Naïve human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandable naïve extra-embryonic endoderm.

Embryonic stem cells (ESCs) exist in at least two states that transcriptionally resemble different stages of embryonic development. Naïve ESCs resemble peri-implantation stages and primed ESCs the pre-gastrulation epiblast. In mouse, primed ESCs give rise to definitive endoderm in response to the pathways downstream of Nodal and Wnt signalling. However, when these pathways are activated in naïve ESCs, they differentiate to a cell type resembling early primitive endoderm (PrE), the blastocyst-stage progenitor of the extra-embryonic endoderm. Here, we apply this context dependency to human ESCs, showing that activation of Nodal and Wnt signalling drives the differentiation of naïve pluripotent cells toward extra-embryonic PrE, or hypoblast, and these can be expanded as an in vitro model for naïve extra-embryonic endoderm (nEnd). Consistent with observations made in mouse, human PrE differentiation is dependent on FGF signalling in vitro, and we show that, by inhibiting FGF receptor signalling, we can simplify naïve pluripotent culture conditions, such that the inhibitor requirements closer resemble those used in mouse. The expandable nEnd cultures reported here represent stable extra-embryonic endoderm, or human hypoblast, cell lines.This article has an associated 'The people behind the papers' interview.

Long-Range Signaling Activation and Local Inhibition Separate the Mesoderm and Endoderm Lineages.

Specification of the three germ layers by graded Nodal signaling has long been seen as a paradigm for patterning through a single morphogen gradient. However, by exploiting the unique properties of the zebrafish embryo to capture the dynamics of signaling and cell fate allocation, we now demonstrate that Nodal functions in an incoherent feedforward loop, together with Fgf, to determine the pattern of endoderm and mesoderm specification. We show that Nodal induces long-range Fgf signaling while simultaneously inducing the cell-autonomous Fgf signaling inhibitor Dusp4 within the first two cell tiers from the margin. The consequent attenuation of Fgf signaling in these cells allows specification of endoderm progenitors, while the cells further from the margin, which receive Nodal and/or Fgf signaling, are specified as mesoderm. This elegant model demonstrates the necessity of feedforward and feedback interactions between multiple signaling pathways for providing cells with temporal and positional information.

FoxH1 mediates a Grg4 and Smad2 dependent transcriptional switch in Nodal signaling during Xenopus mesoderm development.

In the vertebrate blastula and gastrula the Nodal pathway is essential for formation of the primary germ layers and the organizer. Nodal autoregulatory feedback potentiates signaling activity, but mechanisms limiting embryonic Nodal ligand transcription are poorly understood. Here we describe a transcriptional switch mechanism mediated by FoxH1, the principle effector of Nodal autoregulation. FoxH1 contains a conserved engrailed homology (EH1) motif that mediates direct binding of groucho-related gene 4 (Grg4), a Groucho family corepressor. Nodal-dependent gene expression is suppressed by FoxH1, but enhanced by a FoxH1 EH1 mutant, indicating that the EH1 motif is necessary for repression. Grg4 blocks Nodal-induced mesodermal gene expression and Nodal autoregulation, suggesting that Grg4 limits Nodal pathway activity. Conversely, blocking Grg4 function in the ectoderm results in ectopic expression of Nodal target genes. FoxH1 and Grg4 occupy the Xnr1 enhancer, and Grg4 occupancy is dependent on the FoxH1 EH1 motif. Grg4 occupancy at the Xnr1 enhancer significantly decreases with Nodal activation or Smad2 overexpression, while FoxH1 occupancy is unaffected. These results suggest that Nodal-activated Smad2 physically displaces Grg4 from FoxH1, an essential feature of the transcriptional switch mechanism. In support of this model, when FoxH1 is unable to bind Smad2, Grg4 occupancy is maintained at the Xnr1 enhancer, even in the presence of Nodal signaling. Our findings reveal that FoxH1 mediates both activation and repression of Nodal gene expression. We propose that this transcriptional switch is essential to delimit Nodal pathway activity in vertebrate germ layer formation.

The transcription factor Vox represses endoderm development by interacting with Casanova and Pou2.

