ABSTRACT
Both Pax1 and Pax9 belong to the important paired box gene family (PAX), which mainly participates in animal development and sclerotome differentiation. To date, the precise molecular mechanism and related signaling pathway of Pax1 remain unclear. In our study, microinjection of morpholino- (MO-) modified antisense oligonucleotides against pax1b induced pectoral fin bud defects. Furthermore, we demonstrate that the phenotypes caused by the knockdown of Pax1b in zebrafish could not be phenocopied by pax9 MO and could not be rescued by either Pax1a or Pax9 overexpression. We further find that Pax1b affects the expression of col2a1, Uncx4.1, Noggin3, and aggrecan, confirming the role of Pax1b in chondrocyte differentiation and bone maturation. Moreover, we identify an interaction between PAX1 and FOXO1 and find that the interaction was enhanced under hypoxia stress. Together, this evidence for cell death caused by pax1b knockdown provides new insight into the role of the Pax protein family in cell fate determination and tissue specification.
Subject(s)
Animal Fins/embryology , Animal Fins/metabolism , Embryonic Development , PAX9 Transcription Factor/metabolism , Paired Box Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Animal Fins/abnormalities , Animals , Bone Development , Cell Death , Embryonic Development/genetics , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Humans , Morphogenesis , PAX9 Transcription Factor/antagonists & inhibitors , PAX9 Transcription Factor/genetics , Paired Box Transcription Factors/genetics , Phenotype , Protein Binding , Stress, Physiological , Tail/abnormalities , Zebrafish/genetics , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/geneticsABSTRACT
We examined the expression and functions of Pax1 and Pax9 in a teleost fish, the medaka Oryzias latipes. While Pax1 and Pax9 show distinct expression in the sclerotome in amniotes, we could not detect the differential expression of Pax1 and Pax9 in the developing sclerotome of the medaka. Furthermore, unlike the mouse, in which Pax1 is essential for development of the vertebral body, and where the neural arch is formed independent of either Pax1 or Pax9, our morpholino knockdown experiments revealed that both Pax1 and Pax9 are indispensable for the development of the vertebral body and neural arch. Therefore, we conclude that after gene duplication, Pax1 and Pax9 subfunctionalize their roles in the sclerotome independently in teleosts and amniotes. In Stage-30 embryo, Pax9 was strongly expressed in the posterior mesoderm, as was also observed for mouse Pax9. Since this expression was not detected for Pax1 in the mouse or fish, this new expression in the posterior mesoderm likely evolved in Pax9 of ancestral vertebrates after gene duplication. Two-month-old fish injected with Pax9 morpholino oligonucleotide showed abnormal morphology in the tail hypural skeletal element, which may have been related to this expression.
