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Retinoic acid has spectacular teratogenic effects on the development of vertebrate embryos, but whether this has significance for the normal mechanisms of development remains an open question. Recent results from the analyses of Hox gene regulation and the phenotypes of mice mutant for the components of the retinoid signaling pathway suggest that retinoic acid is involved in the genesis of pattern in vivo.

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.

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 mater-nal administration of teratogenic doses of RA on embry-onic 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 RAβ 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 hind-brain to posterior hindbrain, specifically, a transforma-tion of rhombomeres 1 to 3 towards rhombomere 4 iden-tity 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 mediat-ing 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.

Recent evidence from studies in both amphibians and mammals suggest that the fibroblast growth factor (FGF) family of signaling molecules and their receptors may play regulatory roles during early embryogenesis. We have used both standard and whole-mount in situ hybridization techniques to analyze the temporal and spatial expression patterns of the murine fibroblast growth factor receptor-1 (FGFR-1) in order to help define the role of FGFs in the processes of gastrulation and segmentation. FGFR-1 transcripts were detected in the primitive ectoderm of the egg cylinder embryo but not in the primitive endoderm or ectoplacental cone. During gastrulation, FGFR-1 mRNA were expressed at high levels in the migrating embryonic mesoderm of the mid-streak-stage embryo. Late-streak-stage embryos displayed strong expression in both the embryonic ectoderm and mesoderm. Within the ectodermal lineage, FGFR-1 mRNA later became localized to the neural ectoderm during its formation and continued to be expressed at high levels throughout neural development. In the mesodermal lineage, FGFR-1 transcripts became concentrated in the posterior medial mesoderm of the embryo as it condensed to form paraxial mesoderm. The most striking expression patterns were observed before and during segmentation when FGFR-1 was strongly expressed in the presomitic mesoderm and the rostral half of the newly formed somites. The patterns of expression are consistent with a role for FGFR-1 in posterior mesoderm formation. FGFR-1 may also play significant roles in the formation of neural ectoderm and the early events that establish compartments within the developing somites.

The N-myc proto-oncogene encodes a putative transcription factor that has been postulated to be involved in the control of differentiation in a number of lineages at various stages during mammalian embryogenesis. We have generated a leaky mutation in N-myc by gene targeting in embryonic stem cells. In this allele, the neo(r) gene was inserted into the first intron of N-myc, in such a way that alternative splicing around this insertion could result in the generation of a normal N-myc transcript in addition to a mutant transcript. Mice homozygous for this mutation died immediately after birth owing to an inability to oxygenate their blood. Histological examination revealed a marked underdevelopment in the lung airway epithelium, resulting in a decreased respiratory surface area. Analysis of N-myc expression in wild-type and homozygous mutant embryonic lungs suggests that N-myc is required for the proliferation of the lung epithelium in response to local inductive signals emanating from the lung mesenchyme. Homozygous mutant embryos were slightly smaller than normal and also had a marked reduction in spleen size, whereas other tissues that normally express N-myc appeared to be unaffected by the mutation. Molecular analysis revealed that normal N-myc transcripts were found in tissues from homozygous mutant embryos. Different tissues expressed the normal N-myc transcript at different levels relative to those observed in wild-type embryos, with the lowest levels being observed in the lungs. These results illustrate one way in which gene targeting can be used to generate partial loss-of-function mutations and support the importance of generating a series of alleles at a given locus to elucidate the various different functions of a gene during development.

Background The metabolic function of PEPCK-C is not fully understood; deletion of the gene for the enzyme in mice provides an opportunity to fully assess its function. Methods The gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C) was deleted in mice by homologous recombination (PEPCK-C-/- mice) and the metabolic consequences assessed. Results PEPCK-C-/- mice became severely hypoglycemic by day two after birth and then died with profound hypoglycemia (12 mg/dl). The mice had milk in their stomachs at day two after birth and the administration of glucose raised the concentration of blood glucose in the mice but did not result in an increased survival. PEPCK-C-/- mice have two to three times the hepatic triglyceride content as control littermates on the second day after birth. These mice also had an elevation of lactate (2.5 times), β-hydroxybutyrate (3 times) and triglyceride (50%) in their blood, as compared to control animals. On day two after birth, alanine, glycine, glutamine, glutamate, aspartate and asparagine were elevated in the blood of the PEPCK-C-/- mice and the blood urea nitrogen concentration was increased by 2-fold. The rate of oxidation of [2-14C]-acetate, and [5-14C]-glutamate to 14CO2 by liver slices from PEPCK-C-/- mice at two days of age was greatly reduced, as was the rate of fatty acid synthesis from acetate and glucose. As predicted by the lack of PEPCK-C, the concentration of malate in the livers of the PEPCK-C-/- mice was 10 times that of controls. Conclusion We conclude that PEPCK-C is required not only for gluconeogenesis and glyceroneogenesis but also for cataplerosis (i.e. the removal of citric acid cycle anions) and that the failure of this process in the livers of PEPCK-C-/- mice results in a marked reduction in citric acid cycle flux and the shunting of hepatic lipid into triglyceride, resulting in a fatty liver.

The distribution of cytoplasmic poly(A)+RNA between subribosomal (

Dorsal dermis and epaxial muscle have been shown to arise from the central dermomyotome in the chick. En1 is a homeobox transcription factor gene expressed in the central dermomyotome. We show by genetic fate mapping in the mouse that En1-expressing cells of the central dermomyotome give rise to dorsal dermis and epaxial muscle and, unexpectedly, to interscapular brown fat. Thus, the En1-expressing central dermomyotome normally gives rise to three distinct fates in mice. Wnt signals are important in early stages of dermomyotome development, but the signal that acts to specify the dermal fate has not been identified. Using a reporter transgene for Wnt signal transduction, we show that the En1-expressing cells directly underneath the surface ectoderm transduce Wnt signals. When the essential Wnt transducer β-catenin is mutated in En1 cells, it results in the loss of Dermo1-expressing dorsal dermal progenitors and dermis. Conversely, when β-catenin was activated in En1 cells, it induces Dermo1 expression in all cells of the En1 domain and disrupts muscle gene expression. Our results indicate that the mouse central dermomyotome gives rise to dermis, muscle, and brown fat, and that Wnt signalling normally instructs cells to select the dorsal dermal fate.