both changes in the localization and / or distribution of a newly produced collagen IVα1 protein or collagen IVα2 protein in the subject and changes in the localization and / or distribution of a pre-existing photoconverted collagen IVα1 protein or collagen IVα2 protein.

[0069] <Production of the fusion protein of the present disclosure and the cells, tissues, organs, organoids, or animals containing the fusion protein> The fusion protein of the present disclosure can be produced using genetic engineering techniques. Animals containing the fusion protein of the present disclosure can also be produced using genetic engineering techniques. Modification of a collagen-encoding gene in the genome can be performed using a genome editing system (e.g., a CRISPR / Cas9 system). Donor DNA is prepared, which includes an upstream homology region, a nucleic acid encoding a labeled protein, and a downstream homology region. Here, the upstream homology region has a sequence capable of homologous recombination with the upstream of the labeled protein insertion site in the genome, and the downstream homology region has a sequence capable of homologous recombination with the downstream of the labeled protein insertion site in the genome. When the genome editing system cuts the region near the labeled protein insertion site in the presence of donor DNA, the labeled protein is inserted between the upstream and downstream regions when the cut region is repaired. In this way, for example, ES cells can be modified, and animals, preferably mammals, having the modified genome produced above can be obtained from the ES cells. ES cells are not required. The genome of a somatic cell can also be edited and the nucleus of the edited cell introduced into an enucleated egg cell to produce an animal, preferably a mammal, with a modified genome.

[0070] An animal, preferably a mammal, having a modified genome can be subjected to sexual reproduction (e.g., mating) to obtain progeny having the modified genome. Sexual reproduction can be achieved by fertilizing an egg with a sperm.

[0071] The cells or extracellular matrix (particularly basement membrane), tissues and organs of the present disclosure can be induced in vitro or obtained by isolating from the above-mentioned animals.The organoids of the present disclosure can be prepared in vitro from the cells of the present disclosure (for example, pluripotent stem cells).The induction and preparation can be carried out by using well-known conventional techniques.

[0072] <Testing Method for Physiologically Active Substances of the Present Disclosure> The present disclosure provides a method for testing a physiologically active substance. The testing method of the present disclosure includes contacting the isolated cells, tissues, organs, organoids, or animals with a test physiologically active substance. The testing further includes observing the effect of the test physiologically active substance on the labeled collagen IVα1 protein or labeled collagen IVα2 protein (preferably at the contact site). This allows the effect of the test physiologically active substance on the collagen IVα1 protein or collagen IVα2 protein to be tested. The test results can be useful, for example, for evaluating the effect of the test physiologically active substance on the extracellular matrix and tissues containing collagen IVα1 protein or collagen IVα2 protein. The evaluation results can be used, for example, to examine side effects of the test physiologically active substance. The evaluation results can be used, for example, to search for test physiologically active substances that act on collagen IVα1 protein or collagen IVα2 protein, or to evaluate their activity.

[0073] [Materials and Methods] Mice All experimental procedures were approved by the Institutional Animal Care and Use Committee of the RIKEN Kobe Branch. The ethical guidelines of the RIKEN Kobe Branch were also followed for the care and handling of mice. To visualize basement membrane dynamics, we generated Col4a2-eGFP knock-in mice by fusing eGFP to endogenous collagen IVα2 (Accession No. CDB0100E). A 717-bp eGFP sequence (without a stop codon) was inserted into the amino acid sequence A within the putative 7S domain of the gene Col4a2 (GenBank: AAI38041.1) encoding mouse collagen IVα2. ２８ and Q ２９ The fluorescent protein was inserted in frame between A and Q of mouse COL4A2 corresponding to the COL4A2 gene. To minimize structural interference of the inserted fluorescent protein with COL4A2 function, a flexible linker rich in Gly and Ser (GGS) was used. ４were added to both ends of the eGFP gene. To create a donor vector containing homologous arms (319 bp and 286 bp), the Col4a2 genomic sequence surrounding the eGFP insertion site was cloned into pBluescript II. The eGFP cDNA sequence and linker were incorporated into the insertion site in the donor vector. C57BL / 6 zygotes were microinjected with a mixture of Cas9 protein (100 ng / μl), crRNA (50 ng / μl) / tracrRNA (100 ng / μl), and donor vector (10 ng / μl), and the zygotes were then implanted into pseudopregnant females to generate founder knock-in mice. ３３ obtained.

[0074] crRNA (5'-ACU CCU GGC UCA GAG CGU CUG UUU UAG AGC UAU GCU GUU UUG-3'; SEQ ID NO: 6) and tracrRNA (5'-AAA CAG CAU AGC AAG UUA AAA UAA GGC UAG UCC GUU AUC AAC UUG AAA AAG UGG CAC CGA GUC GGU GCU-3'; SEQ ID NO: 7) were purchased from FASMAC. Knock-in mice were screened by PCR-based genotyping using three primer pairs (LF+LR, RF+RR, LF+RR) shown in Figure 7.

[0075] The primers were LF (5'-GCTGCTGCTAGCAACTGACAG-3'; SEQ ID NO:8), LR (5'-GTGCAGATGAACTTCAGGGTCAGC-3'; SEQ ID NO:9), RF (5'-GTCCTGCTGAGTTCGTGACC-3'; SEQ ID NO:10), and RR (5'-AGCACACCTACAATGCACACG-3'; SEQ ID NO:11). The LF and RR primer pair was designed based on the Col4a2 allele, and the RF and LR pair was designed based on the eGFP sequence. Three primer pairs were used for PCR genotyping to distinguish between the WT allele (LF-RR; 182 bp) and the Col4a2-eGFP knock-in alleles (LF-LR; 222 bp, RF-RR; 242 bp, and LF-RR; 971 bp) (Fig. 7B). Genomic sequencing confirmed the correct insertion of the linker and eGFP construct into the Col4a2 gene (Fig. 7C).

[0076] To examine basement membrane turnover, Col4a2-mKikGR mice (accession number CDB0126E) were generated using the same strategy. The mKikGR plasmid was obtained from addgene (#54656). ６７ Purchased from.

[0077] Knockin mice were screened by PCR-based genotyping using three primer pairs (LF1 + LR1, RF1 + RR, and LF1 + RR). The primers were LF1 (5'-AAGACTGGGATCATGGACCG-3'; SEQ ID NO:12), LR1 (5'-TGGCTCCCATTTGACGGTCTTCC-3'; SEQ ID NO:13), RF1 (5'-ACAGTCATAGAGGGCGGACCTCT-3'; SEQ ID NO:14), and RR (5'-AGCACACCTACAATGCACACG-3'; SEQ ID NO:11). The LF1 and RR primer pair was designed based on the Col4a2 allele, and the RF1 and LR1 pair was designed based on the mKikGR sequence. Three primer pairs were used for PCR genotyping to distinguish between the WT allele (LF1-RR; 363 bp) and the Col4a2-mKikGR knock-in alleles (LF1-LR1; 689 bp, RF1-RR; 751 bp, LF1-RR; 1134 bp).

