Genesis最新文献

Paraxial protocadherin (PAPC) is a cell adhesion molecule that marks cells undergoing convergence-extension cell movements in Xenopus and zebrafish gastrulating embryos. Here a mouse homologue (mpapc) was identified and characterized. During early- to mid-gastrulation, mpapc is expressed in the primitive streak as the trunk mesoderm undergoes morphogenetic cell movements. At head-fold stage mpapc expression becomes localized to paraxial regions in which somites are formed in the segmental plate. At later stages, mpapc displays a complex expression pattern in cerebral cortex, olfactory bulb, inferior colliculus, and in longitudinal stripes in hindbrain. To analyze the effect of the loss of PAPC function during mouse development, a null allele of the mouse papc gene was generated. Homozygous animals show no defects in their skeleton and are viable and fertile.

Dad1 is a putative anti-apoptosis gene identified in several distantly related organisms. Expression of Dad1 in transfected cells inhibits apoptosis in vitro. To determine whether Dad1 has a similar function in vivo, we used gene targeting to delete Dad1. Heterozygous adult mice (+/-) show no obvious phenotype or abnormalities, but genotype analysis of over 100 offspring from heterozygous matings detected no weanling, homozygous Dad1 null (-/-) mice. Subsequent analysis of embryos from heterozygous matings detected Dad1 null (-/-) embryos at E3.5 but no later, suggesting Dad1 is required for development beyond the late blastocyst stage. Increased levels of apoptosis were observed in cultured embryos lacking a functional copy of the gene, consistent with an anti-apoptotic role for Dad1.

ENU is a powerful germline mutagen in the mouse, providing the opportunity to analyze the functions of large numbers of genes in the mammalian genome. In many mutagenesis experiments, it would be beneficial to exploit the advantages of inbred mouse strains. To perform an effective ENU mutagenesis screen using inbred mice, a dosage regimen is required to determine the optimal dose of ENU for that inbred strain, a time-consuming preliminary process. We have carried out dosage regimens for mutagenizing doses of ENU in ten inbred strains of mouse: 129X1/SvJ, 129S6/SvEv, A/J, BALB/cJ, BTBR/N, C3He/J, C3HeB/FeJ, C57BL/6J, C57BR/cdJ, and CBA/CaJ, and determined an optimal dose for each strain, defined by length of sterile period and number of males to survive treatment. Three strains: A/J, BALB/cJ and C57BL/6J, are able to tolerate high doses, up to 300 mg/kg body weight, and are highly recommended for mutagenesis studies.

Mouse cerberus-like (cer-l) is a member of the Cerberus/Dan family of secreted factors. As other members of this family of proteins, Cer-l functions in the extracellular space, inhibiting signaling molecules. Here we show that the neural-inducing and mesoderm-inhibiting activities of Cer-l result from specific binding to BMP and Nodal molecules, respectively. These properties resemble the ones from the related factor Xenopus Cerberus. However, Xenopus Cerberus in addition to BMP4 and Nodal also binds to and inhibits Wnt proteins. We show that Cer-l does not directly inhibit Wnt signals. A null allele of the mouse Cer-l gene was generated by targeted inactivation in ES cells. Homozygous embryos show no anterior patterning defects, are born alive, and are fertile. Since mouse Cer-l and Xenopus Cerberus differ in biochemical activities, we propose the existence of additional members of this family of inhibitors, which may compensate for the loss of cer-l.

Mice with a loss of function of prx1, a paired-related homeobox gene formerly called Mhox, showed craniofacial defects, limb shortening, and incompletely penetrant spina bifida. To investigate the mechanisms that regulate prx1 expression, we analyzed a 2.4-kb prx1 genomic flanking region in transgenic mice. This region of the prx1 gene contains an enhancer element that directs expression of a LacZ reporter gene in limb bud mesenchyme and a subset of craniofacial mesenchyme. Deletional analysis in transgenic founders identified a necessary 530-bp core element. Comparison of this core element with human Prx1 sequence showed two highly conserved cassettes that also contained a prx recognition element. Moreover, transgene expression was diminished in posterior handplate of prx1; prx2 double mutant mice. Our data reveal that the prx1 limb enhancer is proximally located within the prx1 gene and suggest that prx1 may have an autoregulatory function in limb mesenchyme.

