Abstract
In the post genomic era, the study of molecular biology has expanded into the study of interactions between genes and molecules. Genomic regulatory networks concerns the transcription factors as well as signal transduction components and the interaction between them, those interaction forms a networks like architecture which explain fully the detail mechanism underlying the embryogenesis. The gene regulatory networks for early development have become so important in recent study; the GRN has been published for endomesoderm in sea urchin, mesoderm in Xenopus and dorsal-ventral specification in Drosophila. Gbx class transcriptional factors regulate the cell fate of cephalic primordium to cause neuromeres segmentation in zebrafish hindbrain. Previous study had demonstrated midbrain-hindbrain boundary (MHB) as well as isthmus organizer is controlled by the interaction between Gbx2 and Otx2. Recently, Gbx1 had been proved to act on the upstream genes of Gbx2 and might play a crucial role in establishment of MHB. However, it is still unclear about how the hindbrain patterns and what signals activate Gbx family to promote brain regionalization. To gain a detail understanding about the mechanism, we apply gene regulatory network (GRN) to broadly constitute the relationship between transcriptional factors and signal transduction component, those interaction forms a sub-network which explain the detail mechanism about how brain develop into several segments. We search the downstream outputs of Gbx family by systematically screening the Gbx morphants coupling with RT-QPCR. At the same time, we also knock down several endomesoderm genes to screen if they act on Gbx family. Our data coupling with literature search can provide many outputs of Gbx family and some inputs acting on them. To further analyze the Gbx1 and Gbx2 regulatory region, we compare the evolutionary conserved elements from seven different species ranged from fugu, tetraodon, xenopus, opossum, and mouse to human, and find several important cis-regulatory modules on zebrafish Gbx1 and Gbx2 genome. In the conserved regions, there are predicted binding sites of Pbx2, Sox 17 and Otx2, which are known to regulate Gbx2 and Gbx1 transcription. We are now performing in vivo reporter assay to find real cis-regulatory modules and direct transcription factor binding sites on Gbx1 and Gbx2 locus. This study of the regulatory elements on Gbx1 and Gbx2 will allow us to identify the important transcription factor binding sites involved in the regulation of Gbx1 and Gbx2, and further enable us to link the gene regulatory networks on the cis-regulatory elements. This information could be useful for establish the underlying mechanisms of how the Gbx1 and Gbx2 are regulated by other transcription factors and how they regulate other transcription factors in order to determine the brain fate.