[0078] To visualize cells and basement membranes simultaneously, Col4a2-eGFP mice were crossed with mTmG mice (The Jackson Laboratories, JAX Strain no. 007576). All mice except Col4a2-mKikGR were crossed with FVB / NJcl mice (CLEA Japan) to avoid interference with melanin deposition.

[0079] For fluorescent immunohistochemistry, tissues were embedded in OCT and frozen for cryosectioning (10-16 μm). Sections were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 5 minutes at 4°C, blocked and permeabilized with blocking buffer (0.5% skim milk / 0.25% fish skin gelatin / 0.5% Triton X-100 / PBS) for 1 hour at room temperature (RT), and then incubated with primary antibodies overnight at 4°C. Sections were then washed with PBS and incubated with fluorescently labeled secondary antibodies for 2 hours at room temperature (RT). Sections were then washed with PBS and mounted with DAKO Fluorescent Mounting Medium. Fluorescence signals were captured under a TCS SP8X (Leica) confocal microscope using LAS X software (version [BETA] 3.5.7.23723).

[0080] For whole-mount immunostaining, tissues were fixed with 4% PFA in PBS for 5 to 60 minutes at 4°C. The tissues were blocked with blocking buffer (0.5% skim milk / 0.25% fish skin gelatin / 0.5% Triton X-100 / PBS) for 1 hour at 4°C, permeabilized, and then incubated with primary antibodies overnight at 4°C. The sections were then washed with PBT (0.2% Tween-20 in PBS, pH 7.4) and incubated with fluorescently labeled secondary antibodies for 2 hours. Sections were then washed three times for 30 minutes in PBT (0.2% Tween-20 in PBS, pH 7.4) and dehydrated in 50% methanol / PBS for 10 minutes, 100% methanol for 5 minutes, 100% methanol for 10 minutes, 50% benzyl alcohol and benzyl benzoate (1:2) (BABB) / methanol for 5 minutes, 100% BABB for 5 minutes, and 100% BABB for 10 minutes. Fluorescent signals were captured under a TCS SP8X (Leica) confocal microscope using LAS X software (version [BETA] 3.5.7.23723). The antibodies used in this study are listed in Supplementary Table 1.

[0081] In vitro culture of embryonic dorsal skin was performed using the previously described method using pregnant mice. ３５The embryos were euthanized by cervical dislocation, and embryos at embryonic day (E) 12.5-13.5 were collected. Under a stereomicroscope, the entire dorsal skin of the embryo was peeled off with a 25G needle, and a fragment approximately 1 / 4 the size of the entire skin was excised for culture. Approximately 10 μl of collagen type IA (Nitta Gelatin) gel solution was added to an empty 35 mm Lumox dish (Sarstedt) on ice, and the skin fragment was embedded in the collagen gel with the dermis facing up. The skin / collagen gel drop was incubated at 37°C and 5% CO to solidify the collagen gel. ２ Humidification CO ２ The tissue was incubated in an incubator for 30 minutes, and then immersed in a medium containing L-glutamine and sodium pyruvate (Wako)-supplemented DMEM / Ham's F12 (1 ml), 20% fetal bovine serum (GIBCO), 100 units / mL penicillin and 100 μg / mL streptomycin (GIBCO, Grand Island, NY, USA), 1× GlutaMAX (GIBCO), 10 mM HEPES (GIBCO), and 100 μg / mL ascorbic acid (Sigma). ２ The cells were cultured at 37°C under humidified conditions containing

[0082] To inhibit MMP enzymatic activity, the medium was replaced with the same medium containing 3 μM batimastat (Abcam; ab142087) on day 3. All subsequent experiments were performed after a minimum of 16 hours of incubation following the addition of the MMP inhibitor.

[0083] EdU incorporation assay On day 4 of culture, Click-iT ＴＭ10 μM EdU was introduced into the culture medium of skin excisions using a 10 mM stock solution from the Plus EdU Cell Proliferation Kit (Thermo Fisher Scientific; C10640). In the MMP inhibitor-treated group, EdU was added 16 hours after the addition of batimastat. After incubation with EdU solution for 2 hours (Figure 2F) or 3 hours (Figure 5C), the skin excisions were fixed and permeabilized according to the manufacturer's protocol. EdU was detected using Click-iT Plus reaction, followed by nuclear counterstaining with Hoechst 33342 or DAPI. EdU-positive nuclei were counted using 3D images of hair follicles acquired at a thickness of 30 μm. To quantify EdU-positive nuclei, Imaris (version 8.4.2, Bitplane) was used to draw freehand lines along the BM (Col4a2-eGFP signal). The tip region was defined as the region between the two lowest curvature points (shown as blue lines in Figure 2F). A line was drawn from the interfollicular junction to the lowest protrusion point, and its midpoint was determined. The section from the curvature point to the midpoint was defined as the lower stalk region (shown as orange lines in Figure 2F), and the section from the interfollicular junction to the midpoint was defined as the upper stalk region (shown as gray lines in Figure 2F).

[0084] Live imaging of hair follicles in the dorsal skin of embryos. After skin tissue detachment, CO ２The cells were cultured in an incubator for 3 days to induce hair follicle formation. Live imaging of dorsal hair follicles was performed using a TCS SP8X (Leica) confocal microscope equipped with LAS X software (version [BETA] 3.5.7.23723), a stage-top incubator (Tokai Hit), and a 25x water-immersion objective (Leica, HC FLUOTAR 25x / 0.95 W VISIR), or an LSM980 (Carl Zeiss) equipped with a two-photon laser unit Chameleon Discovery NX (Coherent), Zen software (version 2.3), a stage-top incubator, and a 32x multi-immersion objective (Zeiss, C-Achroplan 32x / 0.85 W Corr M27). For two-photon laser imaging, a 920 nm laser and a 1040 nm laser were used to excite eGFP and tdTomato, respectively. For long-term live-cell imaging, immersion water was dispensed onto the objective lens using a Water Immersion Micro Dispenser (Leica) or Liquid Dispenser (Merzhauser Wetzlar). Sequential optical section images were stacked in the x-y plane with 512 × 512 pixels and a z-stack step size of 1–1.5 μm, covering a total tissue depth of approximately 30 μm. These image stacks were acquired at 10–30 min intervals.

