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SUMMARYDistal Visceral Endoderm (DVE) cells show a stereotypic unidirectional migration essential for correct orientation of the anterior-posterior axis. They migrate within a simple epithelium, the Visceral Endoderm (VE). It is unknown how DVE cells negotiate their way amongst the surrounding VE cells, what determines the bounds of DVE migration within the VE, and the relative contributions of different cell behaviours to this migration. To address these questions, we used lightsheet microscopy to generate a multi-embryo, singlecell resolution, longitudinal dataset of cell behaviour and morphology. We developed a machine learning based pipeline to segment cells and a data-informed systematic computational framework to extract and compare select morphological, behavioural and molecular parameters of all VE cells in a unified coordinate space. Unbiased clustering of this single-cell ‘phenomic’ dataset reveals considerable patterned phenotypic heterogeneity within the VE and a previously unknown sub-grouping within the DVE. While migrating, DVE cells retain regular morphology, do not exchange neighbours and are crowded, all hallmarks of the jammed state. In contrast, VE cells immediately ahead of them deform and undergo neighbour exchange. We show that DVE cells are characterised by higher levels of apical F-actin and elevated tension relative to the VE cells immediately ahead of them through which they migrate, but stop migrating upon reaching a region of the VE with matching elevated tension.Lefty1mutants, known to show abnormal over-migration of DVE cells, show disruption to this patterned tension in the VE. Our findings provide novel insights into the control of cell behaviour during the remodelling of curved epithelia, indicating that the collective migration of sub-sets of cells can be circumscribed by modulating the mechanical properties of surrounding cells and that migrating cells in this context remain as a jammed solid flock, with surrounding cells facilitating their movement by becoming unjammed.Graphical Abstract

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’.

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.

SUMMARYDuring early post-implantation development of the mouse embryo, the Anterior Visceral Endoderm (AVE) differs from surrounding visceral endoderm (VE) in its migratory behaviour and ability to restrict primitive streak formation to the opposite side of the egg cylinder. In order to characterise the molecular basis for the unique properties of the AVE, we combined single-cell RNA-sequencing of the VE prior to and during AVE migration, with high-resolution imaging, short-term lineage labelling, phosphoproteomics and pharmacological intervention. This revealed the transient nature of the AVE, the emergence of heterogeneities in AVE transcriptional states relative to position of cells, and its prominence in establishing gene expression asymmetries within the spatial constraints of the embryo. We identified a previously unknown requirement of Ephrin- and Semaphorin-signalling for AVE migration. These findings point to a tight coupling of transcriptional state and position in the AVE and reveal molecular heterogeneities underpinning its migratory behaviour and function.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common reason for chronic kidney disease in children. Although more than 30 monogenic causes have been implicated in isolated forms of human CAKUT so far, the vast majority remains elusive. To identify novel monogenic causes of CAKUT we applied homozygosity mapping, together with whole exome sequencing, in a patient from consanguineous descent with isolated CAKUT. We identified a homozygous missense mutation (p.Arg415His) of the Ets Translocation Variant Gene 4 (ETV4). The transcription factor ETV4 is a downstream target of the GDNF/RET signaling pathway that plays a crucial role in kidney development. We show by means of electrophoretic mobility shift assay that the Arg415His mutant causes loss of the DNA binding affinity of ETV4 and fails to activate transcription in a cell-based luciferase reporter assay. We furthermore investigated the impact of the mutant protein on cell migration rate. Unlike wildtype ETV4, the Arg415His mutant failed to rescue cell migration defects observed in two ETV4 knock-down cell-lines. We therefore identified and functionally characterized a recessive mutation in ETV4 in a human patient with CAKUT. We hypothesize that the pathomechanism of this mutation could be via loss of the transcriptional function of ETV4, and a resulting abrogation of GDNF/RET/ETV4 signaling pathway.

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.

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/EBPα 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/EBPα binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.

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.

Hepatocyte transplantation provides an alternative cellular therapy to orthotopic liver transplantation for the treatment of fulminant hepatic failure and hereditary liver disease. Unfortunately there is a serious limitation to the treatment of liver diseases by both liver and hepatocyte transplantation, namely the shortage of organ donors. Therefore, to overcome the problem of organ shortage, additional sources of hepatocytes must be found. Alternative sources of hepatocytes for transplantation have been proposed and these include immortalized cells, stem cells (both embryonic and adult) and differentiated cell types from non-hepatic sources (e.g. the pancreas). The aim of this review is to summarize current knowledge of embryonic and adult stem cell differentiation towards the hepatic phenotype and to examine sources of hepatocytes from developmentally related tissues (e.g. the pancreas).

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.

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).

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.

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.

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.

Betacellulin, a member of the epidermal growth factor family, is expressed in the pancreas and is thought to regulate differentiation of beta cells during development. The aim of the present study was to investigate the effects of exogenous betacellulin on the development of the mouse embryonic pancreas.We used an in vitro culture model system based on the isolation and culture of the dorsal embryonic pancreas from day 11.5 embryos. Cultures were treated for up to 10 days with 10 ng/ml betacellulin and then analysed for changes in the expression of pancreatic exocrine, endocrine and ductal markers.Pancreases developed in culture and expressed the full complement of exocrine (both acinar and ductal) and endocrine cell types. Betacellulin enhanced branching morphogenesis and the proliferation of mesenchyme, increased Pdx1 and insulin production and inhibited the production of the exocrine cell marker amylase and the endocrine hormone glucagon.These results suggest betacellulin has distinct and separate effects on exocrine, endocrine and ductal differentiation. In the future, betacellulin could perhaps be utilised to increase the production of beta cells from embryonic pancreatic tissue for therapeutic transplantation.