The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1(-/-) embryos and we detected no obvious phenotypic defects in adult Cerr1(-/-) mice. However, in embryonic tissue layer recombination assays, Cerr1(-/-) presomitic/somitic mesoderm, unlike Cerr1-expressing wild-type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors.

Summary: We have obtained a novel family of LTR-retrotransposons in Xenopus laevis, named Xretpos, from cDNA and genomic clones. Its long terminal repeats (LTRs) can be subdivided into U3, R, and U3 to U5 region, and are bounded by 6 bp inverted repeats. Xretpos contains primer binding site and polypurine tract, and multiple copies of Xretpos-related element are present in the genome. A long open reading frame (ORF) encodes the CCHC motif conserved in retroviral gag proteins and leucine zipper motif capable of forming the coiled-coil. However, no amino acid homology to usually conserved retroviral pol gene was revealed. We report that in vitro synthesized Xretpos complementary RNAs are translated to produce a predicted size of protein. We also show that zygotically activated Xretpos transcripts are restricted to ventro-posterior specific regions and induced by UV-irradiation and BMP-4 overexpression in cycloheximide-dependent way. genesis 26:198-207, 2000.

Vertebrates employ varied strategies, both chromosomal and nonchromosomal, to determine the sex of the developing embryo. Among reptiles, temperature-dependent sex determination (TSD) is common. The temperature of incubation during a critical period preceding sexual differentiation determines the future sex of the embryo, presumably by altering the activity or expression of a temperature-dependent regulatory factor(s). Here we examine the expression of the Dmrt1 gene, a candidate regulator of mammalian and avian sexual development, in the turtle. During the sex-determining period, Dmrt1 mRNA is more abundant in genital ridge/mesonephros complexes at male-promoting than at female-promoting temperatures. Dmrt1 is the first gene found to show temperature-dependent expression prior to sexual differentiation, and may play a key role in sexual development in reptiles. genesis 26:174-178, 2000.

The mechanism(s) by which localized vascular permeability and angiogenesis occur at the sites of implantation is not clearly understood. Vascular endothelial growth factor (VEGF) is a key regulator of vasculogenesis during embryogenesis and angiogenesis in adult tissues. VEGF is also a vascular permeability factor. VEGF acts via two tyrosine kinase family receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Recent evidence suggests that neuropilin-1 (NRP1), a receptor involved in neuronal cell guidance, is expressed in endothelial cells, binds to VEGF(165) and enhances the binding of VEGF(165) to VEGFR2. We examined the spatiotemporal expression of vegf isoforms, nrp1 and vegfr2 as well as their interactions in the periimplantation mouse uterus. We observed that vegf(164) is the predominant isoform in the mouse uterus. vegf(164) mRNA accumulation primarily occurred in epithelial cells on days 1 and 2 of pregnancy. On days 3 and 4, the subepithelial stroma in addition to epithelial cells exhibited accumulation of this mRNA. After the initial attachment reaction on day 5, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited distinct accumulation of vegf(164) mRNA. On days 6-8, the accumulation of this mRNA occurred in both mesometrial and antimesometrial decidual cells. These results suggest that VEGF(164) is available in mediating vascular changes and angiogenesis in the uterus during implantation and decidualization. This is consistent with coordinate expression of vegfr2, and nrp1, a VEGF(164)-specific receptor, in uterine endothelial cells. Their expression was low during the first 2 days of pregnancy followed by increases thereafter. With the initiation and progression of implantation (days 5-8), these genes were distinctly expressed in endothelial cells of the decidualizing stroma. Expression was more intense on days 6-8 at the mesometrial pole, the presumptive site of heightened angiogenesis and placentation. However, the expression was absent in the avascular primary decidual zone immediately surrounding the implanting embryo. Crosslinking experiments showed that (125)I-VEGF(165) binds to both NRP1 and VEGFR2 present in decidual endothelial cells. These results suggest that VEGF(164), NRP1 and VEGFR2 play a role in VEGF-induced vascular permeability and angiogenesis in the uterus required for implantation. genesis 26:213-224, 2000.