Method for identifying limb regeneration heterogeneity between salamander and xenopus laevis
Transcriptome analysis and high-throughput sequencing were used to identify the heterogeneity of limb regeneration in salamanders and Xenopus, revealing its molecular mechanisms. This provides guidance for regenerative medicine research, solves the problem of identification in existing technologies, and identifies key molecules affecting limb regeneration capabilities.
Patent Information
- Application Number
- CN202511496229.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-20
- Publication Date
- 2026-01-16
AI Technical Summary
There is a lack of effective methods in the current technology to identify the causes of heterogeneity in limb regeneration between salamanders and clawed frogs, and identification can only be done by observation, with a lack of corresponding research.
Transcriptome analysis and high-throughput sequencing were used to collect limb tissues from salamanders and Xenopus at different developmental stages. RNA extraction and data quality control were performed, a genome index was constructed, gene alignment and expression analysis were conducted, and key molecules were identified using principal component analysis and correlation analysis.
This study revealed the molecular mechanisms underlying the heterogeneity of limb regeneration capabilities between salamanders and Xenopus laevis, providing a new direction for regenerative medicine research. By using gene markers of oxidative metabolism, reactive oxygen species, and DNA damage and repair pathways, the study further identified the molecular mechanisms influencing limb regeneration heterogeneity.
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Figure CN121344208A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more specifically, to a method and application for identifying heterogeneity in limb regeneration between salamanders and Xenopus. Background Technology
[0002] Limb regeneration is not present in all species; it exists only in a few. Both salamanders and clawed frogs possess limb regeneration abilities. Salamanders retain this ability throughout their entire life cycle, but clawed frogs only exhibit it before metamorphosis, losing it afterward. This phenomenon, where limb regeneration ability varies significantly between different species, and even between different life stages or individuals of the same species, is called limb regeneration heterogeneity. Previously, this limb regeneration heterogeneity in salamanders and clawed frogs could only be identified through observation; however, research on the underlying causes of this regeneration heterogeneity between the two species is lacking. Summary of the Invention
[0003] The purpose of this invention is to provide a method for identifying limb regeneration heterogeneity between salamanders and Xenopus, which identifies key molecules related to limb regeneration capacity through transcriptome analysis, providing a new direction for regenerative medicine research.
[0004] The technical problem solved by this invention is achieved by the following technical solution.
[0005] This application provides a method for identifying limb regeneration heterogeneity between salamanders and Xenopus, comprising the following steps: S1: Sampling, extracting limb tissues from salamanders and Xenopus at different stages, extracting RNA and performing high-throughput sequencing to obtain raw data; S2: Use FastOC to assess the quality of the raw data and use Trimmomatic to perform quality control on the raw data to remove low-quality bases or adapter contamination sequences; S3: Use the hisat2-build command to build a salamander reference genome index, compare the quality-controlled data with the reference genome, and output the comparison data; S4: Use featureCounts to output the raw count matrix from the alignment data, and calculate the TPM value using the raw count matrix in the R language environment; S5: Using sequence alignment, common genes between salamanders and Xenopus were extracted; S6: Perform principal component analysis and correlation analysis on the data obtained in step S5.
[0006] Furthermore, the samples were limb tissues from salamanders and clawed frogs during three stages of development: early development, metamorphosis, and post-metamorphosis.
[0007] Furthermore, the high-throughput sequencing workflow includes RNA quality assessment, conventional transcriptome library construction, library quality testing, and library clustering and sequencing.
[0008] Furthermore, in step S2, assessing the quality of the raw data includes examining the number and length of reads, GC content, base quality and distribution, proportion of repetitive sequences, and adapter residues.
[0009] Furthermore, in step S3, sequences with a quality score less than 30 are selected for comparison.
