Ronald A. Conlon

Members of the Notch family of transmembrane receptors mediate a number of developmental decisions in invertebrates. In order to study Notch function in a vertebrate organism, we have mutated the Notch1 gene of the mouse. Notch1 gene function is required for embryonic survival in the second half of gestation. In the first half of gestation, we have found no effect of the mutation on the normal programs of neurogenesis, myogenesis or apoptosis. We conclude that Notch1 function is not essential for these processes, at least in early postimplantation development. However, we have found that somitogenesis is delayed and disorganized in Notch1 mutant embryos. We propose that Notch1 normally coordinates the process of somitogenesis, and we provide a model of how this might occur.

ABSTRACT We have used RT-PCR to screen pluripotent murine embryonic stem cells to identify receptor tyrosine kinases (RTKs) potentially involved in the determination or differentiation of cell lineages during early mouse development. Fourteen different tyrosine kinase sequences were identified. The expression patterns of four RTKs have been examined and all are expressed in the mouse embryo during, or shortly after, gastrulation. We report here the detailed expression pattern of one such RTK, the flt-related gene flk-1. In situ hybridization analysis of the late primitive streak stage embryo revealed that flk-1 was expressed in the proximallateral embryonic mesoderm; tissue fated to become heart. By headfold stages, staining was confined to the endocardial cells of the heart primordia as well as to the blood islands of the visceral yolk sac and the developing allantois. Patchy, speckled staining was detected in the endothelium of all the major embryonic and extraembryonic blood vessels as they formed. During early organogenesis, expression was detected in the blood vessels of highly vascularized tissues such as the brain, liver, lungs and placenta. Since flk-1 was expressed in early mesodermal cells prior to any morphological evidence for endothelial cell differentiation (vasculogenesis), as well as in cells that form blood vessels from preexisting ones (angiogenesis), it appears to be a very early marker of endothelial cell precursors. We have previously reported that another novel RTK, designated tek, was expressed in differentiating endothelial cells. We show here that flk-1 transcripts are expressed one full embryonic day earlier than the first tek transcripts. The expression of these two RTKs appear to correlate with the specification and early differentiation of the endothelial cell lineage respectively, and therefore may play important roles in the establishment of this lineage.

Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. Here, we show that neural stem cells are generated independent of RBP-Jkappa, a key molecule in Notch signaling, by using RBP-Jkappa(-/-) embryonic stem cells in an embryonic stem cell-derived neurosphere assay. However, Notch pathway molecules are essential for the maintenance of neural stem cells; they are depleted in the early embryonic brains of RBP-Jkappa(-/-) or Notch1(-/-) mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1(+/-) adult brains. Both neuronal and glial differentiation in vitro were enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch.

The Notch pathway functions in multiple cell fate determination processes in invertebrate embryos, including the decision between the neuroblast and epidermoblast lineages in Drosophila. In the mouse, targeted mutation of the Notch pathway genes Notch1 and RBP-Jk has demonstrated a role for these genes in somite segmentation, but a function in neurogenesis and in cell fate decisions has not been shown. Here we show that these mutations lead to altered expression of the Notch signalling pathway homologues Hes-5, Mash-1 and Dll1, resulting in enhanced neurogenesis. Precocious neuronal differentiation is indicated by the expanded expression domains of Math4A, neuroD and NSCL-1. The RBP-Jk mutation has stronger effects on expression of these genes than does the Notch1 mutation, consistent with functional redundancy of Notch genes in neurogenesis. Our results demonstrate conservation of the Notch pathway and its regulatory mechanisms from fly to mouse, and support a role for the murine Notch signalling pathway in the regulation of neural stem cell differentiation.

Exogenous retinoic acid (RA) has teratogenic effects on vertebrate embryos and alters Hox-C gene expression in vivo and in vitro. We wish to examine whether RA has a role in the normal regulation of Hox-C genes, and whether altered Hox-C gene expression in response to RA leads to abnormal morphology. The expression of 3' Hox-2 genes (Hox-2.9, Hox-2.8, Hox-2.6 and Hox-2.1) and a 5' gene (Hox-2.5) were examined by whole-mount in situ hybridization on embryos 4 hours after maternal administration of teratogenic doses of RA on embryonic day 7 to 9. The expression of the 3' Hox-2 genes was found to be ectopically induced in anterior regions in a stage-specific manner. The Hox-2.9 and Hox-2.8 genes were induced anteriorly in the neurectoderm in response to RA on day 7 but not at later stages. Expression of Hox-2.6 and Hox-2.1 was ectopically induced anteriorly in neurectoderm in response to RA on day 8. Hox-2.1 remained responsive on day 9, whereas Hox-2.6 was no longer responsive at this stage. The expression of the 5' gene Hox-2.5 was not detectably altered at any of these stages by RA treatments. We also examined the response of other genes whose expression is spatially regulated in early embryos. The expression of En-2 and Wnt-7b was not detectably altered by RA, whereas RAR beta expression was induced anteriorly by RA on day 7 and 8. Krox-20 expression was reduced in a stage- and region-specific manner by RA. The ectopic anterior expression of Hox-2.8 and Hox-2.9 induced by RA on day 7 was persistent to day 8, as was the altered expression of Krox-20. The altered pattern of expression of these genes in response to RA treatment on day 7 may be indicative of a transformation of anterior hindbrain to posterior hindbrain, specifically, a transformation of rhombomeres 1 to 3 towards rhombomere 4 identity with an anterior expansion of rhombomere 5. The ectopic expression of the 3' Hox-2 genes in response to RA is consistent with a role for these genes in mediating the teratogenic effects of RA; the rapid response of the Hox-C genes to RA is consistent with a role for endogenous RA in refining 3' Hox-C gene expression boundaries early in development.