Functional involvement of Pax‐1 in somite development: Somite dysmorphogenesis in chick embryos treated with Pax‐1 paired‐box antisense oligodeoxynucleotide

Abstract

The metameric pattern of the vertebrate axial skeleton, defined by structures such as the vertebral bodies and ribs, is a result of segmentation events that occur during embryogenesis. The key event in axial segmentation is somite formation. This study examines the role of Pax‐1, a member of the paired‐box containing Pax gene family, in chick somite development. To investigate whether misexpression of Pax‐1 during somite development is functionally related to abnormal axial patterning, antisense methodologies were used to perturb Pax‐1 expression. An antisense, phosphorothioate‐modified oligodeoxynucleotide (ODN) was designed based on the mouse Pax‐1 paired‐box sequence, and was either injected into or directly applied topically to early, somitic stage chick embryos. Abnormalities in somite structure and pattern were subsequently observed and scored, including loss of somites (18% of injected embryos, 35% of embryos treated by topical application), fusion of somites (25% of injected, 6% with topical application), and shortened body axis (0% of injected, 11% with topical application). Control embryos receiving sense ODN or non‐sense ODN (a scrambled sequence with base composition identical to the antisense ODN) showed substantially fewer somite anomalies, indicating that the effects were sequence‐specific. These developmental abnormalities were analyzed using standard histological methods. Whole mount in situ hybridization was carried out to analyze the three‐dimensional pattern of Pax‐1 expression in whole embryos. In control, untreated embryos, the expression was localized to the entire epithelial somite, and as the somite matured, the expression was limited to its ventromedial region. With Pax‐1 antisense ODN treatment, embryos with fused somites retained expression over the entire fused somite, and embryos that had complete loss of somites had greatly reduced expression of Pax‐1 transcript. The results presented here provide strong evidence that Pax‐1 is functionally important during somitogenesis and morphogenesis of the vertebral column. The spatial pattern of gene expression appears to delineate different populations of cells in the developing embryo (i.e., somite from somite, sclerotome from dermomyotome), and is consistent with the hypothesis that Pax‐1 is involved in forming or maintaining boundaries at specific times and locations during development.