[0010] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. This invention, through transcriptome analysis and sequencing, elucidates the molecular mechanism underlying the heterogeneity of limb regeneration capabilities between salamanders and Xenopus laevis, providing guidance for research on other regeneration capabilities; 2. This invention further identifies the molecular mechanisms affecting limb regeneration heterogeneity by labeling genes involved in oxidative metabolism, reactive oxygen species, and DNA damage and repair pathways. Attached Figure Description
[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0012] Figure 1 This is a diagram showing the growth state of a newt at three different stages in an embodiment of the present invention; Figure 2 This is a diagram showing the growth state of Xenopus laevis at three different stages in an embodiment of the present invention; Figure 3 PCA analysis of three stages of limb development in salamanders and clawed frogs in this embodiment of the invention; Figure 4 This is a Pearson correlation analysis diagram of limb development in three stages of salamander and clawed frog in an embodiment of the present invention; Figure 5 Venn diagram analysis of gene expression in three stages of limb development in salamanders and clawed frogs in this embodiment of the invention; Figure 6 This is a GO analysis diagram of the three stages of limb development in salamanders and clawed frogs in an embodiment of the present invention; Figure 7 This is a diagram showing the expression analysis of genes related to oxidative metabolism during the limb development of a newt in this embodiment of the invention. Figure 8This is a diagram showing the expression analysis of related genes during the development of reactive oxygen species in salamander limbs in an embodiment of the present invention. Figure 9 This is a diagram showing the expression analysis of relevant genes during the injury process of a salamander limb at three different stages in an embodiment of the present invention; Figure 10 This is a graph showing the expression analysis of relevant genes during the repair process of salamander limbs after damage at three different stages in an embodiment of the present invention; Figure 11 This is a diagram showing the expression analysis of genes related to oxidative metabolism during limb development in Xenopus laevis according to an embodiment of the present invention. Figure 12 This is a diagram showing the expression analysis of related genes during the expression of reactive oxygen species in the limb development of Xenopus laevis in this embodiment of the invention. Figure 13 This is a diagram showing the expression analysis of relevant genes during the injury process of Xenopus limbs at three different stages in an embodiment of the present invention; Figure 14 This diagram illustrates the expression analysis of relevant genes during the repair process of Xenopus limbs damaged at three different stages in an embodiment of the present invention. Detailed Implementation
[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0014] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.
[0015] Example This embodiment provides a detailed method for identifying limb regeneration heterogeneity between salamanders and Xenopus, including the following steps: S1: Limb tissue samples were collected from salamanders and clawed frogs at three stages: early development, metamorphosis, and post-metamorphosis. For salamanders, the sampling sites were the limbs. Figure 1 As shown, limb tissues were collected during the early developmental stage (stage 44) to analyze the initial developmental characteristics; limb samples were collected during the metamorphosis stage (stage 48) to observe the morphological reconstruction and tissue changes of the limbs; and limb samples were collected again after the metamorphosis was completed (stage 50, i.e., about 3 months after birth) to assess the structural and functional characteristics after full development.
[0016] For Xenopus laevis, the sampling site is also the limbs. For example... Figure 2 As shown, sampling was conducted at period 54 during the early developmental stage to obtain physiological information on early limb development; sampling was conducted at period 64 during the metamorphosis stage to study the morphological and molecular changes of the limbs during the transformation process; and sampling was conducted at period 66 (approximately 4 months after birth) after the completion of metamorphosis to analyze the state of the limbs after they have reached maturity.
[0017] In addition, three sets of biological replicates were set up for the early development, metamorphosis, and metamorphosis of salamanders and Xenopus.
[0018] RNA was then extracted from limb tissues at each stage using the RNAsimple Total RNA Kit. The transcriptome sequencing workflow included RNA quality assessment, standard transcriptome library construction, library quality checks, and library clustering and sequencing. Paired-end sequencing was used, ultimately yielding raw RNA-Seq data with read lengths of 150 bp.
[0019] S2: First, use FastQC to check the quality of the raw RNA-Seq data, including the number and length of reads, GC content, base quality and distribution, repetitive sequence ratio, and adapter residues. Then, use FastQC to check if the cleandata after quality control meets all standards before proceeding with further analysis.
[0020] FastQC is a core tool for assessing the quality of RNA-Seq data. It uses visual reports to quickly identify potential problems in the data and gain a preliminary understanding of the data's basic information.
[0021] Then, Trimmomatic (Bolger et al., 2014) was used. The raw data was quality controlled using the parameters "SLIDINGWINDOW:5:20 TRAILING:5 HEADCROP:15 MINLEN:20 ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10" to remove low-quality bases or adapter contaminants. Trimmomatic was used to further quality control the raw data, removing low-quality bases, adapter sequences, and excessively short reads.
[0022] S3: The hisat2-build command was used to construct reference genome indexes for salamanders and Xenopus to prepare for data alignment. Then, cleandata was aligned to the reference genomes, and SAM files and statistical alignment information were output (Kim et al., 2019). Additionally, to save computer resources and facilitate subsequent analysis, the SAM files were converted to BAM files, and aligned sequences with an alignment quality score less than 30 were filtered. The BAM files were then sorted and indexed.
