Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo

Exogenous retinoic acid (RA) has teratogenic effects on vertebrate embryos and alters Hox-C gene expression in vivo and in vitro. We wish to examine whether RA has a role in the normal regulation of Hox-C genes, and whether altered Hox-C gene expression in response to RA leads to abnormal morphology. The expression of 3' Hox-2 genes (Hox-2.9, Hox-2.8, Hox-2.6 and Hox-2.1) and a 5' gene (Hox-2.5) were examined by whole-mount in situ hybridization on embryos 4 hours after maternal administration of teratogenic doses of RA on embryonic day 7 to 9. The expression of the 3' Hox-2 genes was found to be ectopically induced in anterior regions in a stage-specific manner. The Hox-2.9 and Hox-2.8 genes were induced anteriorly in the neurectoderm in response to RA on day 7 but not at later stages. Expression of Hox-2.6 and Hox-2.1 was ectopically induced anteriorly in neurectoderm in response to RA on day 8. Hox-2.1 remained responsive on day 9, whereas Hox-2.6 was no longer responsive at this stage. The expression of the 5' gene Hox-2.5 was not detectably altered at any of these stages by RA treatments. We also examined the response of other genes whose expression is spatially regulated in early embryos. The expression of En-2 and Wnt-7b was not detectably altered by RA, whereas RAR beta expression was induced anteriorly by RA on day 7 and 8. Krox-20 expression was reduced in a stage- and region-specific manner by RA. The ectopic anterior expression of Hox-2.8 and Hox-2.9 induced by RA on day 7 was persistent to day 8, as was the altered expression of Krox-20. The altered pattern of expression of these genes in response to RA treatment on day 7 may be indicative of a transformation of anterior hindbrain to posterior hindbrain, specifically, a transformation of rhombomeres 1 to 3 towards rhombomere 4 identity with an anterior expansion of rhombomere 5. The ectopic expression of the 3' Hox-2 genes in response to RA is consistent with a role for these genes in mediating the teratogenic effects of RA; the rapid response of the Hox-C genes to RA is consistent with a role for endogenous RA in refining 3' Hox-C gene expression boundaries early in development.