The Tbx4 transcription factor is crucial for normal hindlimb and vascular development, yet little is known about how its highly conserved expression patterns are generated. We have used comparative genomics and functional scanning in transgenic mice to identify a dispersed group of enhancers controlling Tbx4 expression in different tissues. Two independent enhancers control hindlimb expression, one located upstream and one downstream of the Tbx4 coding exons. These two enhancers, hindlimb enhancer A and hindlimb enhancer B (HLEA and HLEB), differ in their primary sequence, in their precise patterns of activity within the hindlimb, and in their degree of sequence conservation across animals. HLEB is highly conserved from fish to mammals. Although Tbx4 expression and hindlimb development occur at different axial levels in fish and mammals, HLEB cloned from either fish or mouse is capable of driving expression at the appropriate position of hindlimb development in mouse embryos. HLEA is highly conserved only in mammals. Deletion of HLEA from the endogenous mouse locus reduces expression of Tbx4 in the hindlimb during embryogenesis, bypasses the embryonic lethality of Tbx4-null mutations, and produces viable, fertile mice with characteristic changes in the size of bones in the hindlimb but not the forelimb. We speculate that dual hindlimb enhancers provide a flexible genomic mechanism for altering the strength and location of Tbx4 expression during normal development, making it possible to separately modify the size of forelimb and hindlimb bones during vertebrate evolution.
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