[0023] S4: First, gene quantification was performed. The annotation file corresponding to the salamander reference genome was downloaded from the NCBI database. The featureCounts function was used to count paired reads from the BAM file and output the raw count matrix (Liao et al., 2014). To eliminate the influence of sequencing depth and gene length, the TPM values of the whole genome were calculated in batches using R language and the raw count matrix for subsequent analysis.
[0024] After calculation, the longest CDS gene FASTA file was extracted from the FASTA files of Xenopus and salamanders. Then, the formatdb command was used to format the Xenopus FASTA file, and blastn was run for alignment. The transcriptome IDs were converted into gene IDs using the GTF annotation file. Furthermore, the corresponding genes of salamanders and Xenopus were extracted based on the case-insensitive gene names of newts and Xenopus.
[0025] S5: Reference Figure 3 and Figure 4 We performed PCA principal component analysis and correlation analysis using the DESeq2-standardized DDS matrix. First, we used the vst command to perform variance stability transformation on the DDS matrix, then used the R package ggplot2 to plot the PCA graph, and used the cor command with the spearman parameter to perform correlation analysis and plot a heatmap.
[0026] PCA revealed that salamander limb samples from the early developmental stages formed a distinct cluster, while samples from the metamorphosis and post-metamorphosis stages showed partial overlap. This finding highlights the progressive transcriptomic remodeling during metamorphosis. In contrast, Xenopus limb samples exhibited clear separation across the early, metamorphosis, and post-metamorphosis groups, with tight clusters within each group, emphasizing a dramatic limb development process characterized by significant transcriptomic changes throughout metamorphosis. Correlation analysis reinforced these observations: Xenopus samples showed strong intra-group correlations and weak inter-group correlations across all stages, highlighting significant transcriptomic differences during development. Conversely, salamander samples showed higher overall inter-stage correlations. While the correlation between the early developmental stages and the merged metamorphosis and post-metamorphosis stages was relatively low, the correlation between the metamorphosis and post-metamorphosis stages was high, reflecting a smoother transitional metamorphosis process characterized by a relatively stable post-metamorphotic transcriptomic profile.
[0027] Then, using the R package and employing early development and metamorphosis data of salamanders and Xenopus, the mean was calculated for three replicates at each stage. The mean for the metamorphosis stage was divided by the mean for the early development stage to obtain the relative fold change during metamorphosis. By comparing the relative fold changes during metamorphosis in salamanders and Xenopus, the upregulated and downregulated genes in salamanders relative to Xenopus were identified.
[0028] Use the `enrichGO` function in the `clusterProfiler` R package again, with the parameters "pvalueCutoff = 0.05, ont ="all", OrgDb = org.Pwaltl.eg.db". For example... Figure 6 As shown, the enrichment analysis results were visualized using the barplot command (Yu et al., 2012), and a bidirectional GO pathway map was plotted. It can be seen that a total of 3600 genes were consistently expressed across all three developmental stages in both species. Functional enrichment analysis of differentially expressed genes revealed significant pattern changes: compared to salamanders, the upregulated pathways in Xenopus were mainly related to oxidative metabolism, ROS metabolism, and DNA damage responses. Conversely, the downregulated pathways in Xenopus were significantly associated with DNA repair mechanisms.
[0029] Result Validation Please refer to Figures 7-14 In this diagram, the black line represents the mean relative expression level of genes in this pathway, the gray line represents the relative expression level of all genes in this pathway, and the other colored lines represent representative genes. Figures 7-10 This diagram illustrates the expression analysis of genes related to oxidative metabolism, reactive oxygen species expression, DNA damage, and repair in salamander limb tissues during three different stages. Figures 11-14This diagram shows the expression analysis of genes related to oxidative metabolism, reactive oxygen species (ROS) expression, DNA damage, and repair in Xenopus limb tissues during three different stages. It can be seen that during oxidative metabolism, the genes shared by both salamanders and Xenopus are atp6v0e1, atp6v1f, cox11, and cox17. In salamanders, only atp6v1f showed expression levels higher than the mean relative expression level after metamorphosis, while the others were lower than the mean relative expression level. In Xenopus, cox17 showed expression levels higher than the mean relative expression level during and after metamorphosis, while the other genes also showed expression levels lower than the mean.
