A Novel Homeobox Gene, Irxl1, is Involved in Pharyngeal Arch and Muscle Morphogenesis During Zebrafish Embryonic Development

• Main Authors: Han-Ni, 莊函霓
• Other Authors: Huichin Pan
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/09284252838211765287

碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 97 === Iroquois homeobox-like 1 (Irxl1) is a novel member of the TALE superfamily of homeobox genes that is most-closely related to the Iroquois class. We have identified the zebrafish Irxl1 gene and found that it encodes two splicing variants. Whole-mount in situ hybridization (WISH) analysis revealed Irxl1 expression mainly in the telencephalon of the brain and the first two pharyngeal arches. Expression in the somitic region was also detected by RT-PCR. Antisense morpholino knockdown of Irxl1 resulted in deformed head and jaw in the larvae, which only survived for 5 to 7 days. The phenotype can be partially rescued by coinjection of Irxl1 cRNA. The most prominent defect was observed in the mesoderm and cartilage in pharyngeal arches. The number of pharyngeal muscles and cranial neural crest cells was reduced. In addition, the muscle fibers of Irxl1 morphants became disorganized as revealed by phalloidin staining. These results suggest that Irxl1 can regulate pharyngeal arch and muscle development. Because the phenotype of Irxl1 knockdown morphants is somewhat similar to that of Mef2ca, a myocyte enhancing factor that was recently shown to be involved in craniofacial development, and the promoter region of the Irxl1 gene contains several consensus Mef2 binding sites, we thus examined whether Irxl1 can be directly regulated by Mef2ca. By WISH and RT-PCR analysis, we observed that Irxl1 expression was decreased in Mef2ca mutants and morpholino- knockdown morphants. Furthermore, co-injection of Mef2ca into zebrafish embryos up-regulates the promoter activity of Irxl1 in a dual luciferase assay. Meanwhile, myoD expression was increased in the somites of Irxl1-knockdown embryos. Co-injecting myoD promoter- driven luciferase construct with Irxl1 into zebrafish embryos revealed a dramatic inhibition of myoD promoter activity by Irxl1. Analysis of a series of promoter deletion mutants suggested that Irxl1 binding site may locate at -144/-42 nt in the myoD promoter. These results suggest that Irxl1 is an important regulator of brain, muscle and arch morphogenesis, and its function in arch development may be mediated by Mef2ca. Moreover, Irxl1 protein may regulate muscle development through myoD-dependent pathways.