Antisense attenuation of Wnt‐1 and Wnt‐3a expression in whole embryo culture reveals roles for these genes in craniofacial, spinal cord, and cardiac morphogenesis

Abstract

Wnt-1 and Wnt-3a proto-oncogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attenuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurally related to Wnt-1, targeted the forebrain and midbrain region, which were hypoplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nucleotides) make this antisense oligonucleotide/whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development.

Connexin43 gap junction protein plays an essential role in morphogenesis of the embryonic chick face

“…Thus, antisense to mRNA for the cell adhesion molecule E-cadherin (Chen and Hale, 1995) and for signalling molecules implicated in cell coupling, Wnt-1 and Wnt-3a (Augustine et al, 1993), all resulted in hypoplasia of facial primordia. These results are fur- There is failure of fusion between maxillary and mandibular primordia (bottom arrowhead).…”

Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure

“…There is some earlier evidence that the Wnt pathway plays an important role in cardiac morphogenesis, beginning with studies using antisense attenuation of Wnt1 and Wnt3a expression in whole embryo cultures (Augustine et al, 1993). Recent reports demonstrate that Wnt inhibition induces cardiogenesis in Xenopus (Schneider and Mercola, 2001) and in chick (Marvin et al, 2001), but the role of Wnt signaling in mammalian systems was unclear.…”

Glial cells: Old cells with new twists

“…These transcription factors are induced by signaling from receptors stimulated by another group of specific trophic factors and/or morphogens (Augustine et al, 1993;Mehler et al, 1997).…”