Endoderm and mesoderm are both formed upon activation of Nodal signaling but how endoderm differentiates from mesoderm is still poorly explored. The sox-related gene casanova (sox32) acts downstream of the Nodal signal, is essential for endoderm development and requires the co-factor Pou2 (Pou5f1, Oct3, Oct4) in this process. Conversely, BMP signals have been shown to inhibit endoderm development by an as yet unexplained mechanism. In a search for Casanova regulators in zebrafish, we identified two of its binding partners as the transcription factors Pou2 and Vox, a member of the Vent group of proteins also involved in the patterning of the gastrula. In overexpression studies we show that vox and/or Vent group genes inhibit the capacity of Casanova to induce endoderm, even in the presence of its co-factor Pou2, and that Vox acts as a repressor in this process. We further show that vox, but not other members of the Vent group, is essential for defining the proper endodermal domain size at gastrulation. In this process, vox acts downstream of BMPs. Cell fate analysis further shows that Vox plays a key role downstream of BMP signals in regulating the capacity of Nodal to induce endoderm versus mesoderm by modulating the activity of the Casanova/Pou2 regulatory system.

Subnuclear development of the zebrafish habenular nuclei requires ER translocon function.

The dorsal habenular nuclei (Dh) of the zebrafish are characterized by significant left-right differences in gene expression, anatomy, and connectivity. Notably, the lateral subnucleus of the Dh (LsDh) is larger on the left side of the brain than on the right, while the medial subnucleus (MsDh) is larger on the right compared to the left. A screen for mutations that affect habenular laterality led to the identification of the sec61a-like 1(sec61al1) gene. In sec61al1(c163) mutants, more neurons in the LsDh and fewer in the MsDh develop on both sides of the brain. Generation of neurons in the LsDh occurs more rapidly and continues for a longer time period in mutants than in WT. Expression of Nodal pathway genes on the left side of the embryos is unaffected in mutants, as is the left sided placement of the parapineal organ, which promotes neurogenesis in the LsDh of WT embryos. Ultrastructural analysis of the epithalamus indicates that ventricular precursor cells, which form an epithelium in WT embryos, lose apical-basal polarity in sec61al1(c163) mutants. Our results show that in the absence of sec61al1, an excess of precursor cells for the LsDh exit the ventricular region and differentiate, resulting in formation of bilaterally symmetric habenular nuclei.

Signalling pathways in vasculogenic mimicry.

Solid tumour growth is dependent on the development of an adequate blood supply. For years, sprouting angiogenesis has been considered an exclusive mechanism of tumour vascularization. However, over the last years, several other mechanisms have been identified, including vessel-co-option, intussusception, recruitment of endothelial precursor cells (EPCs) and even mechanisms that do not involve endothelial cells, a process called vasculogenic mimicry (VM). The latter describes a mechanism by which highly aggressive tumour cells can form vessel-like structures themselves, by virtue of their high plasticity. VM has been observed in several tumour types and its occurrence is strongly associated with a poor prognosis. This review will focus on signalling molecules and cascades involved in VM. In addition, we will discuss the presence of VM in relation to ongoing cancer research. Finally, we describe the clinical significance of VM regarding anti-angiogenesis treatment modalities.

Left-right asymmetric expression of Pitx is regulated by the asymmetric Nodal signaling through an intronic enhancer in Ciona intestinalis.

Left-right asymmetric expression of Pitx as well as of Nodal has been observed in some ascidian species, but a mechanism that regulates the asymmetric expression of Pitx remains largely unclear. We addressed the transcription regulatory mechanism of the left-right asymmetric expression of Pitx gene in the ascidian, Ciona intestinalis. We first identified an intronic enhancer that drives Ci-Pitx left-sided expression in the epidermis and found that a single FoxH1 binding site present in this enhancer is essential for its activity. Also, we have shown that the enhancer requires Nodal signaling to drive the Ci-Pitx expression in the left epidermis. In C. intestinalis, left-sided expression of the Nodal gene has not been reported so far. We have confirmed that Ci-Nodal is expressed in the left epidermis of embryos at the tailbud stage only when they are allowed to develop within the chorion. Then, to test the importance of the FoxH1 binding site, we carried out knock down experiments using morpholino antisense oligonucleotides against Ci-FoxHa, the only FoxH gene that is expressed in the same stage as the left-sided expression of Ci-Pitx is observed. Knocking down of the function of Ci-FoxHa led to the down regulation of the expression of Ci-Pitx in the left epidermis. The present results suggest that the regulatory mechanism controlling the left-right asymmetric expression of the Pitx genes is well conserved between C. intestinalis and vertebrates.

Xnrs and activin regulate distinct genes during Xenopus development: activin regulates cell division.

BACKGROUND: The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type beta family such as Vg1, activin B, the nodal-related proteins and derrière. METHODOLOGY AND PRINCIPLE FINDINGS: Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17alpha/beta, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited. CONCLUSIONS/SIGNIFICANCE: These observations reveal distinct functions for these two classes of the TGF-beta family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation.