Altered hox gene expression and cellular pathogenesis of 5-aza-2'-deoxycytidine-induced murine hindlimb...

The DNA demethylating agent, 5-aza-2'-deoxycytidine (d-AZA), elicits temporally related morphological defects and altered gene expression in mouse hindlimbs. Segmental formation of limb regions (stylopod, zeugopod. and autopod) is partially dependent on Hox gene activation. The objective of this study was to understand the role of altered expression of key hox genes in the early pathogenesis of d-AZA-induced hindlimb defects in mice. Semiquantitative RT-PCR was used to analyze hox gene expression (Hox C-11 and Hox A and D homologs, paralogs 9-13). Untreated and treated fore and hindlimb buds were collected 12 and 24 hours after IP injection (1 mg/kg) of d-AZA at 9 am on gestational (GD) 10 and processed for RT-PCR. Additional pregnant mice were treated similarly and whole embryos collected 12 and 24 hours posttreatment and processed for histopathological analysis. No changes in hox gene expression were detected in the forelimb tissue. There was a 2-fold down-regulation of hoxA-11 and C-11 in the 12-hour hindlimb bud tissue. No changes in the HoxD series were detected in the hindlimb bud tissue. The 12- and 24-hour untreated mice exhibited some of the morphological features consistent with physiological apoptosis. Most tissues of the treated mice exhibited cellular changes consistent with cell death associated with the cytotoxicity of the compound. The data reported supports the hypothesis that altered gene expression and not cytotoxicity alone is associated with d-AZA-induced hindlimb dysmorphogenesis.