Comprehensive genomic and transcriptomic studies of the sea cucumber <em>Holothuria glaberrima</em>: from the genome to single cell sequencing

Abstract

Regeneration is one of the most fascinating and yet least understood biological processes. Echinoderms, one of the closest related invertebrate groups to humans, can contribute to our understanding of the genetic basis of regenerative processes. Chapter 1 provides a review of the available data aimed to determine the genes and signaling pathways that have been proposed to be involved in regenerative processes. By analyzing these data, we provide a curated a list of genes and gene signaling pathways and match them with the different cellular processes of the regenerative response. In this way, the molecular basis of echinoderm regenerative potential begins to be revealed. Amongst echinoderms, sea cucumbers have the most advanced regenerative capabilities with the potential to regenerate complete organs. These organisms have the ability to grow back most of their body parts following injury, including the intestine and nervous tissue. The cellular and molecular events underlying these abilities in sea cucumbers have been most extensively studied in the species <em>Holothuria glaberrima</em>. However, research into the regenerative abilities of this species have been impeded due to the lack of adequate genomic resources. Aiming to overcome this hurdle, this thesis focuses on the development of genomic data of <em>H. glaberrima</em>.<br /> <br /> In Chapter 2, we describe the first draft genome assembly of <em>H. glaberrima</em>, which totals 1.1 gigabases over 89,105 scaffolds. In addition, transcriptomic data was incorporated to annotate 51,415 genes. To demonstrate the usefulness of the draft genome, we fully annotated the melanotransferrin (<em>Mtf</em>) gene family, which has been proposed to be expanded in the sea cucumber. Using these same data, we extracted the mitochondrial genome, showing high conservation to that of other holothuroids. The draft genome was then further improved by the development of a genome assembly at the chromosome level, which is described in Chapter 3. This assembly with higher contiguity resulted in 1.23 gigabases with a total of 2,619 scaffolds, of which the 23 largest scaffolds contain 94% of the complete genome of <em>H. glaberrima</em>. In addition, through the incorporation of transcriptomic data 34,720 genes were annotated. Various quality assessments based on comparisons to other species were performed including macrosynteny, orthogroup identification, Hox gene cluster annotation, and Sox gene family annotation. The development of this genome led us to new findings including: (1) absence of correlation between transposable elements and regeneration and potential relationship of the Gypsy elements with nearby genes; (2) the expansion of the THAP transcription factor family in the sea cucumber and other echinoderm species and (3) identification and characterization of the novel gene family vNatt, which was observed to be conserved among species of distinct taxonomic lineages.<br /> <br /> Having a chromosome level genome of <em>H. glaberrima</em> opens the possibilities to advance the molecular studies of regeneration in this organism. For instance, in Chapter 4, we employed state-of-the-art single cell RNA sequencing (scRNA-seq) and high-resolution RNA fluorescence in situ hybridiziation (HCR-FISH) analyses to discern the distinct cellular populations associated with the regeneration anlage (i.e., blastema). This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. Through scRNA-seq, we successfully identified thirteen distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.