Analysis of the regulatory regions of the Hox genes has revealed a complex array of positive and negative cis-acting elements that control the spatial and temporal pattern of expression of these genes during embryogenesis. In this study we show that normal expression of the murine Hoxa4 gene during development requires both autoregulatory and retinoic acid-dependent modes of regulation. When introduced into a Hoxa4 null background, expression of a lacZ reporter gene driven by the Hoxa4 regulatory region (Hoxa4/lacZ) is either abolished or significantly reduced in all tissues at E10. 5-E12.5. Thus, the observed autoregulation of the Drosophila Deformed gene is conserved in a mouse homolog in vivo, and is reflected in a widespread requirement for positive feedback to maintain Hoxa4 expression. We also identify three potential retinoic acid response elements in the Hoxa4 5′ flanking region, one of which is identical to a well-characterized element flanking the Hoxd4 gene. Administration of retinoic acid to Hoxa4/lacZ transgenic embryos resulted in stage-dependent ectopic expression of the reporter gene in the neural tube and hindbrain. When administered to Hoxa4 null embryos, however, persistent ectopic expression was not observed, suggesting that autoregulation is required for maintenance of the retinoic acid-induced expression. Finally, mutation of the consensus retinoic acid response element eliminated the response of the reporter gene to exogenous retinoic acid, and abolished all embryonic expression in untreated embryos, with the exception of the neural tube and prevertebrae. These data add to the evidence that Hox gene expression is regulated, in part, by endogenous retinoids and autoregulatory loops.

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