[0030] In addition, regarding the expression of reactive oxygen species (ROS), the genes shared by salamanders and Xenopus are atg9a, bcl2l13, gabarapl2, map1lc3a, phb2, sqstm1, tbcld15, and tbc1d17. In salamanders, the expression level of gabarapl2 is higher than the mean relative expression level in all three stages, and the expression level of map1lc3a is higher than the mean relative expression level after metamorphosis, while the others are lower than the mean relative expression level. In Xenopus, the expression levels of phb2 and gabarapl2 are higher than the mean relative expression level in all three stages, but the expression level of map1lc3a is lower than the mean relative expression level in the later stages of metamorphosis.
[0031] During DNA damage, salamanders and Xenopus share the following expressed genes: ern1, mapk11, mcl1, nfkbia, and sqstm1. In salamanders, only sqstm1 shows slightly higher expression levels than the mean relative expression level after metamorphosis, while the others are lower. In Xenopus, however, ern1 expression levels are higher than the mean relative expression level at all three stages, but nfkbia and sqstm1 expression levels decrease to below the mean relative expression level in the later stages of metamorphosis.
[0032] During DNA damage repair, salamanders and Xenopus share the following expressed genes: axin2, cops6, pcna, pold3, rnf169, smc2, smc4, and smc1. In salamanders, the expression levels of axin2, cops6, pcna, pold3, rnf169, smc2, smc4, and smc1 are all higher than the mean relative expression levels at all three stages. Furthermore, except for pold3, the expression levels of these genes show a trend of first increasing and then decreasing. Additionally, the expression level of rnf169 is lower than the average expression level after metamorphosis.
[0033] In Xenopus laevis, the expression levels of smg1 and rnf169 were higher than the mean relative expression levels in all three stages, but showed a decreasing trend. The expression level of smg1 was higher than the mean relative expression level in the later stages of metamorphosis.
[0034] Based on the above results, time-expression profiling reveals distinct patterns in oxidative metabolism, reactive oxygen species (ROS) metabolism, and DNA damage-related gene activity between Xenopus and newts. In Xenopus, these genes increase dramatically during metamorphosis and then decrease. In contrast, newts exhibit a gradual downregulation of these genes throughout development. Furthermore, genes related to DNA repair are persistently or transiently upregulated in newts but persistently downregulated during Xenopus development. These observations suggest that Xenopus undergoes a significant surge in oxidative metabolic activity and oxidative damage during metamorphosis, which is associated with a significant reduction in limb regeneration capacity. On the other hand, newts experience a milder oxidative metabolic response without affecting their regenerative potential.
[0035] In summary, the present invention provides a method for identifying limb regeneration heterogeneity between salamanders and Xenopus, which analyzes the molecular mechanism of limb regeneration heterogeneity between salamanders and Xenopus through transcriptome analysis and sequencing, providing guidance for research on other regeneration abilities; This invention further identifies the molecular mechanisms affecting limb regeneration heterogeneity by labeling genes involved in oxidative metabolism, reactive oxygen species, and DNA damage and repair pathways.
[0036] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
Claims
1. A method of identifying inter-species heterogeneity in limb regeneration between axolotls and Xenopus, comprising, The method comprises the following steps: S1: sampling, extracting samples of newts and frogs at different stages respectively, performing RNA extraction and high-throughput sequencing on the samples, and obtaining raw data; S2: using FastOC to evaluate the quality of the raw data, and using Trimmomatic to perform quality control on the raw data to remove low-quality bases or adapter contamination sequences; S3: using the hisat2-build command to construct a newt reference genome index, aligning the quality-controlled data and the reference genome, and outputting alignment data; S4: using featureCounts to output the original count matrix from the alignment data, and using the original count matrix to calculate TPM values in the R language environment; S5: using sequence alignment to extract common genes of newts and frogs; S6: performing principal component analysis and correlation analysis on the data obtained in step S5.
2. The method of claim 1, wherein the method is for identifying the heterogeneity of limb regeneration between axolotls and Xenopus. The samples are limb tissues of newts and frogs at three stages of early development, metamorphic development, and post-metamorphic development.
3. The method of identifying interlimb regeneration heterogeneity between axolotls and frogs of claim 2, wherein, The high-throughput sequencing process comprises RNA quality evaluation, general transcriptome library construction, library quality inspection, and library clustering and sequencing.
4. The method of identifying interlimb regeneration heterogeneity between axolotls and Xenopus of claim 3, wherein, In step S2, evaluating the quality of the raw data comprises checking the number and length of reads, GC content, base quality and distribution, repeat sequence proportion, and adapter residues of the raw data.
5. The method of identifying interlimb regeneration heterogeneity between axolotls and Xenopus of claim 4, wherein, In step S3, sequences with a quality score less than 30 are selected for alignment.