Endodermal expression of Nkx6 genes depends differentially on Pdx1

Nkx family members are essential for normal development of many different tissues such as the heart, lungs, thyroid, prostate, and CNS. Here, we describe the endodermal expression pattern of three Nkx6 family genes of which two shows conserved expression in the early pancreatic epithelium. In chicken, Nkx6.1 expression is not restricted to the presumptive pancreatic area but is more broadly expressed in the endoderm. In mice, expression of Nkx6.1 is restricted to the pancreatic epithelium. In both mice and chicken, Nkx6.2 and Pdx1 are expressed in very similar domains, identifying Nkx6.2 as a novel marker of pancreas endoderm. Additionally, our results show that Nkx6.3 is expressed transiently in pancreatic endoderm in chicken but not mouse embryos. At later stages, Nkx6.3 is found in the caudal stomach and rostral duodenum in both species. Finally, we demonstrate that Pdx1 is required for Nkx6.1 but not Nkx6.2 expression in mice and that ectopic Pdx1 can induce Nkx6.1 but not Nkx6.2 or Nkx6.3 expression in anterior chicken endoderm. These results demonstrate that Nkx6.1 lies downstream of Pdx1 in a genetic pathway and that Pdx1 is required and sufficient for Nkx6.1 expression in the early foregut endoderm.     

Ptf1a-mediated control of Dll1 reveals an alternative to the lateral inhibition mechanism

Neurog3-induced Dll1 expression in pancreatic endocrine progenitors ostensibly activates Hes1 expression via Notch and thereby represses Neurog3 and endocrine differentiation in neighboring cells by lateral inhibition. Here we show in mouse that Dll1 and Hes1 expression deviate during regionalization of early endoderm, and later during early pancreas morphogenesis. At that time, Ptf1a activates Dll1 in multipotent pancreatic progenitor cells (MPCs), and Hes1 expression becomes Dll1 dependent over a brief time window. Moreover, Dll1, Hes1 and Dll1/Hes1 mutant phenotypes diverge during organ regionalization, become congruent at early bud stages, and then diverge again at late bud stages. Persistent pancreatic hypoplasia in Dll1 mutants after eliminating Neurog3 expression and endocrine development, together with reduced proliferation of MPCs in both Dll1 and Hes1 mutants, reveals that the hypoplasia is caused by a growth defect rather than by progenitor depletion. Unexpectedly, we find that Hes1 is required to sustain Ptf1a expression, and in turn Dll1 expression in early MPCs. Our results show that Ptf1a-induced Dll1 expression stimulates MPC proliferation and pancreatic growth by maintaining Hes1 expression and Ptf1a protein levels.     

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.     

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.     

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.     

Fate maps of ventral and dorsal pancreatic progenitor cells in early somite stage mouse embryos

The origins of liver progenitor cells have been extensively studied, but evidence on the origin of pancreatic precursor cells is currently limited. Pancreatic and duodenal homeobox gene 1 (Pdx1) is one of the earliest known markers for the pancreas. A transgenic mouse line expressing green fluorescent protein (GFP) under the control of the Pdx1 promoter showed that Pdx1/GFP expression was first observed in the mid-region of the anterior intestinal portal (AIP) lip at embryonic day (E) 8.5 at the 5-6 somite stage (ss). The liver progenitors were confirmed to originate from separate domains at the lateral endoderm and the inner part of the medial AIP as previously reported (Tremblay and Zaret, 2005), which turned out to lie caudally to the Pdx1/GFP-expressing domain. To confirm if the early Pdx1/GFP-positive cells give rise to the pancreatic bud, we labeled the cells on the lip of the AIP using the carbocyanine dye CM-DiI and traced their fates in 1-4 ss, 5-6 ss and 7-9 ss E8.5 embryos using an ex utero whole embryo culture method. At 1 ss, the ventral pancreas progenitors were observed in the lateral endoderm, not yet being segregated from the liver or gut progenitors. Cells that contributed solely to the ventral pancreas first appeared at the AIP lip from 5 ss. At 5-6 ss, cells from the medial of the AIP lip contributed to the ventral pancreas. The pancreas fate region become narrower as development progresses. At 7-9 ss, the cells contributing to the ventral pancreas resided in a narrow region of the AIP lip. From 5 ss, the right flanking region contributes to the posterior gut, and the left flanking region contributes to the anterior gut. Dorsal pancreatic progenitors originate from the dorsal endoderm at the 3-6 somite level at 7-9 ss, though they have not yet diverged from the dorsal gut progenitors at this stage.