Identification of signaling pathways in early mammary gland development by mouse genetics

The mammary gland develops as an appendage of the ectoderm. The prenatal stage of mammary development is hormone independent and is regulated by sequential and reciprocal signaling between the epithelium and the mesenchyme. A number of recent studies using human and mouse genetics, in particular targeted gene deletion and transgenic expression, have identified some of the signals that control specific steps in development. This process involves cell specification and proliferation, reciprocal tissue interactions and cell migration. Since some of these events are recapitulated during tumorigenesis, an understanding of these signaling pathways may contribute to the development of targeted therapies and novel drugs

Actions of parathyroid hormone related peptide in mouse parietal endoderm formation

Summary Since about a decade, several reports have strongly suggested a role for parathyroid hormone related peptide (PTHrP) in the formation of parietal endoderm (PE) in the mouse embryo. This thesis is aimed first at elucidating the biological significance of parathyroid hormone related peptide (PTHrP) signaling via its type I receptor (PTH(rP)-RI) in PE formation, and secondly at finding new molecules downstream of this receptor that induce PE formation. In the first chapter a literature review about the formation of PE from primitive and visceral endoderm (PrE and VE) in the mouse embryo is given. In that chapter it is also illustrated that the changes in cell contacts, morphology and secretory properties, as well as the acquisition of migration of the cells during PE formation are indicative for an epithelio-mesenchymal transition (EMT). We therefore postulate that PE formation is the first EMT in mouse embryogenesis. In chapter 2, we demonstrate that PTHrP signaling is sufficient to induce the transformation of an epithelial to mesenchymal cell type during PE formation in the F9 model system. In addition, we demonstrate that PTHrP enhances the migration of endoderm away from F9 embryoid bodies. A detailed analysis of the PE of receptor null mutants is described here, revealing that this tissue has an aberrant, epithelial instead of mesenchymal morphology, and lacks expression of the PE marker thrombomodulin in contrast to an apparently normal phenotype reported previously. These results indicate that PTHrP signaling, although not required for migration, is necessary for the proper differentiation of PE. In that chapter the downregulation of connexin43 (Cx43), the most abundant gap junctional molecule in F9 cells and the extraembryonic endoderm in vivo, is shown as a result of PTHrP signaling in PE formation. In chapter 3 we further investigated the impor-tance of Cx43 and the function of gap junctions in the formation of PE, and found that their downregulation is a consequence of rather than a prerequisite for PE differentiation. We postulate that the high expression of Cx43 and high extent of gap junctional coupling in VE serves a function in metabolite exchange between the mother and the early embryo. Chapter 4 describes the identification of the zinc finger transcription factor snail as a direct target of PTHrP signaling in F9 PrE signaling. It is furthermore shown there, that snail is also upregulated during the earliest formation of PE ex vivo and in vivo, clearly suggesting an important role in PE formation. This is further investigated in chapter 5, where it is described that high levels of expression of snail are sufficient to induce the dis-integration of adherens junctions in F9 PrE, by loss of membrane localized expression of E-cadherin and ß-catenin. Moreover, it is demonstrated that F9 VE cells overexpressing snail display enhanced migration, more cell dispersion and assume a rounded morphology, sug-gesting that elevated expression of snail is sufficient to at least induce the morphological transition of an epithelial (PrE or VE) to a mesenchymal cell with typical PE morphology. In situ hybridization results demonstrate a lack of Sna expression in the distal PE region in PTH(rP)-RI knockout embryos, strongly suggesting that it is due to a lack of Sna function that these cells have not acquired a mesenchymal phenotype. Finally, in chapter 6, prelimi-nary data of a promoter analysis of the snail gene are shown, which are suggestive for a pu-tative functional cyclic AMP responsive element in the promoter, which may confer the sig-nal induced by PTHrP. In this chapter, all results are discussed and a model for a function of PTHrP in the downregulation of the cell-cell adhesion system in the earliest phase of PE formation is presented. Our finding that the PE of PTH(rP)-RI null mutant embryos displays an epithelial instead of mesenchymal morphology, and does not express PE-specific enhanced levels of thrombomodulin, suggests that this PE does not function properly. This may explain the growth retardation and mortality of the embryos. Given the similarities between PE formation and (breast) tumor progression with respect to the presence of PTHrP, expression of Sna and lack of E-cadherin expression at the cell membrane, our findings may also open avenues to better understand (breast) tumor progression