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Collagenous connective tissue, found throughout the bodies of metazoans, plays a crucial role in maintaining structural integrity. This versatile tissue has the potential for numerous biomedical applications, including the development of innovative collagen-based biomaterials. Inspiration for such advancements can be drawn from echinoderms, a group of marine invertebrates that includes sea stars, sea cucumbers, brittle stars, sea urchins, and sea lilies. Through their nervous system, these organisms can reversibly control the pliability of their connective tissue components (i.e., tendons and ligaments) that are composed of mutable collagenous tissue (MCT). The variable tensile properties of the MCT allow echinoderms to perform unique functions, including postural maintenance, reduction of muscular energy use, autotomy to avoid predators, and asexual reproduction through fission. The changes in the tensile strength of MCT structures are specifically controlled by specialized neurosecretory cells called juxtaligamental cells. These cells release substances that either soften or stiffen the MCT. So far, only a few of these substances have been purified and characterized, and the genetic underpinning of MCT biology remains unknown. Therefore, we have conducted this research to identify MCT-related genes in echinoderms as a first step towards a better understanding of the MCT molecular control mechanisms. Our ultimate goal is to unlock new biomaterial applications based on this knowledge. In this project, we used RNA-Seq to identify and annotate differentially expressed genes in the MCT structures of the brittle star Ophiomastix wendtii. As a result, we present a list of 16 putative MCT modulator genes, which will be validated and characterized in forthcoming functional analyses.
Fig. 1. Ophiomastix wendtii individuals, from which tissue samples were collected. Animal 1 is male, and animals 2 and 3 are female
Fig. 2. Two types of tissue samples harvested from the arm. A “Whole arm segments” with all anatomical components left intact. B The “inner arm core” with the peripheral arm plates and associated structures removed. The oral side is shown for both specimens in A and B. C Diagram of the cross-section through the arm. The faded region indicates the peripheral tissues that are surgically removed when the inner arm core samples are prepared for RNA extraction. The dotted line indicates where the peripheral and inner arm core tissues are separated. Abbreviations: ac – arm coelom; am – aboral muscle; as – aboral skeletal shield; il – intervertebral ligament; ls – lateral skeletal shield; om – oral muscle; os – oral skeletal shield; p – podium; rnc – radial nerve cord; s – spine
Fig. 3. Representative micrographs showing the microanatomical and histological organization of the stomach (A) and the whole arm segments (B – B3) tissue samples used as a starting material for RNA-Seq. A High-magnification view of the stomach wall. B A general view of the whole arm segments in a cross-section. Boxed areas indicate the regions that are shown at a higher magnification in B1 – B3. B1 – lateral ligament. B2 – oral part of the intervertebral ligament. B3 – aboral part of the intervertebral ligament. Abbreviations: ac – arm coelom; am – aboral intervertebral muscle; ctl – connective tissue layer; de – digestive epithelium; gl – gut lumen; il – intervertebral ligament; jn – juxtaligamental node; ll – lateral ligament; ls – lateral shield; me – mesothelium; om – oral intervertebral muscle; rnc – radial nerve cord; vo – vertebral ossicle. Dotted line in B indicates where the separation of the peripheral tissues took place when preparing the inner core arm tissue samples for RNA-Seq. Arrowheads in B1 – B3 indicate bundles of neurites extending from juxtaligamental nodes into the extracellular matrix of ligaments. Plastic semithin sections stained with toluidine blue
Fig. 4. Representative TEM micrographs showing the ultrastructural organization of the connective tissue compartments in the stomach (A) and whole arm segments (B – D) tissue samples used as a starting material for RNA-Seq. A Connective tissue layer of the stomach wall. B Juxtaligamental cells in the aboral juxtaligamental node. C A bundle of neurites projecting from the aboral juxtaligamental node into the aboral part of the intervertebral ligament. D Neurites of the juxtaligamental cells in the aboral part of the intervertebral ligament. Abbreviations: il – intervertebral ligament; jn – aboral juxtaligamental node; np – neurites. Arrowheads in B – D indicate secretory granules in juxtaligamental cells
Fig. 5. Differential gene expression analysis in A whole arm segments vs stomach and in B the inner arm core vs whole arm in O. wendtii. Volcano plots show the \documentclass[12pt]{minimal}
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\begin{document}$$-\log _{10}\text {FDR}$$\end{document}-log10FDR (Y-axis). The significantly upregulated transcripts (red) are to the right, and the significantly downregulated transcripts (green) are to the left. The differentially expressed transcripts were defined as those with at least a two-fold difference in expression (\documentclass[12pt]{minimal}
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\begin{document}$$< -1$$\end{document}<-1) and the associated adjusted p-value (FDR) below 0.05. Red dots indicate significantly upregulated genes (FDR \documentclass[12pt]{minimal}
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\begin{document}$$\log _{10}\text {FC}>$$\end{document}log10FC> 1.0), and green dots represent significantly downregulated genes (FDR < 0.05, \documentclass[12pt]{minimal}
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\begin{document}$$\log _{10}\text {FC} < -1.0$$\end{document}log10FC<-1.0). Black dots represent transcripts whose expression was not statistically different in the respective pairwise comparisons. The numbers on the plots refer to the number of up or downregulated transcripts. C Venn diagram showing the common set of 94 upregulated genes in both comparisons
Fig. 6. Bar plot showing the significantly enriched GO terms in the shared set of 94 transcripts (blue bars) that were upregulated in both the whole arm vs stomach and inner arm core vs whole arm comparisons. Red bars represent the reference gene set (the entire annotated transcriptome)
Fig. 7. Identification and annotation of the putative MCT-related candidate gene set. A BLAST annotation status of the whole set of the 94 genes that were identified as overexpressed in both the whole arm (in comparison to the stomach) and in the inner arm core (in comparison to the whole arm). The box indicates novel genes with unknown function. B Signal peptide presence (as predicted by the SignalP tool [43]) in a subset of 46 novel genes (those that had no associated BLAST hit or were recovered as either uncharacterized proteins or hypothetical proteins boxed are in (A)). C Cellular/extracellular localization of the protein products encoded by the subset of 46 novel candidate genes with unknown function (i.e., the same subset as in (B) and boxed area in (A)), as predicted by the DeepLoc tool [44]
