Inducing the liver: Understanding the signals that promote murine liver budding

The endoderm emerges as an epithelial sheet that covers the surface of the developing murine embryo. This tissue will produce the entire gut tube as well as associated digestive and respiratory organs including the thyroid, thymus, lung, liver, and pancreas. The emergence of each endodermal organ occurs in a temporally distinct manner that is dependant upon reciprocal inductive interactions between the endoderm and the underlying mesoderm. The emergence of the hepatic endoderm, which occurs using a morphological process termed liver budding, initiates during early somitogenesis in the mouse at approximately 8.25 days post‐coitum (dpc). Explant and transplant studies performed in chicken and mouse have demonstrated that secreted signals from adjacent mesodermal tissues initiate the hepatic gene program from ventral‐fated endoderm. Here, we review the data in support of the roles of members of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and Wnt signaling pathways in liver budding and discover that little is known about the precise endogenous signals involved in the molecular and morphological induction of liver budding in the mouse. J. Cell. Physiol. 226: 1727–1731, 2011. © 2010 Wiley‐Liss, Inc.     

Essential roles of zebrafish bmp2a, fgf10, and fgf24 in the specification of the ventral pancreas

In vertebrates, pancreas and liver arise from bipotential progenitors located in the embryonic gut endoderm. Bone morphogenic protein (BMP) and fibroblast growth factor (FGF) signaling pathways have been shown to induce hepatic specification while repressing pancreatic fate. Here we show that BMP and FGF factors also play crucial function, at slightly later stages, in the specification of the ventral pancreas. By analyzing the pancreatic markers pdx1, ptf1a, and hlxb9la in different zebrafish models of BMP loss of function, we demonstrate that the BMP pathway is required between 20 and 24 h postfertilization to specify the ventral pancreatic bud. Knockdown experiments show that bmp2a, expressed in the lateral plate mesoderm at these stages, is essential for ventral pancreas specification. Bmp2a action is not restricted to the pancreatic domain and is also required for the proper expression of hepatic markers. By contrast, through the analysis of fgf10(-/-); fgf24(-/-) embryos, we reveal the specific role of these two FGF ligands in the induction of the ventral pancreas and in the repression of the hepatic fate. These mutants display ventral pancreas agenesis and ectopic masses of hepatocytes. Overall, these data highlight the dynamic role of BMP and FGF in the patterning of the hepatopancreatic region.     

An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells

While particular combinations of mesodermal signals are known to induce distinct tissue-specific programs in the endoderm, there is little information about the response pathways within endoderm cells that control their specification. We have used signaling inhibitors on embryo tissue explants and whole-embryo cultures as well as genetic approaches to reveal part of an intracellular network by which FGF signaling helps induce hepatic genes and stabilize nascent hepatic cells within the endodermal epithelium. Specifically, we found that hepatic gene induction is elicited by an FGF/MAPK pathway. Although the PI3K pathway is activated in foregut endoderm cells, its inhibition does not block hepatic gene induction in explants; however, it does block tissue growth. We also found that at the onset of hepatogenesis, the FGF/MAPK and PI3K pathways do not crossregulate in the endoderm. The finding of separate pathways for endoderm tissue specification and growth provides insights for guiding cellular regeneration and stem cell differentiation.     

Interplay between Wnt2 and Wnt2bb controls multiple steps of early foregut-derived organ development

The vertebrate liver, pancreas and lung arise in close proximity from the multipotent foregut endoderm. Tissue-explant experiments uncovered instructive signals emanating from the neighbouring lateral plate mesoderm, directing the endoderm towards specific organ fates. This suggested that an intricate network of signals is required to control the specification and differentiation of each organ. Here, we show that sequential functions of Wnt2bb and Wnt2 control liver specification and proliferation in zebrafish. Their combined specific activities are essential for liver specification, as their loss of function causes liver agenesis. Conversely, excess wnt2bb or wnt2 induces ectopic liver tissue at the expense of pancreatic and anterior intestinal tissues, revealing the competence of intestinal endoderm to respond to hepatogenic signals. Epistasis experiments revealed that the receptor frizzled homolog 5 (fzd5) mediates part of the broader hepatic competence of the alimentary canal. fzd5 is required for early liver formation and interacts genetically with wnt2 as well as wnt2bb. In addition, lack of both ligands causes agenesis of the swim bladder, the structural homolog of the mammalian lung. Thus, tightly regulated spatiotemporal expression of wnt2bb, wnt2 and fzd5 is central to coordinating early liver, pancreas and swim bladder development from a multipotent foregut endoderm.     