[0085] Fluorescence Recovery After Photobleaching (FRAP) Photobleaching was performed using a TCS SP8X (Leica) confocal microscope or an LSM980 (Carl Zeiss) with the same accessory settings as described above. A 488 nm laser (100% power) was used to induce photobleaching of COL4A2-eGFP in an ROI measuring 15 ± 2 μm in length and 2–3 μm in width. Fluorescence intensity values ​​of the basement membrane region were acquired using ImageJ (version 1.53). First, lines were drawn on the basement membrane of the pre-bleached and bleached regions in three single z-plane images, including the z-stack plane of interest and the stacks above and below it, and the fluorescence intensity values ​​of each pixel were calculated. To generate a line graph, the fluorescence intensity values ​​of each line in the three z-planes were averaged and divided by the average fluorescence intensity value for the time before photobleaching. The specific protein dynamics of COL4A2, particularly its very low diffusivity within the BM and its slow turnover rate in the lower and upper stalk regions, necessitate long-term live imaging to document the complete recovery process. Such long-term observations allow us to accurately capture the plateau of Col4a2-eGFP recovery intensity. However, morphological changes in embryonic skin tissue, including the BM itself, make it increasingly difficult to track the photobleached BM zone throughout the recovery period. The possibility that the total amount of Col4a2-eGFP within the BM may increase or decrease during this long-term recovery process suggests that this experimental system is not static. As a result, due to the difficulty in reaching a clear plateau in the FRAP data, we used simple linear regression to analyze the fluorescence recovery data, which provided a more consistent analysis under these conditions.

[0086] Photoconversion. mKikGR has an excitation maximum at 505 nm and a fluorescence maximum at 520 nm, emitting green fluorescence upon excitation. However, upon irradiation with ultraviolet light, it changes to a red fluorescence with excitation maxima at 360 nm and 580 nm, and a fluorescence maximum at 590 nm (see WO 2004 / 111236A). COL4A2-mKikGR-expressing tissues were imaged under a TCS SP8X (Leica) confocal microscope using a 488 nm laser with the same accessory settings as described above. Photoconversion of a rectangular ROI (15 × 15 μm) was performed using a 405 nm diode laser (20% power, repeated five times). Photoconverted mKikGR was imaged with a 552 nm laser.

[0087] Measurement of changes in basement membrane length and cell position To ensure accurate and consistent measurements of BM length, an appropriate z-plane was carefully selected and monitored at each time point (Figure 11). For consistency, a z-plane was selected where a clear, sharp BM signal was observed in the target region located near the midline of the hair follicle, with the BM oriented vertically (Figure 11A). On the other hand, a tilted BM off the midline could be identified as an unclear, blurred BM signal. This selection allowed us to measure the same tissue region over time.

[0088] Next, the BM was photobleached to establish a reference point for measuring BM length. Photobleaching was performed using a 488 nm laser at 100% power, with three sets of 20 repetitions. ROIs were photobleached at 1 μm intervals across 10 ± 3 consecutive z-planes (covering z-planes where the BM region was observed as a distinct linear structure across the z-stack, centered on the target z-plane) to form a 3D cubic bleached region. As the Col4a2-eGFP signal recovered over time, it became difficult to identify the reference point (the edge of the photobleached edge) in regions with short rotation times. Therefore, every 2–3 h after each photobleaching, the edge of the photobleached region was identified and a new ROI was drawn along that edge. The size (x-y axes) and position of the ROI were adjusted according to the border between the photobleached and non-photobleached BM regions; this procedure ensured that the ROI was consistently located near the original BM region, allowing accurate measurements of BM length at different time points corresponding to the same z-axis.

[0089] After 7 hours, the z-plane where a clear BM could be observed in each section was selected for measuring BM length (Figure 10B). Since no significant morphological changes were observed at the boundary of the 3D cubic bleached area during the imaging period, we conclude that the BM maintained a consistent shape and elongation rate despite variations in the peripheral position of the hair follicle (Figure 10C). This suggests that peripheral positional variations do not affect the accuracy of the measurements.

[0090] To simultaneously analyze changes in basement membrane length and cell position, Col4a2-eGFP mice were crossed with mTmG mice (JAX stock # 007576). The basement membrane was photobleached to create a reference point for measurements. 3D live imaging allowed consistent identification of the same cells by capturing images at intervals of 20–35 min (Figure 13). Basement membrane length was measured between the photobleached edges of COL4A2-eGFP in the upper and lower stalk regions. Cell position was determined by measuring from the photobleached top edge of the reference point on the basement membrane to the midpoint of the plasma membrane of target cells in the upper and lower stalk regions.

[0091] Measurement of basement membrane thickness. Using Col4a2-eGFP signal as an indicator of BM structure, we used single z-plane confocal or two-photon microscopy images. To measure thickness, we used the freehand selection tool in ImageJ2 (version 2.14) to draw a line around the Col4a2-eGFP signal, creating a region with a long axis of 5–10 μm. This region was created in the apical, substalk, and suprastalk BM regions (see Figure 2D). The average BM thickness for each region was calculated by dividing the region area by the length of the long axis.

[0092] Measurement of the division angle of epithelial basal cells The direction of division of epithelial basal cells is determined by the line connecting the centers of the two daughter cells and the basement membrane underneath. ６８ The cell diameter was determined by measuring the angle between the cell membrane and a reference line drawn parallel to the axis. The cell membrane and basement membrane were simultaneously visualized with mem-tdTomato and COL4A2-eGFP, respectively. Cells were manually tracked using Imaris (version 8.4.2, Bitplane) by identifying cell shape and position at each time point, while referencing mem-tdTomato. Dividing cells were identified by observing a series of events: an initial increase in cell volume, rounding of the dividing cell, cleft formation, and finally separation of the daughter cells.

[0093] Detection and Quantification of mRNA Expression in Tissues To detect single RNA molecules in mouse embryonic tissues, whole-mount RNAscope was used. Briefly, freshly dissected tissues were embedded in collagen gel and incubated at 5% CO ２ Humidified CO ２The explants were cultured in an incubator at 37°C. On the third day of culture (16 hours before fixation), 3 μM batimastat was added to the MMPi cultures. On the fourth day of culture, the skin explants were fixed with 4% PFA / PBS. All samples were treated with hydrogen peroxide for 1 hour, washed three times with PBT (PBS containing 0.1% (v / v) Tween-20, pH 7.4), and then hybridized overnight at 40°C. After hybridization, the expression patterns of target mRNAs were detected and visualized using the RNAscope® Multiplex Fluorescent Reagent Kit v2 (ACD; 322381) according to the manufacturer's protocol. The stained tissues were washed with PBS, stored in Ultramount Permanent Mounting Medium (DAKO), and then photographed with a TCS SP8X (Leica) confocal microscope.

[0094] For signal quantification, the freehand selection tool in ImageJ2 (version 2.14) was used to define "ROI areas" in the basal layer, papillary dermis, dermal sheath, suprabasal epithelial layer, and hair follicles in the mesenchymal region, and the enclosed areas were drawn. Col4a2-positive signal particles were counted manually.

[0095] Statistical analysis and reproducibility: No statistical methods were used to predetermine sample size. Statistical parameters, such as the number of samples, number of replicates, type of statistical analysis, and statistical significance, were presented in the results, figures, and figure legends. For quantitative analysis, three or more biological replicates were ensured for each experiment.

[0096] All imaging data from this study have been stored in the SSBD repository (https: / / doi.org / 10.24631 / ssbd.repos.2023.04.298).

