To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh.

*Holoprosencephaly (HPE) is a devastating forebrain abnormality with a range of morphological defects characterized by loss of midline tissue. In the telencephalon, the embryonic precursor of the cerebral hemispheres, specialized cell types form a midline that separates the hemispheres. In the present study, deletion of the BMP receptor genes, Bmpr1b and Bmpr1a, in the mouse telencephalon results in a loss of all dorsal midline cell types without affecting the specification of cortical and ventral precursors. In the holoprosencephalic Shh -/-mutant, by contrast, ventral patterning is disrupted, whereas the dorsal midline initially forms. This suggests that two separate developmental mechanisms can underlie the ontogeny of HPE. The Bmpr1a;Bmpr1b mutant provides a model for a subclass of HPE in humans: midline inter-hemispheric HPE.

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...