A fate map of the murine pancreas buds reveals a multipotent ventral foregut organ progenitor

The definitive endoderm is the embryonic germ layer that gives rise to the budding endodermal organs including the thyroid, lung, liver and pancreas as well as the remainder of the gut tube. DiI fate mapping and whole embryo culture were used to determine the endodermal origin of the 9.5 days post coitum (dpc) dorsal and ventral pancreas buds. Our results demonstrate that the progenitors of each bud occupy distinct endodermal territories. Dorsal bud progenitors are located in the medial endoderm overlying somites 2-4 between the 2 and 11 somite stage (SS). The endoderm forming the ventral pancreas bud is found in 2 distinct regions. One territory originates from the left and right lateral endoderm caudal to the anterior intestinal portal by the 6 SS and the second domain is derived from the ventral midline of the endoderm lip (VMEL). Unlike the laterally located ventral foregut progenitors, the VMEL population harbors a multipotent progenitor that contributes to the thyroid bud, the rostral cap of the liver bud, ventral midline of the liver bud and the midline of the ventral pancreas bud in a temporally restricted manner. This data suggests that the midline of the 9.5 dpc thyroid, liver and ventral pancreas buds originates from the same progenitor population, demonstrating a developmental link between all three ventral foregut buds. Taken together, these data define the location of the dorsal and ventral pancreas progenitors in the prespecified endodermal sheet and should lead to insights into the inductive events required for pancreas specification.     

Regulatory phases of early liver development: paradigms of organogenesis

Genetic analysis, embryonic tissue explantation and in vivo chromatin studies have together identified the distinct regulatory steps that are necessary for the development of endoderm into a bud of liver tissue and, subsequently, into an organ. In this review, I discuss the acquisition of competence to express liver-specific genes by the endoderm, the control of early hepatic growth, the coordination of hepatic and vascular development and the cell differentiation that is necessary to generate a functioning liver. The regulatory mechanisms that underlie these phases are common to the development of many organ systems and might be recapitulated or disrupted during stem-cell differentiation and adult tissue pathogenesis.     

Bmp and Fgf signaling are essential for liver specification in zebrafish

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. Here, we provide in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. We utilized transgenic zebrafish that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. We found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated.     

The molecular basis and prospects in pancreatic development

Studies on the signaling mechanism that control the specification of endoderm-derived organs have only recently begun. While many studies revealed genes involved in the differentiation, growth and morphogenesis of the pancreas through studies of mutant mice, still little is known about how endoderm give rise to specific domains. Although many genes are known to have a role in pancreatic differentiation, growth and morphogenesis, few genes are known to take part in the specification of the pancreas so far. Hallmarks as well as gene markers for early development of the pancreas, which are however still very limited, will be useful for dissecting early events in pancreatic specification. Here, I give a summary on the origin of the dorsal and ventral pancreatic progenitors, signals for inductions, and genes so far known to function in pancreatic differentiation. I also give a future prospect in the use of ES cells and other experimental models, towards a comprehensive understanding of gene networks in the progenitor cells or intermediate cell types which arise during various stages of differentiation.     

Protein kinase C isozymes have distinct roles in neural induction and competence in Xenopus.

The restricted ability of embryonic tissue to respond to inductive signals is controlled by a poorly understood phenomenon, termed competence. In Xenopus, dorsal ectoderm is more competent than ventral ectoderm to become induced to neural tissue. We tested whether the Xenopus protein kinase C (PKC) isozymes alpha and beta have a role in neural induction and competence. We found that PKC alpha is predominantly localized in dorsal ectoderm, whereas PKC beta is uniformly distributed. Overexpression of PKC beta conveys a higher propensity for neural differentiation to both dorsal and ventral ectoderm, but their difference in competence remains. However, ectopic expression of PKC alpha elevates the level of neural competence of ventral ectoderm to that of dorsal ectoderm. These data indicate that different PKC isozymes have distinct roles in mediating both neural induction and competence.