Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.ethylnitrosourea ͉ hedgehog ͉ intraflagellar transport ͉ mesoderm ͉ neural tube closure M ammalian embryogenesis differs fundamentally from the development of other animal models. Localized maternal components are not essential for axis specification in mammals (1), whereas the establishment of the body axes in Drosophila, Caenorhabditis elegans, and zebrafish relies on localization of maternal determinants (2-4). Mammalian development has a unique requirement for complex interactions between embryonic and uterine tissues, and the first cell fate decision in the mouse embryo is the choice between embryonic and extraembryonic lineages (5). After implantation, germ layer organization and tissue specification in the mouse embryo depend on coupled morphogenetic movements and intercellular signals, processes not paralleled in invertebrate embryos. A standard approach to study the genetic control of mouse embryogenesis has been to inactivate evolutionarily conserved genes by targeted mutagenesis; this approach may overlook components that are of particular importance in mammals. In contrast, phenotypebased screens in the mouse have the potential to identify the molecules that control mammalian-specific events.Phenotype-based screens depend on the ability to induce a large number of random mutations in germ cells and on rapid identification of mutants of interest. It has been known for 25 years that ethylnitrosourea (ENU) is an extremely potent mutagen in the mouse (6, 7); and a screen of the progeny of only 700 F 1 progeny of ENU-treated animals should yield an average of one allele of each gene in the genome. We previously described a pilot phenotype-based screen to identify recessive mutations that produce easily visible disruptions in the morphology of the midgestation mouse embryo (8). Similar phenotype-based approaches in other laboratories that focused on later stages of embryonic and fet...

Math1 is a basic helix-loop-helix transcription factor

Metazoans use diverse and rapidly evolving mechanisms to determine sex. In Drosophila melanogaster an X-chromosome-counting mechanism determines the sex of an individual by regulating the master switch gene, Sex-lethal (Sxl). The X-chromosome dose is communicated to Sxl by a set of X-linked signal elements (XSEs), which activate transcription of Sxl through its 'establishment' promoter, SxlPe. Here we describe a new XSE called sisterlessC (sisC) whose mode of action differs from that of previously characterized XSEs, all of which encode transcription factors that activate SxlPe directly. In contrast, sisC encodes a secreted ligand for the Drosophila Janus kinase (JAK) and 'signal transducer and activator of transcription' (STAT) signal transduction pathway and is allelic to outstretched (os, also called unpaired). We conclude that sisC works indirectly on Sxl through this signalling pathway because mutations in sisC or in the genes encoding Drosophila JAK or STAT reduce expression of SxlPe similarly. The involvement of os in sex determination confirms that secreted ligands can function in cell-autonomous processes. Unlike sex signals for other organisms, sisC has acquired its sex-specific function while maintaining non-sex-specific roles in development, a characteristic that it shares with all other Drosophila XSEs.

Adhesive properties of cells undergoing morphogenetic rearrangements can be regulated either at the cellular level or by altering the environment in which rearrangements occur. Here, we describe the identification of a mutation (my F11 ) in the mouse extracellular matrix component Frem2, and provide evidence that suggests Frem2 expression creates an environment conducive to morphogenetic events. Loss of Frem2 function results in defects in developmental events associated with morphogenetic rearrangements of the vasculature and of tissues arising from all germ layers. The Frem2 transcript is restricted both spatially and temporally and appears in advance of cell rearrangement events. Thus, expression of Frem2 may dynamically alter the extracellular matrix to provide a substrate for cell migration and rearrangements during embryogenesis. extracellular matrix ͉ hemorrhage ͉ Frem ͉ CALX␤ ͉ neural tube defect

