Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells

The pancreas is derived from a pool of multipotent progenitor cells (MPCs) that co-express Pdx-1 and Ptf1a. To more precisely define how the individual and combined loss of Pdx-1 and Ptf1a affects pancreatic MPC specification and differentiation we derived and studied mice bearing a novel Ptf1a^YFP allele. While the expression of Pdx-1 and Ptf1a in pancreatic MPCs coincides between E9.5 and 12.5 the developmental phenotypes of Pdx-1 null and Pdx-1; Ptf1a double null mice are indistinguishable, and an early pancreatic bud is formed in both cases. This finding indicates that Pdx-1 is required in the foregut endoderm prior to Ptf1a for pancreatic MPC specification. We also found that Ptf1a is neither required for specification of Ngn3-positive endocrine progenitors nor differentiation of mature β-cells. In the absence of Pdx-1 Ngn3-positive cells were not observed after E9.5. Thus, in contrast to the deletion of Ptf1a, the loss of Pdx-1 precludes the sustained Ngn3-based derivation of endocrine progenitors from pancreatic MPCs. Taken together, these studies indicate that Pdx-1 and Ptf1a have distinct but interdependent functions during pancreatic MPC specification.     

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing β-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.     

Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates

We have characterized and mapped the zebrafish ptf1a gene, analyzed its embryonic expression, and studied its role in pancreas development. In situ hybridization experiments show that from the 12-somite stage to 48 hpf, ptf1a is dynamically expressed in the spinal cord, hindbrain, cerebellum, retina, and pancreas of zebrafish embryos. Within the endoderm, ptf1a is initially expressed at 32 hpf in the ventral portion of the pdx1 expression domain; ptf1a is expressed in a subset of cells located on the left side of the embryo posteriorly to the liver primordium and anteriorly to the endocrine islet that arises from the posterodorsal pancreatic anlage. Then the ptf1a expression domain buds giving rise to the anteroventral pancreatic anlage that grows posteriorly to eventually engulf the endocrine islet. By 72 hpf, ptf1a continues to be expressed in the exocrine compartment derived from the anteroventral anlage. Morpholino-induced ptf1a loss of function suppresses the expression of the exocrine markers, while the endocrine markers in the islet are unaffected. In mind bomb (mib) mutants, in which delta-mediated notch signalling is defective [Dev. Cell 4 (2003) 67], ptf1a is normally expressed. In addition, the slow-muscle-omitted (smu) mutants that lack expression of endocrine markers because of a defective hedgehog signalling [Curr. Biol. 11(2001) 1358] exhibit normal levels of ptf1a. This indicates that hedgehog signaling plays a different genetic role in the specification of the anteroventral (mostly exocrine) and posterodorsal (endocrine) pancreatic anlagen.     

Development of the pancreas in medaka,Oryzias latipes,from embryo to adult

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1‐ and ptf1a‐transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior–posterior axis.     

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.     

Conditional control of the differentiation competence of pancreatic endocrine and ductal cells by Fgf10

Fgf10 is a critical component of mesenchymal-to-epithelial signaling during endodermal development. In the Fgf10 null pancreas, the embryonic progenitor population fails to expand, while ectopic Fgf10 expression forces progenitor arrest and organ hyperplasia. Using a conditional Fgf10 gain-of-function model, we observed that the timing of Fgf10 expression affected the cellular competence of the arrested pancreatic progenitors. We present evidence that the Fgf10-arrested progenitor state is reversible and that terminal differentiation resumes upon cessation of Fgf10 production. However, competence towards the individual pancreatic cell lineages depended upon the gestational time of when Fgf10 expression was attenuated. This revealed a competence window of endocrine and ductal cell formation that coincided with the pancreatic secondary transition between E13.5 and E15.5. We demonstrate that maintaining the Fgf10-arrested state during this period leads to permanent loss of competence for the endocrine and ductal cell fates. However, competence of the arrested progenitors towards the exocrine cell fate was retained throughout the secondary transition. Sustained Fgf10 expression caused irreversible loss of Ngn3 expression, which may underlie the loss of endocrine competence. Maintenance of exocrine competence may be attributable to continuous Ptf1a expression in the Fgf10-arrested progenitors. This may explain the rapid induction of Bhlhb8, a normally distalized cell intrinsic marker, following loss of ectopic Fgf10 expression. We conclude that the window for endocrine and ductal cell competence ceases during the secondary transition in pancreatic development.     

Otx2 expression in anterior neuroectoderm and forebrain/midbrain is directed by more than six enhancers

Otx2 plays essential roles in each site at each step of head development. We previously identified the AN1 enhancer at 91 kb 5' upstream for the Otx2 expressions in anterior neuroectoderm (AN) at neural plate stage before E8.5, and the FM1 enhancer at 75 kb 5' upstream and the FM2 enhancer at 122 kb 3' downstream for the expression in forebrain/midbrain (FM) at brain vesicle stage after E8.5. The present study identified a second AN enhancer (AN2) at 88 kb 5' upstream; the AN2 enhancer also recapitulates the endogenous Otx2 expression in choroid plexus, cortical hem and choroidal roof. However, the enhancer mutants indicated the presence of another AN enhancer. The study also identified a third FM enhancer (FM3) at 153 kb 5' upstream. Thus, the Otx2 expressions in anterior neuroectoderm and forebrain/midbrain are regulated by more than six enhancers located far from the coding region. The enhancers identified are differentially conserved among vertebrates; none of the AN enhancers has activities in caudal forebrain and midbrain at brain vesicle stage after E8.5, nor do any of the FM enhancers in anterior neuroectoderm at neural plate stage before E8.5.