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The timing and gene regulatory logic of organ-fate commitment from within the posterior foregut of the mammalian endoderm is largely unexplored. Transient misexpression of a presumed pancreatic-commitment transcription factor, Ptf1a, in embryonic mouse endoderm (Ptf1a(EDD)) dramatically expanded the pancreatic gene regulatory network within the foregut. Ptf1a(EDD) temporarily suppressed Sox2 broadly over the anterior endoderm. Pancreas-proximal organ territories underwent full tissue conversion. Early-stage Ptf1a(EDD) rapidly expanded the endogenous endodermal Pdx1-positive domain and recruited other pancreas-fate-instructive genes, thereby spatially enlarging the potential for pancreatic multipotency. Early Ptf1a(EDD) converted essentially the entire glandular stomach, rostral duodenum and extrahepatic biliary system to pancreas, with formation of many endocrine cell clusters of the type found in normal islets of Langerhans. Sliding the Ptf1a(EDD) expression window through embryogenesis revealed differential temporal competencies for stomach-pancreas respecification. The response to later-stage Ptf1a(EDD) changed radically towards unipotent, acinar-restricted conversion. We provide strong evidence, beyond previous Ptf1a inactivation or misexpression experiments in frog embryos, for spatiotemporally context-dependent activity of Ptf1a as a potent gain-of-function trigger of pro-pancreatic commitment.
© 2014. Published by The Company of Biologists Ltd.
Stem cell differentiation depends on transcriptional activation driven by lineage-specific regulators as well as changes in chromatin organization. However, the coordination of these events is poorly understood. Here, we show that T-box proteins team up with chromatin modifying enzymes to drive the expression of the key lineage regulator, Eomes during endodermal differentiation of embryonic stem (ES) cells. The Eomes locus is maintained in a transcriptionally poised configuration in ES cells. During early differentiation steps, the ES cell factor Tbx3 associates with the histone demethylase Jmjd3 at the enhancer element of the Eomes locus to allow enhancer-promoter interactions. This spatial reorganization of the chromatin primes the cells to respond to Activin signalling, which promotes the binding of Jmjd3 and Eomes to its own bivalent promoter region to further stimulate Eomes expression in a positive feedback loop. In addition, Eomes activates a transcriptional network of core regulators of endodermal differentiation. Our results demonstrate that Jmjd3 sequentially associates with two T-box factors, Tbx3 and Eomes to drive stem cell differentiation towards the definitive endoderm lineage.
Pancreatic agenesis is a human disorder caused by defects in pancreas development. To date, only a few genes have been linked to pancreatic agenesis in humans, with mutations in pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor 1a (PTF1A) reported in only 5 families with described cases. Recently, mutations in GATA6 have been identified in a large percentage of human cases, and a GATA4 mutant allele has been implicated in a single case. In the mouse, Gata4 and Gata6 are expressed in several endoderm-derived tissues, including the pancreas. To analyze the functions of GATA4 and/or GATA6 during mouse pancreatic development, we generated pancreas-specific deletions of Gata4 and Gata6. Surprisingly, loss of either Gata4 or Gata6 in the pancreas resulted in only mild pancreatic defects, which resolved postnatally. However, simultaneous deletion of both Gata4 and Gata6 in the pancreas caused severe pancreatic agenesis due to disruption of pancreatic progenitor cell proliferation, defects in branching morphogenesis, and a subsequent failure to induce the differentiation of progenitor cells expressing carboxypeptidase A1 (CPA1) and neurogenin 3 (NEUROG3). These studies address the conserved and nonconserved mechanisms underlying GATA4 and GATA6 function during pancreas development and provide a new mouse model to characterize the underlying developmental defects associated with pancreatic agenesis.
Sox17 is essential for both endoderm development and fetal hematopoietic stem cell (HSC) maintenance. While endoderm-derived organs are well known to originate from Sox17-expressing cells, it is less certain whether fetal HSCs also originate from Sox17-expressing cells. By generating a Sox17(GFPCre) allele and using it to assess the fate of Sox17-expressing cells during embryogenesis, we confirmed that both endodermal and a part of definitive hematopoietic cells are derived from Sox17-positive cells. Prior to E9.5, the expression of Sox17 is restricted to the endoderm lineage. However, at E9.5 Sox17 is expressed in the endothelial cells (ECs) at the para-aortic splanchnopleural region that contribute to the formation of HSCs at a later stage. The identification of two distinct progenitor cell populations that express Sox17 at E9.5 was confirmed using fluorescence-activated cell sorting together with RNA-Seq to determine the gene expression profiles of the two cell populations. Interestingly, this analysis revealed differences in the RNA processing of the Sox17 mRNA during embryogenesis. Taken together, these results indicate that Sox17 is expressed in progenitor cells derived from two different germ layers, further demonstrating the complex expression pattern of this gene and suggesting caution when using Sox17 as a lineage-specific marker.
Copyright © 2012 AlphaMed Press.
Fluorescent protein (FP) reporter alleles are useful both for identifying and purifying specific cell populations in the mouse. Here, we report the generation of mouse embryonic stem cells that contain a pancreatic and duodenal homeobox 1 (Pdx1) loxed cassette acceptor (Pdx1(LCA)) allele and the use of recombinase-mediated cassette exchange to derive mice that contain a Pdx1(CFP) (Cerulean) reporter allele. Mice with this allele exhibited cyan fluorescence within the previously well-characterized Pdx1 expression domain in posterior foregut endoderm. Immunolabeling showed that endogenous Pdx1 was coexpressed with CFP at all time points examined. Furthermore, fluorescence-activated cell sorting was used to isolate CFP-positive cells from E11.5 and E18.5 embryonic tissues using both 405 and 445 nm lasers, although the latter resulted in a nearly 50-fold increase in emission intensity. The Pdx1(CFP) allele will enable the isolation of specific foregut endoderm and pancreatic cell populations, both alone and in combination with other FP reporter alleles.
