Transcriptomic analysis of pancreatic cells in zebrafish

Pancreas is a mixed gland composed of endocrine and exocrine tissues and plays a crucial role in the metabolism of all vertebrates. The endocrine cells are mainly grouped into the islets of Langerhans and secrete distinct hormones, such as glucagon (α-cell), insulin (β-cell), somatostatin (δ-cell) and ghrelin (ε-cell). Diabetes occurs when insulin production by the β-cells is unable to counteract increase of glycemia.
The goal of the first part of my thesis was to determine the transcriptomic signatures of each pancreatic cell type in zebrafish in order to identify novel cell type-specific regulatory genes that might be crucial for their differentiation and/or physiology. Pancreatic acinar cells, ductal cells as well as the endocrine α-, β- and δ-cells were isolated from different transgenic adult zebrafish using FACS and RNA-seq was performed from these highly purified cell types. Comparison between the RNA-seq datasets allowed us to highlight all genes (protein coding and non-coding genes) with enriched expression in each cell type and to identify new markers of the mature pancreatic cells in zebrafish. In order to establish the expression blueprint of pancreatic endocrine and exocrine cells conserved from fish to mammals, we compared the pancreatic transcriptomes from zebrafish, mouse and human. Using pancreatic RNA-seq data available in databases, we determined the set of genes displaying enriched expression in endocrine and exocrine cells of human and mouse. Comparison of these data with the zebrafish pancreatic endocrine and exocrine data revealed the genes with conserved expression among vertebrates. Most of the transcription factors previously known to be important for pancreatic cell differentiation are included in this set of conserved genes. This interspecies comparative analysis highlighted genes with evolutionary conserved expression whose pancreatic function is still unknown, but also revealed striking differences in gene expression patterns between species.
The goal of the second part of my thesis was to understand the global transcriptional change produced by the loss-of-function of pax6b in pancreatic endocrine cells during pancreas development in zebrafish. We performed RNA-seq from purified pancreatic endocrine cells from wild-type and mutant (pax6b sa0086 null allele) zebrafish embryos at 27 hpf. By comparing the transcriptome of wild-type and mutant endocrine cells, we identified thousands of genes differentially expressed. Notably, we observed that the expression level of the pancreatic hormones was affected as it was reported in murine models. These analyses have revealed the transcriptional network regulated by pax6b in endocrine cells during differentiation. These analyses highlighted many unknown pax6b targets and novel regulators possibly involved in pancreatic function. Future
functional analyses will be needed to further investigate the function of the novel regulators identified by this study.