Project description:Pancreatic endocrine cells arise from a NGN3+ population during pancreas organogenesis. To gain a more thorough understanding of this progenitor pool, we used a reporter mouse - NGN3-EGFP - and sorted EGFP+ cells from e15.5 pancreata of control animals. The data generated from this experiment will allow us to visualize gene expression levels in endocrine progenitors during normal development and can be used to compare against mutant animal gene expression.
Project description:This experiment used RNA-Seq technology to explore gene expression in mouse Ngn3^GFP/+ [het] FACS sorted pancreatic cells at E15.5 (commited endocrine progenitor cells) and in Ngn3^GFP/GFP [null] at E15.5 (defective endocrine progenitor cells). This experiment is designed to understand the gene expression alteration in the endocrine lineage at different embryonic days. The aim is to understand both Ngn3 dependent and independent gene expression profiles so as to reveal the instructive signals that specfy the collective endocrine islet cell fate or specific islet cell type.
Project description:Endoderm cells undergo a sequence of fate choices to generate insulin-secreting M-NM-2 cells. Studies of chromatin transitions during this process have been limited to the pancreatic progenitor stage that can be reconstituted from stem cells in vitro, with a gap in understanding the induction of endocrine cells. To address this, we established conditions for isolating endoderm cells, pancreatic progenitors, and endocrine cells from different staged embryos and performed genome wide analysis of the H3K27me3 mark of the repressive Polycomb complex. During the transition from endoderm to pancreas progenitors and during the transition from pancreas progenitors to endocrine cells, genes that lose the H3K27me3 mark typically encode transcriptional regulators, whereas genes that acquire the mark typically are involved in cell biology morphogenesis. Precocious depletion of the EZH2, a H3K27 methylase, at the pancreas progenitor stage enhanced the production of endocrine cells, leading to a later increase in pancreatic beta cells. Similarly, pharmacologic inhibition of EZH2 in embryonic pancreatic tissue explants and human embryonic stem cell cultures led to an increase in endocrine progenitors in vitro. These findings reveal a repeating target gene pattern in H3K27me3 dynamics and provide a means to modulate M-NM-2 cell development from stem cells. Analyzed five FACS-sorted tissues in early mouse embryo; for each tissue we sequenced H3K27me3 and input; no replicates
Project description:The pancreatic islet contains multiple hormone+ endocrine lineages (alpha, beta, delta, PP and epsilon cells), but the developmental processes that underlie endocrinogenesis are poorly understood. Here, we generated novel mouse lines and combined them with various genetic tools to enrich all types of hormone+ cells for well-based deep single-cell RNA sequencing (scRNA-seq), and gene coexpression networks were extracted from the generated data for the optimization of high-throughput droplet-based scRNA-seq analyses. These analyses defined an entire endocrinogenesis pathway in which different states of endocrine progenitor (EP) cells sequentially differentiate into specific endocrine lineages in mice. Subpopulations of the EP cells at the final stage (EP4-early and EP4-late) show different potentials for distinct endocrine lineages. epsilon cells and an intermediate cell population were identified as distinct progenitors that independently generate both alpha and PP cells. Single-cell analyses were also performed to delineate the human pancreatic endocrinogenesis process. Although the developmental trajectory of pancreatic lineages is generally conserved between humans and mice, clear interspecies differences, including differences in the proportions of cell types and the regulatory networks associated with the differentiation of specific lineages, have been detected. Our findings support a model in which sequential transient progenitor cell states determine the differentiation of multiple cell lineages and provide a blueprint for directing the generation of pancreatic islets in vitro.
Project description:To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation.
Project description:During embryonic development, islet progenitors are specified from pancreatic duct cells by transient expression of Neurog3, a transcription factor necessary and sufficient for initiation of islet development. To understand the dynamics of Neurog3-dependent endocrine cell fate determination, in this study we used ATAC-Seq to identify accessible genomic regions of purified duct, endocrine progenitor, and endocrine cells isolated from mice with varying Neurog3 dosage
Project description:Gene expression profiles from ALDH high cells sorted from expanded adult human pancreatic organoids are more similar to fetal pancreatic tissue and ALDH high cells sorted from expanded fetal human pancreatic organoids than to adult human islets or adult islet-depleted exocrine tissue.
Project description:MicroRNAs (miRNAs) are small non-coding RNA molecules that have the ability to drive cell lineage decisions by regulating the expression of hundreds of genes. Although evidence indicates that miRNAs have roles in pancreas development and endocrine cell function, the role of miRNAs in pancreatic endocrine cell differentiation has not been systematically explored. To address this, we performed genome-wide small RNA sequencing analysis in pancreatic progenitor cells differentiated in vitro from human embryonic stem cells and endocrine cells isolated from whole human islets. This analysis revealed miRNAs that increase in expression during endocrine cell differentiation. Employing gain-of-function experiments, we identified four miRNAs that can repress a large number of genes that are normally down-regulated during endocrine cell differentiation, including genes encoding transcription factors known to regulate endocrine cell development as well as cell cycle regulators. This knowledge about miRNA target genes in conjunction with HITS-CLIP data allowed us to construct an integrated miRNA-gene regulatory network of endocrine cell differentiation. Our integrated analysis indicates a key role for the identified miRNAs in establishing a transcriptional landscape that promotes the differentiation of pancreatic progenitor cells into endocrine cells. This study not only sheds light on the mechanisms that underlie human endocrine cell differentiation, but also has important implications for devising improved protocols for producing replacement beta cells for diabetes cell therapy.
Project description:MicroRNAs (miRNAs) are small non-coding RNA molecules that have the ability to drive cell lineage decisions by regulating the expression of hundreds of genes. Although evidence indicates that miRNAs have roles in pancreas development and endocrine cell function, the role of miRNAs in pancreatic endocrine cell differentiation has not been systematically explored. To address this, we performed genome-wide small RNA sequencing analysis in pancreatic progenitor cells differentiated in vitro from human embryonic stem cells and endocrine cells isolated from whole human islets. This analysis revealed miRNAs that increase in expression during endocrine cell differentiation. Employing gain-of-function experiments, we identified four miRNAs that can repress a large number of genes that are normally down-regulated during endocrine cell differentiation, including genes encoding transcription factors known to regulate endocrine cell development as well as cell cycle regulators. This knowledge about miRNA target genes in conjunction with HITS-CLIP data allowed us to construct an integrated miRNA-gene regulatory network of endocrine cell differentiation. Our integrated analysis indicates a key role for the identified miRNAs in establishing a transcriptional landscape that promotes the differentiation of pancreatic progenitor cells into endocrine cells. This study not only sheds light on the mechanisms that underlie human endocrine cell differentiation, but also has important implications for devising improved protocols for producing replacement beta cells for diabetes cell therapy.