Copyright © 2011 Wiley-Liss, Inc.
Using a flow cytometry-based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.
microRNAs (miRNAs) are a family of 21-23 nucleotide endogenous non-coding RNAs that post-transcriptionally regulate gene expression in a sequence-specific manner. Typically, miRNAs downregulate target genes by recognizing and recruiting protein complexes to 3'UTRs, followed by translation repression or mRNA degradation. miR-92 is a well-studied oncogene in mammalian systems. Here, using zebrafish as a model system, we uncovered a novel tissue-inductive role for miR-92 during early vertebrate development. Overexpression resulted in reduced endoderm formation during gastrulation with consequent cardia and viscera bifida. By contrast, depletion of miR-92 increased endoderm formation, which led to abnormal Kupffer's vesicle development and left-right patterning defects. Using target prediction algorithms and reporter constructs, we show that gata5 is a target of miR-92. Alteration of gata5 levels reciprocally mirrored the effects of gain and loss of function of miR-92. Moreover, genetic epistasis experiments showed that miR-92-mediated defects could be substantially suppressed by modulating gata5 levels. We propose that miR-92 is a critical regulator of endoderm formation and left-right asymmetry during early zebrafish development and provide the first evidence for a regulatory function for gata5 in the formation of Kupffer's vesicle and left-right patterning.
Holoprosencephaly (HPE) is the most common forebrain and craniofacial malformation syndrome in humans. The genetics of HPE suggest that it often stems from a synergistic interaction of mutations in independent loci. In mice, several combinations of mutations in Nodal signaling pathway components can give rise to HPE, but it is not clear whether modest deficits of Nodal signaling along with lesions in other pathways might also cause such defects. We find that HPE results from simultaneous reduction of Nodal signaling and an organizer BMP (bone morphogenetic protein) antagonist, either Chordin or Noggin. These defects result from reduced production of tissues that promote forebrain and craniofacial development. Nodal promotes the expression of genes in the anterior primitive streak that are important for the development of these tissues, whereas BMP inhibits their expression. Pharmacological and transgenic manipulation of these signaling pathways suggests that BMP and Nodal antagonize each other prior to intracellular signal transduction. Biochemical experiments in vitro indicate that secreted Bmp2 and Nodal can form extracellular complexes, potentially interfering with receptor activation. Our results reveal that the patterning of forebrain and medial craniofacial elements requires a fine balance between BMP and Nodal signaling during primitive streak development, and provide a potential mechanistic basis for a new multigenic model of HPE.
PURPOSE - In the classic view of bladder development the trigone originates from the mesoderm derived wolffian ducts while the remainder of the bladder originates from the endoderm derived urogenital sinus. Recent molecular developmental studies have questioned the veracity of this received wisdom, suggesting an endodermal origin for the trigone. To shed further light on this issue we observed mesenchymal-epithelial interactions between trigone epithelium and fetal urogenital sinus mesenchyma to infer the trigonal germ layer of origin.
MATERIALS AND METHODS - Mouse trigone epithelium was recombined with fetal rat urogenital sinus mesenchyma in tissue recombinant grafts that were placed beneath the renal capsule of athymic mouse hosts. Grafts were harvested at 4 weeks. Control grafts with bladder dome and ureteral epithelium were also examined. Tissues were evaluated with hematoxylin and eosin, and Hoechst dye 33258 to confirm cell species origin. Immunohistochemistry was done with androgen receptor, broad spectrum uroplakin, dorsolateral prostate secretions and seminal vesicle secretions to differentiate prostatic and seminal vesicle differentiation.
RESULTS - Grafts of mouse trigone epithelium with fetal rat urogenital sinus mesenchyma yielded epithelial tissue that stained for dorsolateral prostate secretions but not for seminal vesicle secretions. Control grafts of bladder dome epithelium yielded the expected endodermal prostate differentiation. Control grafts of ureteral epithelium yielded the expected mesodermal seminal vesicle differentiation.
CONCLUSIONS - The consistent finding of prostatic epithelium in tissue recombinants of trigone epithelium and fetal urogenital sinus mesenchyma reinforces the hypothesis that the trigone is derived from the endoderm and not from the mesoderm, as commonly accepted.
Although endodermal organs including the liver, pancreas, and intestine are of significant therapeutic interest, the mechanism by which the endoderm is divided into organ domains during embryogenesis is not well understood. To better understand this process, global gene expression profiling was performed on early endodermal organ domains. This global analysis was followed up by dynamic immunofluorescence analysis of key transcription factors, uncovering novel expression patterns as well as cell surface proteins that allow prospective isolation of specific endodermal organ domains. Additionally, a repressive interaction between Cdx2 and Sox2 was found to occur at the prospective stomach-intestine border, with the hepatic and pancreatic domains forming at this boundary, and Hlxb9 was revealed to have graded expression along the dorsal-ventral axis. These results contribute to understanding the mechanism of endodermal organogenesis and should assist efforts to replicate this process using pluripotent stem cells.
Copyright (c) 2008 Wiley-Liss, Inc.