single-cell-transcriptomic-analyses-mouse-pancreatic-endocrine

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Peking University

The Arabidopsis root has a unique cellular pattern in its single-layered epidermis. Cells residing over the intercellular spaces between underlying cortical cells (H position) differentiate into hair cells, whereas those directly over cortical cells (N position) differentiate into non-hair cells. Recent studies have revealed that this cellular pattern is determined by interactions of six patterning genes CPC, ETC, GL2, GL3/EGL3, TTG, and WER, and that the position-dependent expression of the CPC, GL2, and WER genes is essential for their appropriate interactions. However, little is known about how the expressions of the pattern genes are determined. Here we show that trichostatin A (TSA) treatment of germinating Arabidopsis seedlings alters the cellular pattern of the root epidermis to induce hair cell development at nonhair positions. The effects of TSA treatment are rapid, reversible, concentration-dependent, and position-independent. TSA inhibition of histone deacetylase activity results in hyperacetylation of the core histones H3 and H4, and alters the expression levels and cell specific expression of the patterning genes CPC, GL2 and WER. Analysis of histone deacetylase mutant cellular patterning further verified the participation of histone acetylation in cellular patterning, and revealed that HDA18 is a key component in the regulatory machinery of the Arabidopsis root epidermis. We propose a working model to suggest that histone acetylation may function in mediating a positional cue to direct expression of the patterning genes in the root epidermal cells.

Histone acetylation affects expression of cellular patterning genes in the Arabidopsis root epidermis.

Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells. 

Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification.

Insulin secreted from pancreatic β-cells and glucagon secreted from pancreatic α-cells are the two major hormones working in the pancreas in an opposing manner to regulate and maintain a normal glucose homeostasis. How microRNAs (miRNAs), a population of non-coding RNAs so far demonstrated to be differentially expressed in various types of cells, regulate gene expression in pancreatic β-cells and its closely associated α-cells is not completely clear. In this study, miRNA profiling was performed and compared between pancreatic β-cells and their partner α-cells. One novel miRNA, miR-483, was identified for its highly differential expression in pancreatic β-cells when compared to its expression in α-cells. Overexpression of miR-483 in β-cells increased insulin transcription and secretion by targeting SOCS3, a member of suppressor of cytokine signaling family. In contrast, overexpression of miR-483 decreased glucagon transcription and secretion in α-cells. Moreover, overexpressed miR-483 protected against proinflammatory cytokine-induced apoptosis in β-cells. This correlates with a higher expression level of miR-483 and the expanded β-cell mass observed in the islets of prediabetic db/db mice. Together, our data suggest that miR-483 has opposite effects in α- and β-cells by targeting SOCS3, and the imbalance of miR-483 and its targets may play a crucial role in diabetes pathogenesis. 

Differentially Expressed MicroRNA-483 Confers Distinct Functions in Pancreatic β- and α-Cells.

Pancreatic β and α cells play essential roles in maintaining glucose homeostasis. However, the mechanisms by which these distinct cell populations are generated, expand, and mature during pancreas development remain unclear. In this study, we addressed this critical question by performing a single-cell transcriptomic analysis of mouse β and α cells sorted from fetal to adult stages. We discovered that β and α cells use different regulatory strategies for their maturation and that cell proliferation peaks at different developmental times. However, the quiescent and proliferative cells in both the β lineage and α lineage are synchronous in their maturation states. The heterogeneity of juvenile β cells reflects distinct cell-cycling phases, origins, and maturation states, whereas adult β cells are relatively homogeneous at the transcriptomic level. These analyses provide not only a high-resolution roadmap for islet lineage development but also insights into the mechanisms of cellular heterogeneity, cell number expansion, and maturation of both β and α cells.

Deciphering Pancreatic Islet β Cell and α Cell Maturation Pathways and Characteristic Features at the Single-Cell Level.

How bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes remains unclear. Here, using single-cell transcriptomic analysis of hepatoblasts, hepatocytes, and cholangiocytes sorted from embryonic day 10.5 (E10.5) to E17.5 mouse embryos, we found that hepatoblast-to-hepatocyte differentiation occurred gradually and followed a linear default pathway. As more cells became fully differentiated hepatocytes, the number of proliferating cells decreased. Surprisingly, proliferating and quiescent hepatoblasts exhibited homogeneous differentiation states at a given developmental stage. This unique feature enabled us to combine single-cell and bulk-cell analyses to define the precise timing of the hepatoblast-to-hepatocyte transition, which occurs between E13.5 and E15.5. In contrast to hepatocyte development at almost all levels, hepatoblast-to-cholangiocyte differentiation underwent a sharp detour from the default pathway. New cholangiocyte generation occurred continuously between E11.5 and E14.5, but their maturation states at a given developmental stage were heterogeneous. Even more surprising, the number of proliferating cells increased as more progenitor cells differentiated into mature cholangiocytes. Based on an observation from the single-cell analysis, we also discovered that the protein kinase C/mitogen-activated protein kinase signaling pathway promoted cholangiocyte maturation.

