Cloning and Characterization of ChickenParaxis:A Regulator of Paraxial Mesoderm Development and Somite Formation

AbstractTo investigate the molecular regulation of embryonic somite formation and development, we have cloned the full-length cDNA and characterized the embryonic expression profile of chickenParaxis,a member of a novel family of basic helix-loop-helix (bHLH) proteins, which has been suggested to play a role in paraxial mesoderm development. ChickenParaxisencodes a 1.35-kb mRNA and contains a 53-amino-acid residue bHLH domain, identical in sequence to that found in the mammalianParaxisgenes of mouse, hamster, and human. Northern analysis revealed significantParaxisexpression in the early embryo up to the 30- to 35-somite stage, declining from Incubation Day 4 on and becoming undetectable by Day 5. By whole-mountin situhybridization,Paraxisexpression is first seen distinctly in the emerging paraxial mesoderm of the primitive streak stage chick embryo. During gastrulation,Paraxisexpression in the mesoderm defines bilaterally symmetric crescents located immediately rostral to Hensen's node and appears to pre-configure the emerging somitic mesoderm. During somite development,Paraxisexpression is evident in the rostral segmental plate and the newly formed somites, although the level of expression clearly decreases in the more mature somites. By the 10–12th pair of somites, counting from the caudal end,Paraxisexpression appears to be preferentially localized to the medial aspect of individual somites. Histological analysis showed thatParaxisexpression is evenly distributed in the newly formed caudal epithelial somites, then localized to the medial portion of maturing somites, and preferentially localized in the dermomyotome of more rostral somites before diminishing to undetectable levels in the most cranial somites. The functional involvement ofParaxisin somite development was assessed by perturbing its expression in somitic stage chick embryos using aParaxis-specific antisense oligonucleotide. Disruption of somite formation from the paraxial mesoderm was observed in 67% of the surviving topically treated embryos, whereas control embryos treated with sense or random sequence oligonucleotides did not show similar effects. In addition, direct injection ofParaxis-specific antisense oligonucleotide into the paraxial mesoderm produced discrete segmentation anomalies which correlated spatially with the site of injection. Whole-mountin situhybridization revealed that the regions defective in somite formation displayed perturbedParaxisexpression and a reduction ofPax-1expression, a marker for epithelial somites and sclerotome. Histological analysis indicated poor condensation and/or epithelization of the somitic mesoderm. Finally, embryos treated with valproic acid, a known teratogen which affects somite segmentation, showed perturbedParaxisexpression, suggesting that the mechanism of action of this teratogen involves a pathway(s) requiringParaxisactivity. These data provide evidence thatParaxisacts as an important regulator of paraxial mesoderm and somite development and functions in axial patterning of the chick embryo