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Abstract
Vertebrate Pax1 gene is a member of Pax gene family and encodes a transcription factor associated with crucial roles in the development of pharyngeal pouch, scletrotome and limb bud. In zebrafish, the genome contains two Pax1 paralogs, DrPax1a and DrPax1b, which share high sequence similarity with other Pax1 genes. To elucidate the function of zebrafish DrPax1b gene, we first examined the gene expression pattern and found that it was mainly expressed in the endodermal pharyngeal pouch, caudal somites, notochord, and fin bud. Then, we performed knockdown experiments using antisense morpholino oligonucleotides, which lead to the defects in the vertebral column, tail, pharyngeal skeleton, and pectoral fin. Additionally, we also found that the mouse MmPax1 mRNA, but not the amphioxus AmphiPax1/9 mRNA, could rescue the MO-induced defects. Furthermore, sequence alignment revealed that the N-terminal region of vertebrate Pax1 and amphioxus Pax1/9 were highly conserved, whereas their C-terminal regions were relatively divergent. However, the chimeric Am(N)Dr(C)Pax1, Mm(N)Dr(C)Pax1 and Dr(N)Mm(C)Pax1 mRNA could partially rescue the defects, while the Dr(N)Am(C)Pax1 mRNA could not. In conclusion, our data demonstrate a conserved function of DrPax1b in the development of the vertebral column, pectoral fin and pharyngeal skeleton formation in zebrafish and also provide critical insight into the functional evolution of Pax1 gene by changing its C-terminal sequence.
Highlights
Pax proteins are a group of transcription factors that play key roles in cell proliferation, cell differentiation, organogenesis and embryonic pattern formation
We constructed a phylogenetic tree with drosophila Pax1 (Pox meso) as an out-group (Fig. 2B), and the tree again confirmed that two Pax1 genes existed in the zebrafish genome
Two paralogues Danio rerio Pax1a (DrPax1a) and D. rerio Pax1b (DrPax1b) were previously identified in the zebrafish genome (Wang et al, 2010) and one of them DrPax1a was already related to the development of centrums (Clements et al, 2011)
Summary
Pax proteins are a group of transcription factors that play key roles in cell proliferation, cell differentiation, organogenesis and embryonic pattern formation. Total of nine Pax genes have been determined in mammalian and divided into four subfamilies: Pax1/9, Pax2/5/8, Pax3/7 and Pax4/6, based on the sequence homology and structural similarity (Noll et al, 1993). Among those genes, Pax is a member that plays important roles in embryonic development and its homologous gene is widely distributed through animal phyla (Dahl et al, 1997). Since the gene function of DrPax1b has not been detected yet, this report presents the gene spatial-temporal expression pattern and elucidates the gene function by morpholino-mediated knockdown experiment. We perform rescue experiments with amphioxus Pax1/9, mammalian mouse Pax, and chimeric Pax mRNAs to analyse the conservation and diversification of Pax gene function during the evolution
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