Molecular mechanism of the formation of the signaling center in the prospective hindbrain of vertebrate early embryos

1.斑马鱼hoxb 1b，这是在尾神经板中表达，以指定的位置，在后脑中的菱形1的调节机制，通过报告分析，揭示hoxb 1b的表达是专门驱动的两个下游区域，d1和d4的神经板。研究发现，这两个区域通过两个视黄酸反应区域（RAREs）调节hoxb 1b对视黄酸的反应.已知Pou 2在原肠胚晚期的中脑和后脑中瞬时表达，允许中脑-后脑边界（MHB）和后脑的发育。pou 2主要受上游2.1 kb DNA的空间调控，其表达是通过2.1 kb DNA中4个Pou 2结合Octamer序列的协同作用所介导的自调节环来维持的，而pou 2则通过上游DNA中的RARE被RA抑制.通过引入不同的缺失来检查fgf 8的晚期MHB增强子的内部结构，表明MHB增强子通过其中的两个Pax 2结合序列从中脑到后脑中的r5被广泛激活，并且该增强子中的28-bp区域抑制中脑和后脑后部中的表达，从而产生fgf 8的MHB特异性表达。研究还发现，这种MHB增强子在脊椎动物进化过程中是广泛保守的.用ENU处理的斑马鱼进行突变体筛选，导致分离显示中脑和后脑缺陷的突变体系（aa 6 k）。除了大脑缺陷外，突变胚胎还具有触摸反应异常和明显的下巴缺陷。连锁分析将该突变定位于3号染色体，并发现了几个紧密的SSLP标记。可能参与aa 6 k突变的几个候选基因正在调查中。

1. The regulatory mechanism of zebrafish hoxb1b, which is expressed in the caudal neural plate to specify the position of rhombomere 1 in the hindbrain, was examined by the reporter analysis, revealing that hoxb1b expression was specifically driven in the neural plate by two downstream regions, d1 and d4. It was found that these two regions regulate hoxb1b in response to retinoic acid (RA) via two retinoic acid responsive regions (RAREs).2. Pou2 is known to be expressed transiently in the midbrain and hindbrain in late gastrulae, allowing for the development of the midbrain-hindbrain boundary (MHB) and hindbrain. Its regulatory mechanism was examined, showing that pou2 is spatially regulated mainly by the upstream 2.1-kb DNA, that the expression is maintained through an autoregulatory loop mediated by the cooperative function of four Pou2-binding Octamer sequences in the 2.1-kb DNA, and that pou2 is repressed by RA through a RARE in the upstream DNA.3. The internal structure of the late MHB enhancer of fgf8 was examined by introducing different deletions, showing that the MHB enhancer is activated broadly from the midbrain to r5 in the hindbrain by the two Pax2 binding sequences therein, and that a 28-bp region in this enhancer represses expression in the midbrain and posterior hindbrain, giving MHBspecific expression of fgf8. It was also found that this MHB enhancer is broadly conserved during vertebrate evolution.4. Mutant screen was conducted with zebrafish treated with ENU, leading to isolation of a mutant line (aa6k) that shows defects in the midbrain and hindbrain. Besides the brain defect, the mutant embryos have anomalies in touch response and significant jaw defects. The linkage analysis localized the mutation resides in chromosome 3, and revealed several close SSLP markers. Possible involvement of several candidate genes in aa6k mutation is now under investigation.