Smad1 and Smad8 Function Similarly in Mammalian Central Nervous System Development

ABSTRACT Smads 1, 5, and 8 are the intracellular mediators for the bone morphogenetic proteins (BMPs), which play crucial roles during mammalian development. Previous research has shown that Smad1 is important in the formation of the allantois, while Smad5 has been shown to be critical in the process of angiogenesis. To further analyze the BMP-responsive Smads, we disrupted the murine Smad8 gene utilizing the Cre/loxP system. A Smad8 hypomorphic allele (Smad8 Δexon3 ) was constructed that contains an in-frame deletion of exon 3, removing one-third of the MH2 domain and a small portion of the linker region. Xenopus injection assays indicated that this Smad8 deletion allele is still functional but has reduced ventralizing capability compared to the wild type. Although Smad8 Δexon3/Δexon3 embryos are phenotypically normal, homozygotes of another hypomorphic allele of Smad8 (Smad8 3loxP ) containing a neomycin cassette within intron 3, phenocopy an embryonic brain defect observed in roughly 22% of Smad1 +/ − embryos analyzed at embryonic day 11.5. These observations suggest that BMP-responsive Smads have critical functions in the development of the mammalian central nervous system.

Mitochondrial DNA Instability and Peri-Implantation Lethality Associated with Targeted Disruption of Nuclear Respiratory Factor 1 in Mice

ABSTRACT In vitro studies have implicated nuclear respiratory factor 1 (NRF-1) in the transcriptional expression of nuclear genes required for mitochondrial respiratory function, as well as for other fundamental cellular activities. We investigated here the in vivo function of NRF-1 in mammals by disrupting the gene in mice. A portion of the NRF-1 gene that encodes the nuclear localization signal and the DNA-binding and dimerization domains was replaced through homologous recombination by a β-galactosidase–neomycin cassette. In the mutant allele, β-galactosidase expression is under the control of the NRF-1 promoter. Embryos homozygous for NRF-1 disruption die between embryonic days 3.5 and 6.5. β-Galactosidase staining was observed in growing oocytes and in 2.5- and 3.5-day-old embryos, demonstrating that the NRF-1 gene is expressed during oogenesis and during early stages of embryogenesis. Moreover, the embryonic expression of NRF-1 did not result from maternal carryover. While most isolated wild-type and NRF-1+/− blastocysts can develop further in vitro, the NRF-1−/− blastocysts lack this ability despite their normal morphology. Interestingly, a fraction of the blastocysts from heterozygous matings had reduced staining intensity with rhodamine 123 and NRF-1−/− blastocysts had markedly reduced levels of mitochondrial DNA (mtDNA). The depletion of mtDNA did not coincide with nuclear DNA fragmentation, indicating that mtDNA loss was not associated with increased apoptosis. These results are consistent with a specific requirement for NRF-1 in the maintenance of mtDNA and respiratory chain function during early embryogenesis.