Identification of upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5

Nkx2-5 marks the earliest recognizable cardiac progenitor cells, and is act ivated in response to inductive signals involved in lineage specification. Nkx2-5 is also expressed in the developing foregut, thyroid, spleen, stomac h and tongue, One approach to elucidate the signals involved in cardiogenes is was to examine the transcriptional regulation of early lineage markers s uch as Nkx2-5, We generated F-0 transgenic mice, which carry Nkx2-5 flankin g sequences linked to a lacZ reporter gene, We identified multiple regulato ry regions located within the proximal 10.7 kb of the Nkx2-5 gene, In addit ion to a proximal promoter, we identified a second promoter and a novel ups tream exon that could participate in the regulation of Nkx2-5 transcription . Although used rarely in normal development, this novel exon could be spli ced into the Nkx2-5 coding region in several ways, thereby potentially crea ting novel Nkx2-5 protein isoforms, whose transcriptional activity is great ly diminished as compared to wild-type Nkx2-5, An enhancer that directs exp ression in pharynx, spleen, thyroid and stomach was identified within 3.5 k b of exon 1 between the coding exon 1 and the novel upstream exon la, Two o r more enhancers upstream of exon la were capable of driving expression in the cardiac crescent, throughout the myocardium of the early heart tube, th en in the outflow tract and right ventricle of the looped heart tube, A neg ative element was also located upstream of exon1a, which interacted in comp lex ways with enhancers to direct correct spatial expression. In addition, potential autoregulatory elements can be cooperatively stimulated by Nkx2-5 and GATA-4, Our results demonstrate that a complex suite of interacting re gulatory domains regulate Nkx2-5 transcription. Dissection of these element s should reveal essential features of cardiac induction and positive and ne gative signaling within the cardiac field.