An integrated approach identifies the molecular underpinnings of murine anterior visceral endoderm migration.

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

Embryonic and extraembryonic tissues during mammalian development: shifting boundaries in time and space.

The first few days of embryonic development in eutherian mammals are dedicated to the specification and elaboration of the extraembryonic tissues. However, where the fetus ends and its adnexa begins is not always as self-evident during the early stages of development, when the definitive body axes are still being laid down, the germ layers being specified and a discrete form or bodyplan is yet to emerge. Function, anatomy, histomorphology and molecular identities have been used through the history of embryology, to make this distinction. In this review, we explore them individually by using specific examples from the early embryo. While highlighting the challenges of drawing discrete boundaries between embryonic and extraembryonic tissues and the limitations of a binary categorization, we discuss how basing such identity on fate is the most universal and conceptually consistent. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Epithelial dynamics during early mouse development.

The first epithelia to arise in an organism face the challenge of maintaining the integrity of the newly formed tissue, while exhibiting the behavioral flexibility required for morphogenetic processes to occur effectively. Epithelial cells integrate biochemical and biomechanical cues, both intrinsic and extrinsic, in order to bring about the molecular changes which determine their morphology, behavior and fate. In this review we highlight recent advances in our understanding of the various dynamic processes that the emergent epithelial cells undergo during the first seven days of mouse development and speculate what the future holds in understanding the mechanistic bases for these processes through integrative approaches.

Hepatocyte-ductal transdifferentiation is mediated by reciprocal repression of SOX9 and C/EBPα.

Primary hepatocytes rapidly dedifferentiate when cultured in vitro. We have studied the mechanism of hepatocyte dedifferentiation by using two culture media: one that maintains hepatocytes in a differentiated state and another that allows dedifferentiation. We show that dedifferentiation involves partial transformation of hepatocytes into cells that resemble biliary epithelial cells. Lineage labeling and time-lapse filming confirm that the dedifferentiated cells are derived from hepatocytes and not from contaminating ductal or fibroblastic cells in the original culture. Furthermore, we establish that the conversion of hepatocytes to biliary-like cells is regulated by mutual antagonism of CCAAT/enhancer binding protein alpha (C/EBPα) and SOX9, which have opposing effects on the expression of hepatocyte and ductal genes. Thus, hepatocyte dedifferentiation induces the biliary gene expression program by alleviating C/EBPα-mediated repression of Sox9. We propose that reciprocal antagonism of C/EBPα and SOX9 also operates in the formation of hepatocytes and biliary ducts from hepatoblasts during normal embryonic development. These data demonstrate that reprogramming of differentiated cells can be used to model the acquisition and maintenance of cell fate in vivo.

Isolation of high quality RNA from embryonic kidney and cells.

All the mRNAs within a cell and their relative levels are indicative of gene expression within that cell, which is essential for its structure and function in its differentiated state. Therefore, methods for the identification of the specific mRNAs and the quantitation of their levels are invaluable tools for understanding gene expression. Due to high endogenous RNase activity within virtually all living cells, the isolation of good quality RNA with minimal degradation is not a trivial task. This protocol outlines a tried and tested methodology for isolating high quality RNA from embryonic kidneys for various applications including microarray analysis and quantitative reverse transcription PCR (qRT-PCR).

Generation of hepatocyte-like cells from in vitro transdifferentiated human fetal pancreas.

Although the appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology, evidence for the conversion of human pancreatic cells to liver cells is still lacking. We therefore investigated the developmental plasticity between human embryonic pancreatic cells and liver cells. Cells were isolated and expanded from 7-8-week-old human fetal pancreata (HFP) and were characterized for the absence and presence of pancreatic and hepatic markers. In vitro expanded HFP were treated with fibroblast growth factor 2 (FGF2) and dexamethasone (DX) to induce a liver phenotye in the cells. These treated cells in various passages were further studied for their capacity to be functional in hepatic parenchyma following retrorsine-induced injury in nude C57 black mice. Amylase- and EPCAM-positive-enriched cells isolated from HFP and treated with FGF2 and DX lost expression of pancreatic markers and gained a liver phenotype. Hepatic differentiation was based on the expression (both at the mRNA and protein level) of liver markers albumin and cytokeratin 19. When transplanted in vivo into nude mice treated with retrorsine, both cell types successfully engrafted and functionally differentiated into hepatic cells expressing human albumin, glycogen, dipeptidyl peptidase, and gamma-glutamyltranspeptidase. These data indicate that human fetal pancreatic cells have a capacity to alter their gene expression profile in response to exogenous treatment with FGF2 and DX. It may be possible to generate an unlimited supply of hepatocytes in vitro for cell therapy.

Reprogramming of liver to pancreas.

Islet grafts have demonstrated that patients with diabetes would benefit greatly by beta-cell therapy. However, the paucity of available islets for transplantation as well as the immunological barriers faced in allogeneic transplantation represent a tremendous barrier to regenerative approaches to the treatment of diabetes. Here, we present a strategy and protocols to transdifferentiate developmentally related hepatocytes into beta-cells by the ectopic expression of critical beta-cell transcription factors.

Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPalpha.

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPalpha showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPalpha binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.

Transdifferentiation in developmental biology, disease, and in therapy.

Transdifferentiation (or metaplasia) refers to the conversion of one cell type to another. Because transdifferentiation normally occurs between cells that arise from the same region of the embryo, understanding the molecular and cellular events in cell type transformations may help to explain the mechanisms underlying normal development. Here we review examples of transdifferentiation in nature focusing on the possible role of cell type switching in metamorphosis and regeneration. We also examine transdifferentiation in mammals in relation to disease and the use of transdifferentiated cells in cellular therapy.

Stem cells in the adult pancreas and liver.

Stem cells are undifferentiated cells that can self-renew and generate specialized (functional) cell types. The remarkable ability of stem cells to differentiate towards functional cells makes them suitable modalities in cellular therapy (which means treating diseases with the body's own cells). Potential targets for cellular therapy include diabetes and liver failure. However, in order for stem cells to be clinically useful, we must learn to identify them and to regulate their differentiation. We will use the intestine as a classical example of a stem cell compartment, and then examine the evidence for the existence of adult stem cells in two endodermally derived organs: pancreas and liver. We will review the characteristics of the putative stem cells in these tissues and the transcription factors controlling their differentiation towards functional cell types.

Liver specification: a new role for Wnts in liver development.

Secreted Wnt proteins control a diverse array of developmental decisions. A recent analysis of the zebrafish mutant prometheus points to a previously unknown role for Wnts during liver specification.