The HAND1 basic helix-loop-helix transcription factor regulates trophoblast differentiation via multiple mechanisms

The basic helix-loop-helix (bHLH) transcription factor genes Hand1 and Mash 2 are essential for placental development in mice. Hand1 promotes different iation of trophoblast giant cells, whereas Mash2 is required for the mainte nance of giant cell precursors, and its overexpression prevents giant cell differentiation. We found that Hand1 expression and Mash2 expression overla p in the ectoplacental cone and spongiotrophoblast, layers of the placenta that contain the giant cell precursors, indicating that the antagonistic ac tivities of Hand1 and Mash2 must be coordinated. MASH2 and HAND1 both heter odimerize with E factors, bHLH proteins that are the DNA-binding partners f or most class B bHLH factors and which are also expressed in the ectoplacen tal cone and spongiotrophoblast. In vitro, HAND1 could antagonize MASH2 fun ction by competing for E-factor binding. However, the Hand1 mutant phenotyp e cannot be solely explained by ectopic activity of MASH2, as the Hand1 mut ant phenotype was not altered by further mutation of Mash2. Interestingly, expression of E-factor genes (ITF2 and ALF1) was down-regulated in the trop hoblast lineage prior to giant cell differentiation. Therefore, suppression of MASH2 function, required to allow giant cell differentiation, may occur in vivo by loss of its E-factor partner due to loss of its expression and/ or competition from HAND1. In giant cells, where E-factor expression was no t detected, HAND1 presumably associates with a different bHLH partner. This may account for the distinct functions of HAND1 in giant cells and their p recursors. We conclude that development of the trophoblast lineage is regul ated by the interacting functions of HAND1, MASH2, and their cofactors.