Analysis of the gene network patterning the hindhtain in vertebrates.

1.在斑马鱼胚胎后脑形成过程中，hoxb1b在神经板中表达，其前边界位于r3-r4边界，提供位置信息。为了了解hoxb1b转录的调控机制，我以GFP为报告基因，进行了基因转移启动子分析。结果，在上游DNA （-1.8 kb和-4.6 kb）中鉴定出两个调控区域，在后期（生长发育）驱动GFP表达，在下游DNA （+1.5 kb和+4.7）中鉴定出两个调控区域，在早期（原肠期）驱动GFP表达。我们也很清楚，下游的两个区域调节了对维甲酸的反应，维甲酸是脊椎动物胚胎后脑的模式。gbx2和fgf8均表达于早期神经板的原始前后脑，并支配中脑-后脑边界区（MHB）。我对hoxb1b进行了启动子分析。对于gbx2，上游区域-6.9 kb足以在后脑前部和中脑进行转录，而fgf8的两个下游区域+10.6和+15.1 kb除了耳囊和视柄外，还可以驱动后脑前部的报告基因表达。在fgf8的侧翼区域也发现了额外的调控区域，这些区域在胚胎中表达fgf8的许多胚胎区域中驱动表达。为了无偏倚地确定控制脑形成的调控基因，我们对经enu处理的雄性鱼的后代进行了胚胎脑异常筛选作为先导实验，建立了中脑、背间脑和轴形成等表型异常的几个突变系。

1.During the formation of hindbrain in zebrafish embryos, hoxb1b is expressed in the neural plate with its anterior boundary at the r3-r4 border, providing the positional information. In order to know the regulatory mechanism of hoxb1b transcription, I conducted the promoter analysis using gene transfer with GFP as a reporter. As a result, two regulatory regions were identified in the upstream DNA (-1.8 and -4.6 kb) which drove the GFP expression at late stages (somitogenesis) and two regulatory regions in the downstream DNA (+1.5 and +4.7) that drove GFP at earlier stages (gastrula). It also became clear that the downstream two regions mediate the responsiveness to retinoic acid that is known to pattern the hindbrain in vertebrate embryos.2.gbx2 and fgf8 are both expressed in the primordial anterior hindbrain of the early neural plate and pattern the midbrain-hindbrain boundary region (MHB). I conducted the promoter analyses as for hoxb1b. For gbx2, the upstream region at -6.9 kb was shown to be sufficient for transcription in the anterior hindbrain and diencephalon, while two downstream regions of fgf8 at +10.6 and +15.1 kb could drive reporter expression in the anterior hindbrain in addition to the otic vesicle and optic stalk. Additional regulatory regions have also been identified in the flanking region of fgf8 that drove expression in a number of embryonic regions where fgf8 is expressed in embryos.3.In order to identify regulatory genes that govern the brain formation without any bias, progeny fish of ENU-treated male fish were screened for anomalies in the embryonic brain as a pilot experiment, leading to establishment of several mutant lines showing abnormal phenotypes such as in the midbrain, dorsal diencephalon, and axis formation.