Ectoderm

Abstract: The in vitro developmental potential of mouse blastocyst-derived embryonic stem cell lines has been investigated. From 3 to 8 days of suspension culture the cells form complex embryoid bodies with endoderm, basal lamina, mesoderm and ectoderm. Many are morphologically similar to embryos of the 6- to 8-day egg-cylinder stage. From 8 to 10 days of culture about half of the embryoid bodies expand into large cystic structures containing alphafoetoprotein and transferrin, thus being analagous to the visceral yolk sac of the postimplantation embryo. Approximately one third of the cystic embryoid bodies develop myocardium and when cultured in the presence of human cord serum, 30% develop blood islands, thereby exhibiting a high level of organized development at a very high frequency. Furthermore, most embryonic stem cell lines observed exhibit similar characteristics. The in vitro developmental potential of embryonic stem cell lines and the consistency with which the cells express this potential are presented as aspects which open up new approaches to the investigation of embryogenesis.

Abstract: The PATCHED (PTC) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome (BCNS). Functions of PTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation died during embryogenesis and were found to have open and overgrown neural tubes. Two Shh target genes, ptc itself and Gli, were derepressed in the ectoderm and mesoderm but not in the endoderm. Shh targets that are, under normal conditions, transcribed ventrally were aberrantly expressed in dorsal and lateral neural tube cells. Thus Ptc appears to be essential for repression of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation were larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas, abnormalities also seen in BCNS patients.

Abstract: Before gastrulation, the mouse embryo consists of three distinct cell lineages which were established in the blastocyst during the peri-implantation period, that is, epiblast, extraembryonic endoderm, and trophectoderm. The epiblast, from which the entire fetus will form, as well as the extraembryonic mesoderm and amnion ectoderm, is a cup-shaped epithelium apposed on its open end to the extraembryonic ectoderm, a trophectoderm derivative. Both epiblast and extraembryonic ectoderm are covered by visceral endoderm, which is part of the extraembryonic endoderm lineage (Hogan et al. 1994). The primordial germ cells (PGCs) of the mouse embryo are derived from part of the population of epiblast cells that will give rise mainly to the extraembryonic mesoderm. Precursors of the PGCs are located before gastrulation in the extreme proximal region of the epiblast adjacent to the extraembryonic ectoderm, and have descendants not only in the germ line, but also in extraembryonic structures, that is, the allantois, blood islands, and yolk sac mesoderm, as well as both layers of the amnion. At embryonic day (E) 6.0, these precursors lie scattered in a ring that extends up to three cell diameters from the junction with the extraembryonic ectoderm (Lawson and Hage 1994). Early in gastrulation, they converge toward the primitive streak in the posterior of the embryo and translocate through it. Allocation to the germ cell lineage is thought to occur in ∼45 cells around E7.2, after the precursors have passed through the streak and have come to reside in the extraembryonic mesoderm (Lawson and Hage 1994). This is about the time when the putative PGCs can first be identified morphologically in a cluster posterior to the primitive streak in a position that will later become the base of the allantois (Ginsburg et al. 1990). PGCs stain strongly in a characteristic pattern for alkaline phosphatase (AP) activity (Chiquoine 1954), which by this stage is due to tissue nonspecific AP (Hahnel et al. 1990; MacGregor et al. 1995). The PGCs continue to express AP during their proliferation in the developing hindgut and migration into the genital ridges (for review, see Buehr 1997). Transplantation studies have shown that genetically marked distal epiblast cells from pre- and early-primitive streak-stage embryos, which would normally contribute to neuroectoderm and never to the PGCs, can give rise to PGCs and extraembryonic mesoderm when grafted to the proximal epiblast (Tam and Zhou 1996). These results raise the possibility that PGC precursors are induced by extracellular factors and/or cell interactions present locally at the junction between the extraembryonic ectoderm and epiblast. Candidate genes encoding putative germ cell precursor inducing factors are predicted to be expressed in the mouse embryo before and during gastrulation. One such factor is Bone Morphogenetic Protein 4 (Bmp4), a member of the TGFβ superfamily of intercellular signaling proteins (Hogan 1996; Waldrip et al. 1998). Most mouse embryos homozygous for a null mutation in Bmp4 die around gastrulation (∼E6.5) (Winnier et al. 1995). On some genetic backgrounds, however, a proportion of the mutant embryos survive until the early somite stage and show severe defects, particularly in the extraembryonic mesoderm (Winnier et al. 1995). In this paper, we exploit this late phenotype to show that PGC formation absolutely requires Bmp4 signaling. In addition, the size of the founding population of PGCs is significantly reduced in heterozygous mutant embryos. By using a Bmp4–lacZ reporter allele, we have definitively localized Bmp4 expression before gastrulation in the extraembryonic ectoderm and in mid- to late- primitive streak stage embryos in the extraembryonic mesoderm. Thus, Bmp4 is expressed at the right time and in the right place to play a role both in the quantitative induction of PGC precursors in the proximal epiblast and in their allocation to the germ cell lineage in the extraembryonic mesoderm. Furthermore, by analyzing genetic chimeras, we have clearly established a role for Bmp4 in the induction of PGC precursors and demonstrate for the first time that a secreted signal from the extraembryonic ectoderm is required for the normal development of the epiblast.