ANTISENSE OLIGONUCLEOTIDE DOWN-REGULATION OF E-CADHERIN IN THE YOLK-SAC AND CRANIAL NEURAL-TUBE MALFORMATIONS

The cadherins are a family of calcium-dependent cell adhesion molecule s that are regulated both spatially and temporally during development. Epithelial cadherin (E-cadherin) is present in epithelial cells in bo th the embryo and yolk sac during organogenesis. The consequences of d isrupting the expression of E-cadherin at this stage of development ar e poorly understood. We report here our studies on the effects of anti sense oligonucleotides on E-cadherin in the rat whole embryo culture s ystem. Four 18-base single strand phosphorothioate oligodeoxynucleotid es (AS-oligos), complementary to Various regions of the mouse E-cadher in cDNA sequence, were dissolved in saline and injected into the amnio tic cavities of 5-7 somite rat embryos; a sense (S-oligo) to oligo-1, an 18-base random sequence oligo (C-oligo), and PBS were used as contr ols. Embryos were cultured for up to 45 h; embryo morphology and the r elative concentrations of E-cadherin protein were examined. All six ol igonucleotides (AS-oligos and control oligos) induced malformations wh en amounts ranging from 25 to 50 pmol of oligonucleotide were injected per embryo. The malformations induced by all the oligos included cran iofacial hypoplasia, an enlarged pericardium, twisted spinal cord, swe lling of the rhombencephalon, and underdeveloped forelimb. injection o f AS-oligo-1, a sequence starting at the tenth base downstream from th e translation initiation codon (ATG), resulted in malformed embryos wi th a high incidence of cranial neural tube malformations. The effects of AS-oligo-1 on the relative abundance of E- and neural (N)-cadherin proteins were examined by Western blot analysis. In the AS-oligo-1-exp osed malformed embryos, the relative abundance of E-and N-cadherin pro teins was not altered up to 24 h after injection; E- and N-cadherin co ncentrations in the embryo were decreased at 45 h postinjection. In co ntrast, the relative abundance of the E-cadherin protein in the yolk s ac was reduced at 1-2 h after injection of AS oligo-1 and returned to control levels by 4 h. S-oligo-1 did not induce any change in the rela tive abundance of E- or N-cadherins. Thus, there was a tissue-specific and temporary ''knockdown'' of E-cadherin expression in the yolk sac of embryos exposed to antisense (AS-oligo-1); the down-regulation of y olk sac E-cadherin appears to lead to the induction of neural tube def ects in the embryo. The exposure of whole embryos in culture to antise nse oligonucleotides provides a model system in which the roles of dev elopmentally important molecules and their spatial and temporal contri butions to embryogenesis can be elucidated.