Single-cell murine genetic fate mapping reveals bipotential hepatoblasts and novel multi-organ endoderm progenitors.

The definitive endoderm (DE) is the embryonic germ layer that forms the gut tube and associated organs, including thymus, lungs, liver and pancreas. To understand how individual DE cells furnish gut organs, genetic fate mapping was performed using the Rosa26lacZ Cre-reporter paired with a tamoxifen-inducible DE-specific Cre-expressing transgene. We established a low tamoxifen dose that infrequently induced heritable lacZ expression in a single cell of individual E8.5 mouse embryos and identified clonal cell descendants at E16.5. As expected, only a fraction of the E16.5 embryos contained lacZ-positive clonal descendants and a subset of these contained descendants in multiple organs, revealing novel ontogeny. Furthermore, immunohistochemical analysis was used to identify lacZ-positive hepatocytes and biliary epithelial cells, which are the cholangiocyte precursors, in each clonally populated liver. Together, these data not only uncover novel and suspected lineage relationships between DE-derived organs, but also illustrate the bipotential nature of individual hepatoblasts by demonstrating that single hepatoblasts contribute to both the hepatocyte and the cholangiocyte lineage in vivo.

Identification and fate mapping of the pancreatic mesenchyme.

The murine pancreas buds from the ventral embryonic endoderm at approximately 8.75 dpc and a second pancreas bud emerges from the dorsal endoderm by 9.0 dpc. Although it is clear that secreted signals from adjacent mesoderm-derived sources are required for both the appropriate emergence and further refinement of the pancreatic endoderm, neither the exact signals nor the requisite tissue sources have been defined in mammalian systems. Herein we use DiI fate mapping of cultured murine embryos to identify the embryonic sources of both the early inductive and later condensed pancreatic mesenchyme. Despite being capable of supporting pancreas induction from dorsal endoderm in co-culture experiments, we find that in the context of the developing embryo, the dorsal aortae as well as the paraxial, intermediate, and lateral mesoderm derivatives only transiently associate with the dorsal pancreas bud, producing descendants that are decidedly anterior to the pancreas bud. Unlike these other mesoderm derivatives, the axial (notochord) descendants maintain association with the dorsal pre-pancreatic endoderm and early pancreas bud. This fate mapping data points to the notochord as the likely inductive source in vivo while also revealing dynamic morphogenetic movements displayed by individual mesodermal subtypes. Because none of the mesoderm examined above produced the pancreatic mesenchyme that condenses around the induced bud to support exocrine and endocrine differentiation, we also sought to identify the mesodermal origins of this mesenchyme. We identify a portion of the coelomic mesoderm that contributes to the condensed pancreatic mesenchyme. In conclusion, we identify a portion of the notochord as a likely source of the signals required to induce and maintain the early dorsal pancreas bud, demonstrate that the coelomic mesothelium contributes to the dorsal and ventral pancreatic mesenchyme, and provide insight into the dynamic morphological rearrangements of mesoderm-derived tissues during early organogenesis stages of mammalian development.

Patterning of the hepato-pancreatobiliary boundary by BMP reveals heterogeneity within the murine liver bud.

During development, the endoderm initiates organ-restricted gene expression patterns in a spatiotemporally controlled manner. This process, termed induction, requires signals from adjacent mesodermal derivatives. Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) emanating from the cardiac mesoderm and the septum transversum mesenchyme (STM), respectively, are believed to be simultaneously and uniformly required to directly induce hepatic gene expression from the murine endoderm. Using small molecule inhibitors of BMP signals during liver bud induction in the developing mouse embryo, we found that BMP signaling was not uniformly required to induce hepatic gene expression. Although BMP inhibition caused an overall reduction in the number of induced hepatoblasts, the STM-bounded posterior liver bud demonstrated the most severe loss of the essential hepatic transcription factor, hepatocyte nuclear factor 4-α (HNF4α), whereas the sinus venosus-lined anterior liver bud was less affected. We found that the posterior liver bud progenitors were anteriorly displaced and aberrantly activated pancreatobiliary markers, including sex-determining region Y-box 9 (SOX9). Additionally, we found that ectopically expressed SOX9 inhibited HNF4α and that BMP was indirectly required for hepatoblast induction. Finally, because previous studies have demonstrated that FGF signals are essential for anterior but not posterior liver bud induction, we examined synchronous BMP and FGF inhibition and found this led to a nearly complete loss of hepatoblasts. CONCLUSION: BMP signaling is required to maintain the hepato-pancreatobiliary boundary, at least in part, by indirectly repressing SOX9 in the hepatic endoderm. BMP and FGF signals are each required for the induction of spatially complementary subsets of hepatoblasts. These results underscore the importance of studying early inductive processes in the whole embryo. (Hepatology 2018;68:274-288).

Laser-mediated cell ablation during post-implantation mouse development.

BACKGROUND: Laser-mediated cell ablation is a powerful tool that has been used to understand cell fate in a variety of externally developing organisms but has not been used during mammalian post-implantation development. RESULTS: We describe a method pairing laser ablation with murine embryo culture and establish parameters that can be used to precisely ablate cells in the selected field with minimal disruption to adjacent cells or the underlying cell matrix. Ablation of a large domain of endoderm, followed by ~1 day of culture results in a phenotypically normal embryo and gut tube, indicating that laser ablation is compatible with normal development. We next focused on one of the three precursor populations that have been shown to produce the liver bud. Ablations of a single progenitor domain result in a unilateral delay in the liver bud while the contralateral side is unaffected. CONCLUSIONS: We demonstrate that laser ablation is a specific and useful technique for studying cell fate in the mouse embryo. This method represents a powerful advance in developmental studies in the mouse and can be used to provide information on the specification of organs, differentiation, cell migration, and vital tissue interactions during development.

Morphogenetic analysis of peri-implantation development.

BACKGROUND: Although successful implantation is required for development in placental mammals, the molecular and morphogenetic events that define peri-implantation remain largely unexplored. RESULTS: Here we present detailed morphological and immunohistochemical analysis of mouse embryos between embryonic day 3.75 and 5.25 of gestation, during the implantation process in vivo. We examined expression patterns of key transcription factors (Sox2, Oct4, Nanog, Cdx2, Gata6, Sox17, and Yy1) during pre- and postimplantation development. Additionally, we examined morphogenetic changes through analysis of ZO-1, Laminin, and E-Cadherin localization. The results presented reveal novel changes in gene expression and morphogenetic events during peri-implantation in utero. Here we show: (1) molecular and morphological changes in primitive endoderm cells as they transition from a salt and pepper distribution to a sheet covering the inner cell mass; (2) tissue-specific GATA6 levels; and (3) a striking pattern of SOX17 that is suggestive of a functional role either directing or permitting implantation at specific sites in the uterine epithelium. CONCLUSIONS: A growing number of knockout mice display peri-implantation lethality, and the data presented herein identify key morphogenetic landmarks that can be used to characterize mutant phenotypes, as well as further our basic understanding of peri-implantation development.

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.