Sox2(+) adult mouse pituitary cells can self-renew and terminally differentiate in vitro, but their physiological role in vivo and possible contribution to oncogenesis remain largely unknown. Using genetic lineage tracing, we show here that the Sox2(+) cell compartment of both the embryonic and adult pituitary contains stem/progenitor cells that are able to differentiate into all hormone-producing lineages and contribute to organ homeostasis during postnatal life. In addition, we show that targeted expression of oncogenic β-catenin in Sox2(+) cells gives rise to pituitary tumors, but, unexpectedly, the tumor mass is not derived from the Sox2(+) mutation-sustaining cells, suggesting a paracrine role of Sox2(+) cells in pituitary oncogenesis. Our data therefore provide in vivo evidence of a role for Sox2(+) stem/progenitor cells in long-term physiological maintenance of the adult pituitary, and highlight an unexpected non-cell-autonomous role for these cells in the induction of pituitary tumors.

Planar-cell-polarity (PCP) signalling is necessary for initiation of neural tube closure in higher vertebrates. In mice with PCP gene mutations, a broad embryonic midline prevents the onset of neurulation through wide spacing of the neural folds. In order to evaluate the role of convergent extension in this defect, we vitally labelled the midline of loop-tail (Lp) embryos mutant for the PCP gene Vangl2. Injection of DiI into the node, and electroporation of a GFP expression vector into the midline neural plate, revealed defective convergent extension in both axial mesoderm and neuroepithelium, before the onset of neurulation. Chimeras containing both wild-type and Lp-mutant cells exhibited mainly wild-type cells in the midline neural plate and notochordal plate, consistent with a cell-autonomous disturbance of convergent extension. Inhibitor studies in whole-embryo culture demonstrated a requirement for signalling via RhoA-Rho kinase, but not jun N-terminal kinase, in convergent extension and the onset of neural tube closure. These findings identify a cell-autonomous defect of convergent extension, requiring PCP signalling via RhoA-Rho kinase, during the development of severe neural tube defects in the mouse.

Wingless (Wnt)/β-catenin signaling plays an essential role during normal development, is a critical regulator of stem cells, and has been associated with cancer in many tissues. Here we demonstrate that genetic expression of a degradation-resistant mutant form of β-catenin in early Rathke's pouch (RP) progenitors leads to pituitary hyperplasia and severe disruption of the pituitary-specific transcription factor 1-lineage differentiation resulting in extreme growth retardation and hypopituitarism. Mutant mice mostly die perinatally, but those that survive weaning develop lethal pituitary tumors, which closely resemble human adamantinomatous craniopharyngioma, an epithelial tumor associated with mutations in the human β-catenin gene. The tumorigenic effect of mutant β-catenin is observed only when expressed in undifferentiated RP progenitors, but tumors do not form when committed or differentiated cells are targeted to express this protein. Analysis of affected pituitaries indicates that expression of mutant β-catenin leads to a significant increase in the total numbers of pituitary progenitor/ stem cells as well as in their proliferation potential. Our findings provide insights into the role of the Wnt pathway in normal pituitary development and demonstrate a causative role for mutated β-catenin in an undifferentiated RP progenitor in the genesis of murine and human craniopharyngioma.

Little is known about the mechanism by which embryonic liver, lung, and pancreas progenitor cells emerge from the endodermal epithelium to initiate organogenesis. Understanding this process and its genetic control provides insight into ontogeny, developmental abnormalities, and tissue regeneration. We find that shortly after hepatic endoderm cells are specified, they undergo a transition from a columnar, gut morphology to a pseudostratified morphology, with concomitant "interkinetic nuclear migration" (INM) during cell division. INM is a hallmark of pseudostratified epithelia and the process used by neural progenitors to emerge from the neural epithelium. We find that the transition of the hepatic endoderm, but not the neural epithelium, to a pseudostratified epithelium is dependent upon the cell-autonomous activity of the homeobox gene Hex. In the absence of Hex, hepatic endoderm cells survive but maintain a columnar, simple epithelial phenotype and ectopically express Shh and other genes characteristic of the midgut epithelium. Thus, Hex promotes endoderm organogenesis by promoting the transition to a pseudostratified epithelium, which in turn allows hepatoblasts to emerge into the stromal environment and continue differentiating.

SummaryWe generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein -diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells. KeywordsCre; loxP; diphtheria toxin; eGFP; Nkx2.5; Wnt1; genetic ablation; heart; midbrain The role of individual cells in complex tissues or within the whole organism can be examined by specific deletion of appropriate lineages. For example, during embryogenesis specific groups of cells (signalling centres) control the fate of neighbouring cells by emanating diffusible molecules (Meinhardt, 1983;Martinez et al., 1991;Shimamura and Rubenstein, 1997;Placzek and Briscoe, 2005). Mechanical ablation of restricted embryonic regions has been useful in identifying these signalling centres and their role during development (Placzek et al., 1995;Shimamura and Rubenstein, 1997;Thomas and Beddington, 1996). However, these experiments require very precise surgery on the developing embryo, which can often be a difficult task. In adulthood the loss of small numbers of strategically placed cells may result in conditions such as Parkinson's disease (Moore, 2005), Type I diabetes (Butler et al., 2003), and Hirschsprung's disease (Amiel and Lyonnet, 2001). Thus, a versatile system to specifically ablate cells of any lineage during embryogenesis or in adulthood would be of substantial benefit for studies of development, as well as to model human diseases of various aetiologies. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author ManuscriptWith the aim of developing such a system, we generated a ROSA26-eGFP-DTA mouse line, which combines the use of enhanced green fluorescent protein (GFP), diphtheria toxin A subunit (DTA), and Cre recombinase. eGFP is a mutated version of GFP that displays enhanced fluorescence in mammalian cells in vivo and in vitro (Srinivas et al., 2001). Diphtheria toxin is secreted by pathogenic strains of Corynebacterium diphtheriae and is composed of two subunits, A and B. Subunit B is responsible for the internalisation of the toxin upon binding to its receptor. Once inside the cell, subunit A catalyses the inactivation of elongation factor 2, resulting in termination of protein synthesis and apoptosis of the target cell (Maxw...