[0097] [Results] Development of a 4D imaging method to visualize basement membrane dynamics in living tissues To visualize basement membrane dynamics in real time, we first attempted to develop knock-in mice expressing fluorescently tagged endogenous collagen IV. Of the six polypeptide chains of collagen IV, we selected the α2 chain (Col4a2), which is present in the basement membrane of all tissues. The α2 chain is an α1α1α2 heterotrimer. ２８ The cDNA encoding the eGFP protein was inserted into the 7S domain of the collagen IVα2 chain in a mouse zygote using a CRISPR / Cas9-assisted knock-in method via an artificial linker sequence. ３３ The Col4a2-eGFP knock-in mice were inserted into the Col4a2 gene (Figures 1A, 1D, and 7A). PCR screening and genome sequencing confirmed successful introduction of the Col4a2 gene into Col4a2-eGFP knock-in pups (Figures 7B and 7C). Importantly, when knock-in heterozygote mice were bred, the Col4a2-eGFP allele was inherited by offspring in the expected Mendelian ratio of 1:2:1 (Figure 1B and Table 2). Furthermore, adult homozygous knock-in mice were visually indistinguishable from wild-type littermates (Figure 1C). Specifically, the following observations were made: 1. Mice expressing modified collagen IVα2 were indistinguishable in appearance from wild-type littermates; 2. In mice expressing modified collagen IVα2, the modified collagen IVα2 protein was deposited in the basement membrane similar to endogenous collagen IV, without ectopic deposition or defects; 3. 3. Mice expressing modified collagen IVα2 show no obvious abnormalities in organ histology; and 4. Mice expressing modified collagen IVα2 show Mendelian inheritance when bred, and survive normally even when both endogenous collagen IVα2 alleles are replaced with modified collagen IVα2. These results indicate that heterozygous and homozygous Col4a2-eGFP knock-in mice develop normally and are fertile. This is consistent with Jones et al. ６９This is in contrast to a study that reported that homozygous Turq2-Col4a1 knock-in mice were embryonic lethal. Mice with disrupted Col4a1 / 2 locus were embryonic lethal at E10.5-11.5, and mutations in the Col4a1 / 2 locus cause various developmental abnormalities and pathologies. Therefore, the modified collagen IVα1α1α2 produced in this example retains the function and distribution of endogenous collagen IVα1α1α2, which is necessary for ontogeny and homeostasis, and is considered to be useful for analysis and tool utilization.

[0098]

[0099] The same method as for Col4a2-eGFP was used to prepare the vector, and the above linker (GGS) was added to both sides. ４ The eGFP sandwiched between the two was introduced into Col4a1. Specifically, the introduction site was the amino acid sequence D ３０ and C ３１The genotype of the offspring from the cross between Col4a1-eGFP het and Col4a1-eGFP het was determined to be between D and C of mouse Col4a1, corresponding to the genotype of the offspring (see Figure 1E). The genotypes of the offspring from the cross between Col4a1-eGFP het and Col4a1-eGFP het were determined to be 4 wild-type, 9 heterozygotes, and 3 homozygotes, indicating that the genotype was transmitted to the offspring in a roughly Mendelian ratio (1:2:1). Specifically, the offspring obtained by crossing heterozygotes were examined for the presence or absence of EGFP insertion into Col4a1 by junction PCR (heterozygotes are sometimes referred to simply as "het" in this specification). The primer set used for detection was LF2 (SEQ ID NO:15: 5'-TTGAACCCTAAGACCCAGGGA-3'), LF3 (SEQ ID NO:16: 5'-GATCCAGGTTCTGTGGGACC-3'), LR2 (SEQ ID NO:17: 5'-CTTCATGTGGTCGGGGTAGC-3'), LF4 (SEQ ID NO:18: 5'-GAGCAAAGACCCCAACGAGA-3'), and RR2 (SEQ ID NO:19: 5'-TCCAAAAAAGCGCCATTGAGT-3'). LF2, LF3, and RR2 were designed on the Col4a1 allele, and LR2 and LF4 were designed on the eGFP sequence. Three primer pairs were used for PCR genotyping: LF2-RR2 (733 bp) for the wild-type allele, LF3-LR2 (608 bp) and LF4-RR2 (478 bp) for the Col4a1-eGFP knock-in allele, and LF2-RR2 for the wild-type and knock-in alleles (733 bp for the wild-type allele; 1522 bp for the knock-in allele, or no band due to the large amplification band size). As shown in Figure 8A, amplification products specific to the wild-type allele and the knock-in allele were examined in each litter. As shown in Figure 8B, F1, M1, and M3 in the top panel were homozygous, F3-F7 and M2 were heterozygous, and F2 was wild-type. In the lower panel, F2 to F4 were wild-type, and F1, F5, and M1 were heterozygotes, suggesting that the method of the present disclosure avoids embryonic lethality.Skin tissue sections were prepared from Col4a1-eGFP knock-in homozygotes and heterozygotes, and the localization of collagen IV and perlecan was observed by EGFP fluorescent and immunohistochemical staining. As a result, linear patterns of the basement membrane containing COL4A1 were visualized in both homozygotes (see Figure 9A) and heterozygotes (see Figure 9B), and this pattern colocalized with collagen IV and perlecan. Jones et al. reported that a fluorescent protein (mTurquoise2) was introduced at different positions (K) without a linker. ２８ and G ２９ The method of Jones et al. is embryonic lethal in homozygotes, suggesting some kind of dysfunction. COL4A2 forms a trimer with two Col4a1 molecules. Even if all COL4A2 molecules are labeled, only one of the trimers is labeled, allowing for the formation of labeled trimers with less functional interference, and all trimers can be labeled. In this respect, the COL4A2 labeling method of the present disclosure may be more useful than COL4A1.

[0100] eGFP was detected throughout embryos at E13.5, E15.5, and E17.5, and a basement membrane-like linear staining pattern was observed at the tissue boundaries of many organs, including the skin, kidney, and brain (Fig. 1D, Fig. 7D-G). In the adult skin of Col4a2-eGFP heterozygous mice, eGFP colocalized with COL4A2 and perlecan in the basement membranes of the epidermis, hair follicle epithelium, arrector pili muscle, and vascular tissues (Fig. 1E and Fig. 7H), suggesting that the eGFP tag is fused to the COL4A2 protein and that the fusion protein is correctly localized to the basement membrane. In homozygous mice, antibodies against eGFP, pan-collagen IV, and perlecan showed a basement membrane-like staining pattern and colocalized with each other (Fig. 1F and Fig. 7I). This suggests that normal basement membrane formation was achieved even when the endogenous Col4α2 allele was replaced with the knock-in allele. In adult kidneys, previous reports ３４As shown in Fig. 1G and Fig. 7J, eGFP was detected in glomeruli along Bowman's capsule and collecting ducts, and in an amorphous pattern in the mesangial matrix. In the adult brain, eGFP overlapped with perlecan and localized to blood vessels and fracton-like structures that form the blood-brain barrier (Fig. 1H and Fig. 7K). We conclude that the COL4A2-eGFP fusion protein was properly incorporated into the basement membrane and functioned normally.

[0101] Next, we investigated whether the COL4A2-eGFP fusion protein had sufficient fluorescence intensity to visualize hair follicle dynamics in long-term live imaging of 3D tissues. We employed an excised ex vivo culture system of embryonic dorsal skin, which mimics hair follicle development in vivo (Figure 1I). ３５ Confocal 3D live imaging captured spatiotemporal changes in COL4A2-eGFP. For example, changes in the shape of the basement membrane were captured in developing hair follicles and blood vessels without significant photobleaching (Fig. 1I, J). Furthermore, a ring-shaped accumulation of COL4A2-eGFP was observed in the neck region of developing hair follicles (Fig. 1J, closed arrowhead). These results demonstrate that knock-in mice expressing endogenously fluorescently tagged COL4A2 are a viable approach for studying basement membrane dynamics in situ.

[0102] Spatially Differential Basement Membrane Expansion Rates Synchronize with Directional Cell Migration. To investigate how the basement membrane expands spatiotemporally with the elongation and shape changes of developing organs, we first photobleached eGFP fluorescence in several rectangular regions of the basement membrane to compartmentalize basement membrane zones and measured the length changes of these basement membrane compartments (Figures 2A, 2B, and 2C). To maintain the photobleached edges, photobleaching was repeated every 2–3 h (see Methods and Figure 10). After 7 h of culture, the basement membrane showed an approximately 30% increase in length at the tip of the growing hair follicle, and its elongation rate gradually decreased toward the top of the follicle, with no or reduced elongation in the upper stalk region (Figures 2B and 2C). Thus, the basement membrane of developing hair follicles does not expand uniformly like a passively inflating balloon, but rather undergoes spatially different expansion, creating a spatial gradient in the expansion rate. Considering the rapid expansion of the basement membrane (BM) near the tip, we investigated structural differences in different regions of the BM. Using Col4a2-eGFP signal as an indicator of BM structure, we first measured its thickness in different tissue regions. The BM in the junctional / upper stalk region was 55% and 53% thicker than those in the tip and lower stalk regions, respectively (Fig. 2D and 2E).

[0103] While extensive mesh-like micropores have been detected in the salivary glands, lungs, kidneys, and pregastrulation embryos of developing mice (Harunaga, Doyle, and Yamada, 2014; Kyprianou et al., 2020), we did not detect these typical micropores in this study (see Figure 11A). Instead, pores were occasionally present in the BM beneath some developing hair follicles (see Figure 11B). These may be temporary pores allowing melanocytes and immune cells to pass through the BM (Adameyko et al., 2009; Bahr et al., 2022; Kabashima et al., 2019; Vandamme and Berx, 2019). These pores were not observed after treatment with a matrix metalloproteinase inhibitor (batimastat) (see Figure 11C). These observations suggest organ-specific variations in BM remodeling.

[0104] Basement membranes can control tissue shape by inducing changes in cell behavior and creating patterned physical constrictions. ４ Next, we investigated the relationship between the rate of basement membrane expansion and the proliferation of basal epithelial progenitor cells. Immunostaining for the proliferation marker Ki67 and EdU incorporation assays revealed that approximately 75% of basal epithelial progenitor cells were proliferating in the tip region, where basement membrane expansion was prominent, and this percentage gradually decreased toward the top of the hair follicle. Only approximately 25% of cells in the upper shaft region were Ki67-positive (Figures 2F and 2G). A similar trend was observed in the EdU incorporation assay. This suggests that the rate of cell proliferation is related to the degree of basement membrane expansion.

[0105] We further investigated cell movement on this expanded basement membrane to determine how much of the observed cell movement can be explained by active cell-autonomous migration, how much contributes to cell movement relative to the basement membrane, and how much is the result of coordinated movement with the BM. To track the migration of epithelial basal progenitor cells and their relative position to the underlying basement membrane, we photobleached COL4A2-eGFP fluorescence in several regions of the basement membrane and measured changes in both cell position and basement membrane length (Figure 2H). Over a 9.5-hour period, cells in the upper stalk region displaced approximately 4% from their reference point on the basement membrane (the photobleached upper end) toward the tip of the hair follicle, whereas cells in the lower stalk region displaced approximately 32% (Figure 2I). The lower basement membrane contracted approximately 3% in the upper stalk and extended approximately 22% in the lower stalk toward the tip of the hair follicle. To determine the contribution of cell-autonomous movement, including the effect of cell proliferation, to overall cell displacement, we subtracted the contribution of basement membrane extension from cell displacement (Fig. 2J). In the lower stalk region, the contribution of cell-autonomous movement was approximately 69% of total cell displacement. These results indicate that both cells and the basement membrane move cooperatively toward the tip of the hair follicle. Our measurements further support this, showing that the photobleached edge of the basement membrane moved along with the cells (see Fig. 2H and Fig. S13). Taken together, these results suggest that basal epithelial cells move in unison with the directionally expanding underlying basement membrane but do not actively migrate on a stable basement membrane (Fig. 2K). A statistically significant difference was observed between the upper and lower stalks for cell migration in Fig. 2K (n = 7).

[0106] Basement membrane expansion is associated with increased turnover of collagen IV proteins. The molecular mechanisms driving the expansion of pre-existing edgeless basement membranes remain largely unknown. They may be driven by the incorporation of new collagen IV proteins through homeostatic remodeling of the pre-existing collagen IV network. However, the spatiotemporal extent to which the incorporation and turnover of basement membrane molecules occurs during morphogenesis has not been quantified with cellular spatial resolution. Therefore, we measured the kinetics of fluorescence recovery after photobleaching (FRAP) of COL4A2-eGFP as a proxy for the incorporation of collagen IV proteins into the basement membrane (Figure 3A). Surprisingly, given the slow turnover rate of collagen reported in adult animals (days to months), this kinetics is not significantly different from that observed in adult animals. ３６－３８ Unlike the COL4A2-eGFP-containing follicular stalks, the fluorescence in the tip region of the hair follicle recovered 50% in only 3 hours and 25 minutes (205 minutes) after photobleaching, as suggested by curve-fit data (Figures 3A and 3B), demonstrating a significantly faster COL4A2 uptake rate. Fluorescence recovery was observed uniformly throughout the large photobleached region but not from its edge, suggesting that fluorescence recovery occurred via extrafollicular uptake of COL4A2-eGFP rather than via molecular diffusion from adjacent, unphotobleached hair follicle regions. In contrast, COL4A2-eGFP in the lower stalk and junction recovered approximately 20.3% and 5.7%, respectively, indicating a relatively slower COL4A2 uptake rate (Figure 3B). Figure 3B shows results from n = 3, but this fluorescence remained unchanged when the number of samples was increased to n = 6. These observations were supported by statistical tests based on experimental data, which confirmed that fluorescence in the apical region had recovered by 54% at 3 hours and 30 minutes after bleaching, whereas the substalk and junctional regions recovered more slowly, by 23% and 7%, respectively (Fig. 3C).

[0107] Furthermore, to monitor the dynamics of COL4A2 protein at specific locations and time points, we generated a knock-in mouse line expressing a fusion protein, Col4a2-mKikGR, by replacing eGFP in COL4A2-eGFP with the photoconvertible fluorescent protein mKikGR, fusing endogenous COL4A2 with mKikGR. Col4a2-mKikGR emits green fluorescence, but upon UV laser irradiation, it emits red fluorescence. The photoconverted red COL4A2-mKikGR in the basal membrane of the lower stalk region gradually decreased and almost completely disappeared over 8-10 hours of culture. Meanwhile, the green fluorescence gradually increased, replacing the red signal (see Figure 3D and Figure 14). This color change from red to green indicates COL4A2 protein turnover, meaning that existing COL4A2-mKikGR protein is replaced by newly synthesized or recruited protein. In contrast, photoconverted red COL4A2-mKikGR remained in the upper stalk and follicle-to-follicle junction. These results suggest that the turnover rate of COL4A2 is rapid at the tip of developing hair follicles. We also observed that red fluorescence did not diffuse from the photoconverted region. Therefore, the rate of hair follicle area expansion is closely related to the rate of COL4A2 area turnover.

[0108] Matrix metalloproteinases are required for COL4A2 uptake, basement membrane expansion, and hair follicle morphogenesis. Proteolytic cleavage of ECM components plays an important role in ECM remodeling. ９ At the molecular level, proteolysis of basement membrane components cleaves assembled basement membrane proteins, promoting their degradation and metabolism, making the basement membrane more flexible and potentially creating new insertion sites for free basement membrane proteins. This process promotes basement membrane turnover, turnover, and remodeling. The central enzymes in ECM remodeling are matrix metalloproteinases (MMPs). ９Recent advances in ECM imaging have begun to reveal that the protease activity of MMPs is required for molecular and tissue-level dynamics of basement membranes, efficient branching morphogenesis, and early embryonic development. １４，１５，３９ We therefore hypothesized that MMP activity is required at the molecular and tissue level to generate the basement membrane dynamics observed during hair follicle morphogenesis. We tested this by treating excised skin tissue with batimastat (BB-94), a broad-spectrum peptidomimetic MMP inhibitor that binds to the active site of MMPs and inhibits the enzymatic activity of several MMPs, including MMP-2 and MMP-9, which cleave collagen IV. ４０ After 16 hours of treatment with this inhibitor (see Figure 4A), the fluorescence recovery of COL4A2-eGFP in the FRAP assay was significantly delayed in all regions of the hair follicle basement membrane (Figures 4B-4D). The fluorescence in the tip region of control, untreated hair follicles recovered 50% after only 3.5 hours of photobleaching, whereas the fluorescence in the inhibited samples recovered only approximately 5% (Figures 4C-4D). These reduced recovery rates under MMP inhibitor treatment were not due to suppression of Col4a2 mRNA expression (Figures 15A and 15B). Similarly, the recovery rates in the lower stalk and junction were significantly reduced. These quantitative data indicate that MMP enzymatic activity is important for the incorporation of COL4A2 into the basement membrane.

[0109] Next, we examined the changes in the basement membrane expansion rate of hair follicles treated with MMP inhibitors. Quantitative analysis revealed that the BM exhibited distinct elongation patterns over the first 7 hours. The BM in the tip region showed a tendency toward decreased elongation, while the BM in the lower and upper stalk regions tended to shrink further (Figure 4E). However, over the next 7 hours (16–23 hours after MMP inhibitor addition), the tip region of MMP inhibitor-treated hair follicles only increased by approximately 6%, compared with the approximately 30% increase observed in the control follicles (Figure 4F). Little elongation or shrinkage was observed in other regions. These results indicate that MMP enzymatic activity is required for COL4A2 uptake and basement membrane elongation. Although early or partial MMP inhibition allowed some BM elongation, the spatial pattern of BM elongation was altered. Furthermore, these results suggest that collagen IV protein turnover kinetics is closely related to BM tissue-level elongation, generating a spatial gradient in BM elongation rate. The correlation observed under control conditions is further supported by the fact that local COL4A2 turnover rate and BM elongation are suppressed under MMP inhibition, indicating that this relationship promotes BM elongation and plays an important role in establishing spatial gradients in BM dynamics.

[0110] Changes in basement membrane dynamics and structure can profoundly influence organ morphogenesis and shape. １７，４１ Therefore, we investigated the morphological changes of developing hair follicles with and without the addition of MMP inhibitors. Early after the addition of MMP inhibitors (0-16 hours), hair follicles showed abnormal and disproportionate shapes (Fig. 4G). Hair follicles treated with the inhibitors stopped elongating (L). ＨＦ 4J-4J), and the width of the hair follicle increased (W ＨＦ These different tissue structures are referred to as L ＨＦ and W ＨＦThe shape factor S was expressed as the ratio of BM length to BM length (Figure 4H). The high and increasing S values ​​in control hair follicles indicated the formation of elongated, cylindrical structures during normal development (Figures 4M-4O). In contrast, the S values ​​in inhibitor-treated hair follicles did not increase as development progressed, but rather tended to decrease (Figures 4M-O). This indicates the formation of elongated hair follicle structures compared to controls. This elongation effect during the early stage of MMP inhibition, when BM elongation is still possible, suggests that partial BM elongation with an altered spatial pattern may lead to abnormal hair follicle shape. However, during the later stage of MMP inhibitor treatment, collagen IV protein uptake was significantly delayed and BM elongation was almost completely suppressed (Figures 4B-D and 4F), so hair follicles did not show further shape changes compared to controls during this period (Figures 4P-U). These observations suggest that MMP inhibition treatment initially alters the spatial pattern of the BM, allowing elongation and causing follicle enlargement, but later suppresses BM elongation and changes in hair follicle shape. We conclude that MMP enzymatic activity is required for COL4A2 protein uptake and turnover and basement membrane expansion. Inhibition of COL4A2 protein uptake and turnover and basement membrane expansion inhibits hair follicle morphogenesis.

[0111] Inhibition of matrix metalloproteinases alters the direction of daughter cell allocation in epithelial progenitor cells. Spatiotemporal regulation of cell proliferation and spatial arrangement of cells are major determinants of organ shape. ４２To explore possible reasons for the wider shape of developing hair follicles after MMP inhibitor administration, we examined the cell division patterns of epithelial basal progenitor cells by live imaging (Figure 5A). Cell division decreased in MMP-inhibited follicles over the 9.5-hour imaging period (Figure 5B). In inhibitor-treated follicles, EdU-positive basal cells decreased in the tip and lower stalk, but not in the upper stalk, suggesting a small effect of MMP inhibitors on basement membrane dynamics (Figures 5C and 5D). In the lower stalk, a statistically significant difference in cell migration was observed between the control and MMP-inhibited groups. These data indicate that MMP inhibition reduces basal cell proliferation, but a substantial number of basal cells continue to proliferate over the experimental period.

