The long-range regulation of Hoxa13 in the developing limbs and genital bud
The posterior HoxA and HoxD homeodomain transcription factor genes are essential for appendicular development. My thesis research demonstrates that a domain of genes upstream of Hoxa13 (Evx1, Hibadh, Tax1bp1, and Jazf1) are embryonically co-expressed in the distal limb and genital bud during embryogenesis (E11.5–E13.5), suggesting the existence of a common/shared enhancer element influencing their expression. A similar co-expressed gene neighborhood exists at the ancestrally related HoxD locus. Paralogous sequence comparison of 1 Mb genomic DNA upstream of the HoxA and HoxD clusters revealed five highly conserved non-coding sequences. Two of these sequences are clustered within homologously conserved sequences on each chromosome: mmA13CNS, ∼2.25 kb located within the fourth intron of Hibadh upstream of Hoxa13, and mmD13CNS, ∼4 kb located within the previously identified 'distal limb enhancer' critical region upstream of Hoxd13. To test mmA13CNS function, this element was placed in cis with a minimal promoter and shown to be insufficient for precise Hoxa13-like expression in the limb buds or genital bud of LacZ transgenic mice, even though non- Hoxa13-like LacZ expression was reproducibly found in these tissues.
Subsequently, this research demonstrated the sufficiency of a single critical region for mouse Hoxa13-like embryonic genital bud expression, as assayed by β-globin minimal promoter LacZ recombinant BAC transgenes. This research also shows that at least two sequences remote to the HoxA cluster are collectively sufficient to drive expression in the developing distal limbs, similar in timing and domains to the endogenous Hoxa13 expression pattern.