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

1. 报道者分析了斑马鱼在尾侧神经板表达的hoxb1b的调控机制，发现hoxb1b的表达在神经板中受到d1和d4两个下游区域的特异性驱动。发现这两个区域通过两个维甲酸响应区（RAREs）调控hoxb1b对维甲酸（RA）的响应。已知Pou2在原肠晚期的中脑和后脑中短暂表达，允许中脑-后脑边界（MHB）和后脑的发育。对其调控机制进行了研究，结果表明，pou2主要受上游2.1 kb DNA的空间调控，其表达是通过2.1 kb DNA中四个与pou2结合的八聚体序列协同作用介导的自调控环来维持的，而pou2则是通过上游DNA中的一个RARE受到RA的抑制。通过引入不同的缺失对fgf8晚期MHB增强子的内部结构进行了检测，结果表明MHB增强子在中脑至后脑r5处被其中的两个Pax2结合序列广泛激活，并且该增强子中有一个28 bp的区域抑制中脑和后脑后部的表达，使fgf8具有MHB特异性表达。研究还发现，该MHB增强子在脊椎动物进化过程中具有广泛的保守性。对经ENU处理的斑马鱼进行突变体筛选，分离出在中脑和后脑显示缺陷的突变系（aa6k）。除了大脑缺陷外，突变胚胎还存在触摸反应异常和明显的颌骨缺陷。连锁分析将突变定位在3号染色体上，并发现了几个接近的SSLP标记。目前正在研究aa6k突变中可能涉及的几个候选基因。

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.