新穎同源箱基因Irxl1在斑馬魚胚胎發育中參與下顎軟骨及肌肉形態的生成

Iroquois homeobox-like 1 (Irxl1)是一個新穎的同源箱TALE家族成員基因，它與Iroquois的家族蛋白最為相似。我們在斑馬魚中找到此Irxl1基因並發現其有2種剪接的型式。以全胚胎原位雜交法分析，發現Irxl1主要表現在頭部的端腦及咽喉腮弓。利用反股的Morpholino抑制內生性的Irxl1蛋白質，會造成頭部及下顎軟骨的畸形，較嚴重的胚胎只能存活五到七天。若與Irxl1 cRNA一起共同注射到胚胎則可以部份回復這些異常的表型。這些異常的胚胎最顯著的缺失位於咽喉鰓弓的中胚層與軟骨，且咽喉肌與神經脊細胞也同樣的減少。利用phalloidin染色觀察morphant胚胎的肌肉束，其型態也有異常。這些結果顯示Irxl1可參與調節下顎軟骨與肌肉的發育。由於Irxl1 knock down的morphants胚胎下顎軟骨的缺失與Mef2ca knock down胚胎相似，且Irxl1基因promoter有發現數個可能為Mef2ca所結合的區域，因此我們檢測是否Irxl1可被Mef2ca直接調控。藉由全胚胎原位雜交法與RT-PCR分析，我們發現在Mef2ca的突變株與knockdown morphant胚胎中，Irxl1的表現量都下降。同時，在雙冷光分析中共同注射Mef2ca也可以提高Irxl1 promoter的活性。另外，在Irxl1 morphants胚胎的體節中，myoD表現量卻是上升的。共同注射myoD promoter所驅動的冷光酵素基因與Irxl1a至斑馬魚胚胎中發現Irxl1對myoD promoter有顯著的抑制調控，而一系列的promoter刪減突變分析顯示在myoD promoter -144/-42之間可能有Irxl1結合的序列。這些結果顯示Irxl1可能為一個腦部、肌肉與咽喉鰓弓形態發生的重要調節者，而其功能可能是透過Mef2ca和myoD路徑調控下顎軟骨與肌肉的發育。

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.