A single-cell transcriptomic analysis reveals precise pathways and regulatory mechanisms underlying hepatoblast differentiation.

Pancreatic endocrine lineages are derived from pancreatic progenitors that undergo a cell fate transition requiring a switch from low to high Ngn3 expression. However, the underlying chromatin regulatory mechanisms are unclear. Here, we performed epigenomic analysis of gene regulatory loci featuring histone marks in cells with low or high level of Ngn3 expression. In combination with transcriptomic analysis, we discovered that in Ngn3-high cells, the removal of H3K27me3 was associated with the activation of key transcription factors and the establishment of primed and active enhancers. Deletion of , a histone demethylase for H3K27me3, at the pancreatic progenitor stage impaired the efficiency of endocrine cell fate transition and thereafter islet formation. Curiously, single-cell RNA-seq revealed that the transcriptome and developmental pathway of Ngn3-high cells were not affected by the deletion of  Our study indicates sequential chromatin events and identifies a crucial role for Jmjd3 in regulating the efficiency of the transition from Ngn3-low to Ngn3-high cells.

Dynamics of chromatin marks and the role of JMJD3 during pancreatic endocrine cell fate commitment.

The pancreas of vertebrates is separately derived from both the dorsal and ventral endodermal domains. However, the difference between these two programs has been unclear. Here, using a pancreatic determination gene, , driven GFP transgenic mouse strain, we identified Pdx1-GFP highly expressing cells (Pdx1) and Pdx1-GFP lowly expressing cells (Pdx1) in both embryonic dorsal Pdx1-expressing region (DPR) and ventral Pdx1-expressing region (VPR). We analyzed the transcriptomes of single Pdx1 and Pdx1 cells from the DPR and VPR. In the VPR, Pdx1 cells have an intermediate progenitor identity and can generate hepatoblasts, extrahepatobiliary cells, and Pdx1 pancreatic progenitor cells. In the DPR, Pdx1 cells are directly specified as pancreatic progenitors, whereas Pdx1 cells are precocious endocrine cells. Therefore, our study defines distinct road maps for dorsal and ventral pancreatic progenitor specification. The findings provide guidance for optimization of current β-cell induction protocols by following the  dorsal pancreatic specification program.

Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs.

The liver is an indispensable organ for metabolism and drug detoxification. The liver consists of endoderm-derived hepatobiliary lineages and various mesoderm-derived cells, and interacts with the surrounding tissues and organs through the ventral mesentery. Liver development, from hepatic specification to liver maturation, requires close interactions with mesoderm-derived cells, such as mesothelial cells, hepatic stellate cells, mesenchymal cells, liver sinusoidal endothelial cells and hematopoietic cells. These cells affect liver development through precise signaling events and even direct physical contact. Through the use of new techniques, emerging studies have recently led to a deeper understanding of liver development and its related mechanisms, especially the roles of mesodermal cells in liver development. Based on these developments, the current protocols for in vitro hepatocyte-like cell induction and liver-like tissue construction have been optimized and are of great importance for the treatment of liver diseases. Here, we review the roles of mesoderm-derived cells in the processes of liver development, hepatocyte-like cell induction and liver-like tissue construction.

Article	Year
Histone acetylation affects expression of cellular patterning genes in the Arabidopsis root epidermis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 16176989	2005
Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification. The EMBO journal\| PubMed ID: 25107471	2014
Differentially Expressed MicroRNA-483 Confers Distinct Functions in Pancreatic β- and α-Cells. The Journal of biological chemistry\| PubMed ID: 26109062	2015
Deciphering Pancreatic Islet β Cell and α Cell Maturation Pathways and Characteristic Features at the Single-Cell Level. Cell metabolism\| PubMed ID: 28467935	2017
A single-cell transcriptomic analysis reveals precise pathways and regulatory mechanisms underlying hepatoblast differentiation. Hepatology (Baltimore, Md.)\| PubMed ID: 28681484	2017
Dynamics of chromatin marks and the role of JMJD3 during pancreatic endocrine cell fate commitment. Development (Cambridge, England)\| PubMed ID: 29559448	2018
Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs. EMBO reports\| PubMed ID: 30065074	2018
The contributions of mesoderm-derived cells in liver development. Seminars in cell & developmental biology\| PubMed ID: 30193996	2018

Cheng-Ran Xu

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