ENU诱导获得一种短尾小鼠及其突变基因的初步定位

用一种化学诱变剂ENU(乙酰基亚硝基脲 )腹腔注射 3 0只 8～ 1 0周龄C5 7BL 6J(简称B6)雄鼠 (G0代 ) ,6周后与同品系正常母鼠配种繁殖后代 (G1代 )小鼠 3 5 1只。对其后代进行筛选获得一种可遗传的显性短尾突变小鼠。为了定位该突变基因 ,运用平均分布于B6和DBA 2 (简称D2 )小鼠常染色体而在这两者间又有差异的 3 9个微卫星对突变小鼠的 (D2×B6)F1代短尾突变小鼠回交D2得到的有短尾表型的[(B6×D2 )F1×D2 ]F2 代小鼠进行基因组扫描。反向运用经典的位置候选基因法 ,将短尾突变基因定位于 1 7号染色体 ,与D1 7Mit3 3的LOD值为 9 0 8。选用该染色体上与短尾表型相关基因Brachyury (T)最近的微卫星D1 7Mit1 43引物扩增 ,在 1 0 9只F2 代短尾小鼠中未发生一例交换 ,表明Brachyury基因是本例短尾突变强有力的候选基因。     

Brachyury-downstream notochord genes and convergent extension in Ciona intestinalis embryos

Formation of the chordate body is accomplished by a complex set of morphogenetic movements including convergent extension of notochord cells. In the ascidian Ciona intestinalis, Brachyury plays a key role in the formation of the notochord, and more than 30 Bra-downstream notochord genes have been identified. In the present study, we examined the effects of functional suppression of nine Bra-downstream notochord genes, which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Ci-trop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When the function of the first two genes (Ci-PTP and Ci-ACL) was suppressed with specific morpholinos, the notochord cells failed to converge, while functional suppression of Ci-prickle resulted in a failure of intercalation, and therefore the cells in these three types of embryo remained in the mid-dorsal region of the embryo. Functional suppression of the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and Ci-ASAK) resulted in the partial defect of intercalation, and the notochord did not consist of a single row. In addition, when the function of the last two genes (Ci-ERM and Ci-pellino) was suppressed, notochord cells failed to elongate in the embryo, even though convergence/extension took place normally. These results indicate that many Bra-downstream notochord genes are involved in convergence/extension of the embryo.     

aproctous,a locus that is necessary for the development of the proctodeum in Drosophila embryos,encodes a homolog of the vertebrate Brachyury gene

The proctodeum of the Drosophila embryo originates from the posterior end of the blastoderm and forms the hindgut. By enhancer-trap mutagenesis, using a P-element-lacZ vector, we identified a mutation that caused degeneration of the proctodeum during shortening of the germ band and named it aproctous (apro). Expression of the lacZ reporter gene, which was assumed to represent expression of the apro gene, began at the cellular blastoderm stage in a ring that encompassed about 10–15% of the egg's length (EL) and included the future proctodeum, anal pads, and posterior-most part of the visceral mesoderm. In later stages, strong expression of lacZ was detected in the developing hindgut and anal pads. Expression continued in the anal pads and epithelium of the hindgut of larvae; the epithelium of the hindgut of the adult fly also expressed lacZ. The spatial patterns of the expression of lacZ in various mutants suggested that the embryonic expression of apro was regulated predominantly by two gap genes, tailless (tll) and huckebein (hkb): tll is necessary for the activation of apro, while hkb suppressed the expression of apro in the region posterior to 10% EL. Cloning and sequencing of the apro cDNA revealed that apro was identical to the T-related gene (Trg) that is a Drosophila homolog of the vertebrate Brachyury gene. apro appears to play a key role in the development of tissues derived from the proctodeum.     

Analysis of haploid development based on expression patterns of developmental genes in the medaka Oryzias latipes

The abnormalities of haploid medaka embryos were characterized by comparative analysis of histologic sections and expression patterns of some developmental marker genes between haploids and diploids to clarify whether medaka haploids are useful for identifying mutants. During gastrulation, an obvious defect was first observed as a delay of epiboly and involution. This delay was shown to be caused not by the perturbation of mesoderm induction, but by widespread cell death and disorganization of cell arrangement in the blastoderm. This disorganization of cell arrangement was also detected in various organs, such as the brain, somite and notochord, at a late developmental stage. Ten days after fertilization, a small head and a short body axis were formed; these changes were also observed in haploid embryos in other species, but their cause is unknown. Based on the expression patterns of HNF3beta and goosecoid, it was demonstrated that a short and impotent prechordal plate induced near the marginal zone in haploid embryos was responsible for this defect. However, in these experiments it was also demonstrated that many major organs in haploids, such as the somite and notochord, differentiated incompletely but were present. Therefore, it was concluded that haploid screening is suitable for identifying mutations revealed by an obvious phenotype, such as dorsoventral polarity.     

Brachyury (T) expression in embryonal carcinoma P19 cells resembles its expression in primitive streak and tail-bud but not that in notochord

Brachyury (T) is involved in mesoderm induction during early mouse development. T expression in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro, was studied. Endogenous T expression in P19 cells was transiently induced when the cells were allowed to form aggregates. This expression was enhanced by dimethyl sulfoxide. In situ hybridization showed that T expressing cells formed clusters on the aggregates. Transfection of plasmids encoding reporter genes under the control of the upstream region of T showed that the sequence up to -351 bp can resemble the differentiation-dependent expression of T in P19 cells. To define the promoter region regulating T expression, transgenic mice carrying LacZ under the control of the upstream region were prepared. The region up to -351 bp is sufficient to direct the expression in the primitive streak and tail-bud. The upstream region up to -2400 bp does not support expression in the notochord. The sequence between -987 and -585 bp enhances expression in the primitive streak and tail-bud. In the tail-bud where new cells for elongation of the anteroposterior axis were continuously supplied, the sequence up to -987 bp drove lacZ expression in gut endoderm and prospective neuroectoderm as well as in mesoderm derivatives.     

Embryonal effects of <Emphasis Type="Italic">t</Emphasis>-haplotypes in mice

The effects of t-haplotypes on embryonic morphology in house mouse Mus musculus were described. Lethal mutations, t-haplotypes, in homozygotes induce abnormal embryogenesis and zygotic death at different developmental stages, which depends on the time of their action in ontogeny. Death commonly occurs in the first semester of pregnancy from the morula to the mature embryo stage (day 9–10), and the embryogenetic abnormalities and their timing were Specific for each t-haplotype. Such mutations were analyzed to identify the gene products (proteins) affecting the nervous system development. The t-complex proved to contain tandem repeats coding for regulatory factors modulating the expression of specific structural genes in mouse neurons.