The identification and characterization of DNA sequences necessary for proper gene expression have provided insights into gene regulation and generated tools useful for further experimentation. Studies of developmentally regulated genes have demonstrated how transcription factors interact at enhancers to generate restricted patterns of expression during embryogenesis. In vertebrates, the pursuit of such studies has relied on the generation of transgenic mice and thus has been limited by the time and expense required generating and characterizing these mice. The recently developed technique of in ovo electroporation allows the rapid introduction of exogenous DNA into developing chicken embryos. Here we have used this technique to introduce DNA containing murine enhancer/reporter constructs into cells of the chicken neural tube, resulting in appropriate expression of the reporter. This technique has the potential to greatly reduce the effort involved in the study of vertebrate enhancers. Furthermore, we have characterized factors such as timing of electroporation, concentration of DNA, and choice of basal promoters and found that they can influence the degree to which expression of enhancer constructs reflects endogenous gene expression.

Embryonic blood vessel formation is initially mediated through the sequential differentiation, migration, and assembly of endothelial cells (ECs). While many molecular signals that promote vascular development have been identified, little is known about suppressors of this process. In higher vertebrates, including birds and mammals, the vascular network forms throughout the embryonic disk with the exception of a region along the midline. We have previously shown that the notochord is responsible for the generation and maintenance of the avascular midline and that BMP antagonists expressed by this embryonic tissue, including Noggin and Chordin, can mimic this inhibitory role. Here we report that the notochord suppresses the generation of ECs from the mesoderm both in vivo and in vitro. We also report that the notochord diminishes the ability of mature ECs to organize into a primitive plexus. Furthermore, Noggin mimics notochord-based inhibition by preventing mesodermal EC generation and mature EC network formation. These findings suggest that the mesoderm surrounding the midline is competent to give rise to ECs and to form blood vessels, but that notochord derived-BMP antagonists suppress EC differentiation and maturation processes leading to inhibition of midline vessel formation.

The generation of the appropriate types and numbers of mature neurons during the development of the spinal cord requires the careful coordination of patterning, proliferation, and differentiation. In the dorsal neural tube, this coordination is achieved by the combined action of multiple ligands of both the Wnt and TGF-beta families, and their effectors, such as the bHLH proteins. TGF-beta signaling acting through the BMP receptors is necessary for the generation of several dorsal interneuron types. Other TGF-beta ligands expressed in the dorsal neural tube interact with the Activin receptors, which signal via a different set of SMAD proteins than BMPs. The effects of Activin signaling on the developing neural tube have not been described. Here we have activated the Activin signal transduction pathway in a cell-autonomous manner in the developing chick neural tube. We find that a constitutively active Activin receptor promotes differentiation throughout the neural tube. Although most differentiated cell populations are unaffected by Activin signaling, the number of dorsal interneuron 3 (dI3) cells is specifically increased. Our data suggest that Activin signaling may promote the formation of the dI3 precursor cells within a region circumscribed by BMP signaling and that this function is not dependent upon BMP signaling.

In the spinal neural tube, populations of neuronal precursors that express a unique combination of transcription factors give rise to specific classes of neurons at precise locations along the dorsoventral axis. Understanding the patterning mechanisms that generate restricted gene expression along the dorsoventral axis is therefore crucial to understanding the creation of diverse neural cell types. Bone morphogenetic proteins (BMPs) and other transforming growth factor β (TGFβ) proteins are expressed by the dorsal-most cells of the neural tube (the roofplate) and surrounding tissues, and evidence indicates that they play a role in assigning cell identity. We have manipulated the level of BMP signaling in the chicken neural tube to show that BMPs provide patterning information to both dorsal and intermediate cells. BMP regulation of the expression boundaries of the homeobox proteins Pax6, Dbx2 and Msx1 generates precursor populations with distinct developmental potentials. Within the resulting populations, thresholds of BMP act to set expression domain boundaries of developmental regulators of the homeobox and basic helix-loop-helix (bHLH) families, ultimately leading to the generation of a diversity of differentiated neural cell types. This evidence strongly suggests that BMPs are the key regulators of dorsal cell identity in the spinal neural tube.