The pitx2 homeobox protein is required early for endoderm formation and nodal signaling

Nodal and Nodal-related factors play fundamental roles in a number of devel opmental processes, including mesoderm and endoderm formation, patterning o f the anterior neural plate, and determination of bilateral asymmetry in ve rtebrates. pitx2, a paired-like homeobox gene, has been proposed to act dow nstream of Nodal in the gene cascade providing left-right cues to the devel oping organs. Here, we report that pitx2 is required early in the Nodal sig naling pathway for specification of the endodermal and mesodermal germ laye rs. We found that pitx2 is expressed very early during Xenopus and zebrafis h development and in many regions where Nodal signaling is required, includ ing the presumptive mesoderm and endoderm at the blastula and gastrula stag es and the prechordal mesoderm at later stages. In Xenopus embryos, overexp ression of pitx2 caused ectopic expression of goosecoid and sox-17 beta and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus ani mal cap explants partially mimics the effects of Nodal overexpression, sugg esting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction. We fu rther demonstrate that pitx2 is induced by Nodal signaling in Xenopus anima l caps and that the early expression of zebrafish pitx2 is absent when the Nodal signaling pathway is inactive. Inhibition of pitx2 function using a c himeric EnR-pitx2 blocked specification of the mesoderm and endoderm and ca used severe embryonic defects resembling those seen when Nodal signaling is inhibited. Following inhibition of pitx2 function, the fate of ventral veg etal blastomeres was shifted from an endodermal to a more mesodermal fate, an effect that was reversed by wild-type pitx2. Finally, we show that inhib ition of pitx2 function interferes with the response of cells to Nodal sign aling. Our results provide direct evidence that pitx2 function is required for normal specification of the endodermal and mesodermal germ layers. (C) 2000 Academic Press.