[0112] We further investigated the orientation of daughter cells relative to the basement membrane using live imaging data. In control hair follicles, 50% of dividing cells formed an orientation angle below 30% (8 < 30°; horizontal), while the remaining 39.6% formed an orientation angle above 60% (8 > 60°; vertical) (Figures 5A, 5E, and 5G). Surprisingly, with MMP inhibitor treatment, approximately 13.9% of dividing daughter cells were assigned to a horizontal orientation, while 75.0% were assigned to a vertical orientation (above the basement membrane) (Figure 5F). This change was observed across the entire tissue region (Figure 5G). Thus, the orientation of daughter cell allocation of basal epithelial progenitor cells is closely linked to MMP-dependent COL4A2 protein dynamics and basement membrane expansion. These results suggest that increased cell supply toward the center of the epithelial tissue in the tip and lower stalk regions contributes to the generation of the wider hair follicle tissue shape observed under MMP inhibition.

[0113] Discussion: In this study, we successfully generated knock-in mice expressing fluorescently tagged endogenous COL4A2. Through live imaging and quantitative analysis of COL4A2 dynamics during hair follicle formation, we revealed significant spatial gradients in the rates of COL4A2 turnover and basement membrane expansion (Figure 6). Importantly, these two processes are tightly coupled. Furthermore, epidermal progenitor cells migrate along with the expanding basement membrane, and MMP inhibition significantly suppresses both COL4A2 turnover and basement membrane expansion. This inhibition shifts the division angle of epidermal progenitor cells to a completely vertical direction. Meanwhile, epithelial tube elongation ceases, resulting in the emergence of wider tube shapes. Our results elucidate the interplay between COL4A2 turnover, basement membrane expansion, and epidermal progenitor cell behavior, revealing the complex orchestration of tissue morphogenesis via the molecular and functional dynamics of the basement membrane.

[0114] Challenges and Importance of Fluorescent Tagging of Endogenous Basement Membrane Proteins A major challenge in ECM biology is to quantitatively investigate the dynamics of the matrix, which exists as a complex supramolecular assembly, in real time at both the molecular and tissue levels. Traditionally, fluorescently labeled antibodies against ECM proteins have been used to visualize ECM in tissues, providing valuable insights into ECM dynamics during morphogenesis. ３９，４３，４４Fluorescent tagging of endogenous ECM proteins is expected to solve these problems, but this has been difficult to achieve in mammals. This is due to the complex composition of the BM, interactions with cell surface receptors, and the lack of an experimental system that can efficiently confirm whether fluorescently tagged BM proteins are integrated into the BM without impairing their function. In this study, we inserted eGFP or mKikGR near the N-terminus of the 7S domain of the endogenous Col4a2 gene, avoiding the putative N-terminal intermolecular covalent binding site and the collagen triple helical domain of the 7S domain. Our results suggest that the 7S domain plays an important role in intermolecular binding and collagen IV network formation and stability, but its N-terminus may be structurally tolerant to the insertion of exogenous molecules. This site can be used for labeling with fluorescent proteins or biosensors, allowing the introduction of morphogenetic factors, pH sensors, and calcium sensors within the BM.

[0115] In this study, eGFP or mKikGR with a short linker was inserted near the N-terminus of the 7S domain of the endogenous Col4a2 gene, avoiding the putative intermolecular covalent cross-linking site at the N-terminus and the collagenous triple helix domain of the 7S domain. ３２，５４，５５ These results suggest that the 7S domain is intermolecularly cross-linked and plays an important role in the network formation and stability of collagen IV, while its N-terminus may be structurally tolerant to the insertion of foreign molecules. This site could be used for tagging with other fluorescent proteins, bioactive molecules, and biosensors (e.g., morphogens, pH sensors, calcium sensors) in the basement membrane in vivo.

[0116] Two mouse strains carrying fluorescently labeled collagen IV exhibit distinct homozygous phenotypes. Col4a2-eGFP exhibits normal development, while mTurq2-Col4a1 is embryonic lethal (Jones et al., 2024). Several factors may account for this difference. The fluorescent protein insertion sites are different: Col4a2-eGFP is inserted between A28 and Q29 of Col4a2, while mTurq2-Col4a1 is inserted between K28 and G29 of Col4a1. Furthermore, because COL4A1 and COL4A2 form a heterotetramer with an α1α1α2 configuration, differences in the number of fluorescent proteins per tetramer may affect function. Furthermore, differences in the fluorescent proteins and linker design (in this strain, (GGS)) may also contribute to this difference. ４ The use of fluorescent proteins (using linkers on both sides) may also contribute to the different phenotypes. Differences in the properties of fluorescent proteins and the flexibility of linkers may affect the functionality of collagen IV, but the mechanisms are unclear. Further functional and structural studies, as well as the development of effective screening systems, are required to elucidate the causes of these differences and to gain a deeper understanding of the impact of fluorescent labeling on molecular function and animal development.

[0117] Embryonic development involves large-scale cell migration and tissue transformation. The dynamics of cell behavior during this process are thought to be primarily due to the coordinated interplay of active cell shape changes, cell migration, and cell proliferation, with the ECM being viewed as a passive, stable substrate for these cell-autonomous activities. 13,42,56,57 For example, during development and regeneration in mouse hair follicles, key morphogenetic cell dynamics, such as increased cell proliferation and apical migration of epithelial cells, are observed. ３５，５８However, our live imaging and quantitative analysis of basement membranes reveal that basement membranes exhibit a spatial gradient in elongation rate, resulting in directional expansion along the organ's elongation axis. Furthermore, epithelial progenitor cells proliferate and migrate alongside the directionally expanding basement membrane, rather than actively migrating on a stationary basement membrane. When basement membrane extension is restricted by MMP inhibitors, progenitor cells change the daughter cell allocation angle from horizontal to vertical, halting the organ's directional elongation and resulting in a wider organ. These findings suggest that directional basement membrane expansion contributes to the coordinated, directional expansion of tubular epithelia during morphogenesis by controlling daughter cell allocation angles and cell displacement.

[0118] Basement membrane movement has also been observed in other systems, such as avian embryos, where vortex-like movements of fibronectin-containing ECM beneath the epiblast, such as the basement membrane, have been reported. ４７ In hydra, the basement membrane and fibrous matrix were shown to move along with the cells toward the tips of the legs and tentacles. ６３ Furthermore, directional movement of the collagen IV network has been observed during branching morphogenesis of the salivary gland. ３９ Interestingly, in this experimental system, the collagen IV network moved from the branch tips toward the stem, which is opposite to the direction of basement membrane movement in developing hair follicles. Therefore, quantitative measurements of complex tissue movements involving both cellular and matrix components will greatly enhance our understanding of the active role of basement membrane dynamics in various biological phenomena, such as morphogenesis, tissue regeneration, and disease progression.

