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

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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.