Janet Rossant

Placental development: Lessons from mouse mutants

The placenta is the first organ to form during mammalian embryogenesis. Problems in its formation and function underlie many aspects of early pregnancy loss and pregnancy complications in humans. Because the placenta is critical for survival, it is very sensitive to genetic disruption, as reflected by the ever-increasing list of targeted mouse mutations that cause placental defects. Recent studies of mouse mutants with disrupted placental development indicate that signalling interactions between the placental trophoblast and embryonic cells have a key role in placental morphogenesis. Furthering our understanding of mouse trophoblast development should provide novel insights into human placental function.

Interaction between Oct3/4 and Cdx2 Determines Trophectoderm Differentiation

Trophectoderm (TE), the first differentiated cell lineage of mammalian embryogenesis, forms the placenta, a structure unique to mammalian development. The differentiation of TE is a hallmark event in early mammalian development, but molecular mechanisms underlying this first differentiation event remain obscure. Embryonic stem (ES) cells can be induced to differentiate into the TE lineage by forced repression of the POU-family transcription factor, Oct3/4. We show here that this event can be mimicked by overexpression of Caudal-related homeobox 2 (Cdx2), which is sufficient to generate proper trophoblast stem (TS) cells. Cdx2 is dispensable for trophectoderm differentiation induced by Oct3/4 repression but essential for TS cell self-renewal. In preimplantation embryos, Cdx2 is initially coexpressed with Oct3/4 and they form a complex for the reciprocal repression of their target genes in ES cells. This suggests that reciprocal inhibition between lineage-specific transcription factors might be involved in the first differentiation event of mammalian development.

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism.

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.

Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta.

Classical endocrine studies have shown that steroid hormones are required for the maintenance of pregnancy and placental viability. The oestrogen-receptor-related receptor β (ERR-β) is an orphan member of the superfamily of nuclear hormone receptors. Although ERR-β is homologous to the oestrogen receptor and binds the oestrogen response element, it is not activated by oestrogens. Expression of ERR-β during embryogenesis defines a subset of extra-embryonic ectoderm that subsequently forms the dome of the chorion, suggesting that ERR-β may be involved in early placental development. Homozygous mutant embryos generated by targeted disruption of the Estrrb gene have severely impaired placental formation, and die at 10.5 days post-coitum. The mutants display abnormal chorion development associated with an overabundance of trophoblast giant cells and a severe deficiency of diploid trophoblast. The phenotype can be rescued by aggregation of Estrrb mutant embryos with tetraploid wild-type cells, which contribute exclusively to extra-embryonic tissues. Our results indicate that ERR-β has an important role in early placentation, and suggest that an inductive signal originating from or modified by the chorion is required for normal trophoblast proliferation and differentiation.

FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst

Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.

Genomic imprinting of Mash2, a mouse gene required for trophoblast development

The mouse gene Mash2 encodes a transcription factor required for development of trophoblast progenitors. Mash2− homozygous mutant embryos die at 10 days post–coitum from placental failure. Here we show that Mash2 is genomically imprinted. First, Mash2+/− embryos inheriting a wild–type allele from their father die at the same stage as −/− embryos, with a similar placental phenotype. Second, the Mash2 paternal allele is initially expressed by groups of trophoblast cells at 6.5 and 7.5 days post–coitum, but appears almost completely repressed by 8.5 days post–coitum. Finally, we have genetically and physically mapped Mash2 to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin–2, insulin–like growth factor–2 and H19.

Cell and molecular regulation of the mouse blastocyst.

Animals use diverse strategies to specify tissue lineages during development. A common strategy is to partition maternally supplied and localized lineage determinants into progenitor cells. The mouse embryo appears to use a different, more regulative strategy to specify the first three lineages: the epiblast (EPI: future embryo), the trophectoderm (TE: future placenta), and the primitive endoderm (PE: future yolk sac). These lineages are specified during two successive differentiation steps leading to formation of the blastocyst. Here, we review classic and contemporary models of early lineage specification in the mouse, and describe recent efforts to understand the molecular regulation of these events. We describe evidence that trophectoderm differentiation bears resemblance to the process of epithelialization and describe the importance of apical/basal protein complexes in regulating this process. Next, we present a revised model of PE specification, and describe evidence that PE cells in the inner cell mass sort out to occupy their ultimate position on the surface of the EPI. Finally, we describe factors that reinforce these lineages and three distinct stem cell types that can be isolated from them. Together, these mechanisms guide the differentiation of the first lineages of the mouse and thereby set up tissues that will be important for the first steps of embryonic body patterning. Developmental Dynamics 235:2301–2314, 2006.

Stem cells from the Mammalian blastocyst.

Early differentiation of the mammalian embryo leads to the development of two distinct lineages—the inner cell mass (ICM) and the trophectoderm. Cells of the ICM are pluripotent and give rise to all tissues of the fetus, while trophectoderm cells are restricted in their potential to the trophoblast cell layers of the placenta. In the mouse, apparently immortal stem cell lines can be obtained from both cell types. These cell lines, embryonic stem (ES) cells and trophoblast stem (TS) cells, are morphologically and molecularly distinct and depend on different signaling pathways for their maintenance. They also show different cell fates when introduced into early embryos to generate chimeras. However, a change in the levels of expression of a key regulator of pluripotency, Oct4, can push ES cells towards the TS phenotype, when grown in TS cell conditions. Stem cell potential in the early embryo thus appears to depend on a combination of the levels of expression of key intrinsic regulators and the appropriate extrinsic environmental factors. Manipulation of both intrinsic and extrinsic regulators may be needed to reveal the full potential of stem cells from other stages of development and the adult.

Making the blastocyst: lessons from the mouse

Mammalian preimplantation development, which is the period extending from fertilization to implantation, results in the formation of a blastocyst with three distinct cell lineages. Only one of these lineages, the epiblast, contributes to the embryo itself, while the other two lineages, the trophectoderm and the primitive endoderm, become extra-embryonic tissues. Significant gains have been made in our understanding of the major events of mouse preimplantation development, and recent discoveries have shed new light on the establishment of the three blastocyst lineages. What is less clear, however, is how closely human preimplantation development mimics that in the mouse. A greater understanding of the similarities and differences between mouse and human preimplantation development has implications for improving assisted reproductive technologies and for deriving human embryonic stem cells.

Fibroblast growth factors in mammalian development

Polypeptide growth factors are secreted signalling molecules that function as intercellular communicators. Detailed analyses of the expression and function of members of the fibroblast growth factor (FGF) family and their recepotors have demonstrated that the FGF signalling pathways play essential roles in regulating cellular proliferation, differentiation and tissue patterning during vertebrate embryogenesis. Recent studies on the molecular basis of human dysmorphic syndromes have revealed that aberrant FGF signalling during limb and skeletal development can lead to pathogenesis.