[0119] Mechanism of Basement Membrane (BM) Elongation The phenomenon of BM elongation raises important questions about its underlying mechanism. Although MMP activity contributes to BM turnover and homeostatic elongation, it alone does not fully explain the driving force behind elongation. Simply removing and replacing BM components likely results in homeostatic turnover without significant elongation. Mechanical tension generated by cells and the ECM is thought to be important for BM elongation and directional organ growth. As epithelial cells proliferate, anisotropic tension is exerted on the BM, causing it to elongate. This tension creates space within the BM for the incorporation of more free collagen IV molecules, promoting elongation along the major axis of the growing tissue (Chang et al., 2024; Khalilgharibi and Mao, 2021). Indeed, our results demonstrate that BM elongation occurs in conjunction with epithelial cell proliferation and horizontal daughter cell positioning. However, when MMP inhibitors were applied, the orientation of cell alignment shifted from horizontal to perpendicular to the BM, while cell proliferation continued. This suggests that the parallel cell alignment normally associated with elongating tissues is adapted to the properties and dynamics of the surrounding ECM (Box, Joyce, and Devenport, 2019; Dekoninck et al., 2020; Gudipati et al., 2017; Nestor-Bergmann et al., 2019). Therefore, without MMP-dependent matrix remodeling, this proliferation-induced tension alone may be insufficient to drive BM elongation.

[0120] The BM may also actively elongate or contract through the polymerization and cross-linking of its components (Diaz-de-la-Lozá and Stramer, 2024; Serna-Morales et al., 2023). In this study, we observed that BM elongation progressed with increased polymerization of type IV collagen, indicating that this process is not simply passive elongation but involves coordinated remodeling of the BM molecular structure. Detailed spatial and temporal analyses of cell behavior and BM dynamics, such as determining whether BM elongation occurs locally before parallel cell division or vice versa, and specific manipulation of cellular and ECM activities, are essential to understand the role of BM assembly in regulating cell behavior. Alterations in BM components may also promote BM and tissue elongation. For example, the incorporation of different BM components, such as laminin or perlecan, which alters the stiffness and elasticity of the BM, may allow elongation ( Pastor-Pareja and Xu, 2011 ; Topfer et al., 2022 ).

[0121] Finally, tissue-scale BM movement (including elongation) may displace adherent cells and contribute to coordinated cell-BM migration along specific axes (Loganathan et al., 2016). Our findings of BM-coupled cell migration in developing mouse skin further support the idea that BM elongation is tightly integrated with tissue-level mechanical forces and cell dynamics.

[0122] Role of MMPs in BM Dynamics and Morphogenesis Our results indicate that spatiotemporal regulation of MMP activity is important in orchestrating BM remodeling and epithelial morphogenesis. However, the mechanisms underlying the regulation of MMP activity in hair follicles remain largely unknown. On the other hand, studies on mammary glands have shown that MMP2-deficient glands exhibit incomplete terminal end bud invagination and excessive secondary branching, whereas MMP3-deficient glands exhibit normal invagination but lack secondary branching (Wiseman et al., 2003). These findings suggest that MMP activity must be finely regulated to achieve proper tissue morphogenesis. Understanding how MMPs control BM dynamics during hair follicle and other organ development is crucial for elucidating the mechanisms of tissue-specific morphogenesis.

[0123] In summary, the basement membrane live imaging approach established in this study offers the potential to investigate the role of basement membrane dynamics in a wide range of biological phenomena in mammals and other animals, including early development, organogenesis, homeostasis, regeneration, and disease progression such as cancer. Therefore, this imaging technique will significantly contribute to the quest for a deeper understanding of the functional importance of basement membrane dynamics in multicellular systems as a complex of cells and ECM.

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Claims

1. A nucleic acid encoding a modified collagen IVα2 protein, said nucleic acid having an insertion in a region encoding the 7S domain, either directly or via a linker, of a nucleic acid encoding another protein, wherein the nucleic acid encoding the modified collagen IVα2 protein is capable of producing a collagen IVα2 protein fused to said other protein, wherein said modified collagen IVα2 protein is capable of forming a trimer with two collagen IVα1 proteins, and wherein non-human animals having said insertions in all of their collagen IVα2 proteins survive to adulthood and reproduce.

2. The nucleic acid of claim 1, wherein the additional protein comprises a fluorescent protein.

3. A non-human animal having the nucleic acid of claim 1 or 2.

4. The non-human animal according to claim 3, wherein at least one nucleic acid encoding endogenous collagen IVα2 on the genome has the insertion and expresses the fusion protein.

5. A non-human animal described in claim 3 or 4, in which all of the nucleic acids encoding endogenous collagen IVα2 on the genome have the insertion, express the fusion protein, and survive and reproduce to adulthood.

6. The nucleic acid of claim 1 or 2, which is mRNA.

7. A modified collagen IVα2 protein which is the translation product of the nucleic acid of claim 6.

8. A heterotrimeric complex comprising the modified collagen IVα2 protein of claim 7 and two collagen IVα1.

9. An isolated animal cell, wherein at least one or all of the nucleic acids encoding collagen IVα2 contained in said cell are the nucleic acids described in claim 1 or 2.

10. An isolated organoid, tissue or organ comprising the animal cell of claim 9.

11. A nucleic acid encoding a modified collagen IVα1 protein, having an insertion in a region encoding the 7S domain directly or via a linker with a nucleic acid encoding another protein, wherein the nucleic acid encoding the modified collagen IVα1 protein is capable of producing a collagen IVα1 protein fused with said other protein, wherein said modified collagen IVα1 protein is capable of forming a trimer with one collagen IVα1 protein and one collagen IVα2 protein, or two of said modified collagen IVα1 proteins are capable of forming a trimer with one collagen IVα2 protein, and wherein a non-human mammal having said insertions in all of its collagen IVα1 proteins is capable of surviving and reproducing to adulthood.

12. The nucleic acid of claim 11, wherein the additional protein comprises a fluorescent protein.

13. A non-human animal having the nucleic acid of claim 1 or 2.

14. The non-human animal of claim 13, wherein at least one nucleic acid encoding endogenous collagen IVα1 on the genome has the insertion and expresses the fusion protein.

15. A non-human animal described in claim 13 or 14, in which all of the nucleic acids encoding endogenous collagen IVα1 on the genome have the insertion, express the fusion protein, and survive and reproduce to adulthood.

16. The nucleic acid of claim 11 or 12, which is mRNA.

17. A modified collagen IVα1 protein which is the translation product of the nucleic acid of claim 16.

18. A heterotrimeric complex comprising two modified collagen IVα1 proteins of claim 17 and one collagen IVα2.

19. An isolated animal cell, wherein at least one or all of the nucleic acids encoding collagen IVα1 contained in said cell are the nucleic acids described in claim 11 or 12.

20. An isolated organoid, tissue or organ comprising the animal cell of claim 19.

Images