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Open Biol
2024 Aug 28;148:240111. doi: 10.1098/rsob.240111.
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Molecular basis of urostyle development in frogs: genes and gene regulation underlying an evolutionary novelty.
Senevirathne G
,
Shubin NH
.
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Evolutionary novelties entail the origin of morphologies that enable new functions. These features can arise through changes to gene function and regulation. One key novelty is the fused rod at the end of the vertebral column in anurans, the urostyle. This feature is composed of a coccyx and a hypochord, both of which ossify during metamorphosis. To elucidate the genetic basis of these features, we used laser capture microdissection of these tissues and did RNA-seq and ATAC-seq at three developmental stages in tadpoles of Xenopus tropicalis. RNA-seq reveals that the coccyx and hypochord have two different molecular signatures. Neuronal (TUBB3) and muscle markers (MYH3) are upregulated in coccygeal tissues, whereas T-box genes (TBXT, TBXT.2), corticosteroid stress hormones (CRCH.1) and matrix metallopeptidases (MMP1, MMP8 and MMP13) are upregulated in the hypochord. ATAC-seq reveals potential regulatory regions that are observed in proximity to candidate genes that regulate ossification identified from RNA-seq. Even though an ossifying hypochord is only present in anurans, this ossification between the vertebral column and the notochord resembles a congenital vertebral anomaly seen prenatally in humans caused by an ectopic expression of the TBXT/TBXT.2 gene. This work opens the way to functional studies that can elucidate anuran bauplan evolution.
Figure 1. Changes in transcriptomics during anuran urostyle development. (a) The experimental setup and the developmental stages used for laser-capture microdissections. Ten sections of cryosections (10 μM each) were taken from three developmental stages (stage 56: prometamorphosis; stage 61: beginning of the metamorphic climax and stage 65: end of metamorphosis), and the coccygeal and hypochordal tissues were dissected out. (b) Principal component analysis for the urostyle tissues (coccyx and hypochord) used for the transcriptomic assay (n = 4 biological replicates per developmental stage). Each dot represents a tissue sample. (c) Heatmap, highlighting the differentially expressed genes, compared between three developmental stages and two tissue types. The heatmap highlights that the two tissue types possess two distinct sets of genes.
Figure 2. Comparison of the hypochordal and coccygeal sections before metamorphosis (stage 57). (a) A transverse section across the coccyx, highlighting the aggregating mesenchymal cells around the spinal cord. (b) A transverse section across the hypochord, highlighting the embryonic hypochordal cells ventral to the notochord and notochordal sheath. Nuclei stained in blue, using DAPI and neurons stained in red using acetylated tubulin. Abbreviations: CX, coccyx; HY, hypochord; MC, mesenchymal cells; NT, notochord; SC, spinal cord.
Figure 3. Comparative transcriptomic analysis of the two tissue types: coccyx and hypochord. (a) A reactome pathway analysis for up/down regulatory genes in coccyx versus hypochord; the central circles represent a top-level pathway, and the circles away from the centre represents lower levels in each respective pathway. ‘Zoomed-in’ sections show top-level pathways of (i) developmental biology, (ii) neuronal system, (iii) programmed cell death and (iv) extracellular matrix organization. Overrepresented pathways (p < 0.05) are coloured in yellow (coccyx) and purple (hypochord). Pathways that are not significant are shown in light grey lines. Most hypochordal genes are involved in organizing the extracellular matrix, whereas the majority of coccygeal genes are involved in neuronal remodelling and modifications. (b) The total number of urostyle-responsive genes (FDR < 0.01) is between hypochord and coccyx. (c) Volcano plot showing differentially expressed genes across hypochord and coccyx during development (p < 0.05, FDR < 0.01).
Figure 4. Heatmaps showing differentially expressed genes involved in GO functions belonging to the ‘early activation cluster’ of osteocyte differentiation. This cluster includes the GO functions ‘axon guidance’, ‘axon development’, ‘axogenesis’, ‘regulation of axogenesis’ and ‘neuron projection guidance’. Genes of interest that are differentially expressed between the coccyx and hypochord are highlighted in purple.
Figure 5. Urostyle-responsive regulatory regions. (a) Schematic diagram showing the workflow for chromatin profiling experiment. (b) Proportions of developing urostyle ATAC-seq peaks annotated to different genomic regions across development; majority of the peaks fall within the distal intergenic region and beginning (stage 61) and end of metamorphic climatic (stage 65) peaks differ from the prometamorphic (stage 56) ATAC-seq peaks with respect to peaks falling within the exon regions that are not the first exon. (c) Open chromatin regions (depicted using black rectangles) identified from IDR 0.05 called peaks for stage 56, stage 61 and stage 65 at the loci of validated upregulatory genes narrowed down from RNA-seq analyses.
