A GATA-dependent nkx-2.5 regulatory element activates early cardiac gene expression in transgenic mice

nkx-2.5 is one of the first genes expressed in the developing heart of earl y stage vertebrate embryos. Cardiac expression of nkx-2.5 is maintained thr oughout development and nkx-2.5 also is expressed in the developing pharyng eal arches, spleen, thyroid and tongue. Genomic sequences flanking the mous e nkx-2,5 gene were analyzed for early developmental regulatory activity in transgenic mice, Approximately 3 kb of 5' flanking sequence is sufficient to activate gene expression in the cardiac crescent as early as E7.25 and i n limited regions of the developing heart at later stages. Expression also was detected in the developing spleen anlage at least 24 hours before the e arliest reported spleen marker and in the pharyngeal pouches and their deri vatives including the thyroid. The observed expression pattern from the -3 kb construct represents a subset of the endogenous nkx-2.5 expression patte rn which is evidence for compartment-specific nkx-2.5 regulatory modules, A 505 bp regulatory element was identified that contains multiple GATA, NKE, bHLH, HMG and HOX consensus binding sites. This element is sufficient for gene activation in the cardiac crescent and in the heart outflow tract, pha rynx and spleen when linked directly to lacZ or when positioned adjacent to the hsp68 promoter. Mutation of paired GATA sites within this element elim inates gene activation in the heart, pharynx and spleen primordia of transg enic embryos. The dependence of this nkx-2.5 regulatory element on GATA sit es for gene activity is evidence for a GATA-dependent regulatory mechanism controlling nkx-2.5 gene expression, The presence of consensus binding site s for other developmentally important regulatory factors within the 505 bp distal element suggests that combinatorial interactions between multiple re gulatory factors are responsible for the initial activation of nkx-2.5 in t he cardiac, thyroid and spleen primordia.