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The Bmp4 signaling molecule is expressed in ventral splanchnic and branchial-arch mesoderm and outflow-tract (OFT) myocardium, suggesting a role for Bmp4 in OFT development. Inactivation of Bmp4 in the caudal branchial arch and splanchnic mesoderm and OFT myocardium by using a conditional null allele of Bmp4 and the Nkx2.5 cre recombinase allele resulted in abnormal morphogenesis of branchialarch arteries (BAAs) and defective OFT septation. Expression of aortic-sac myocardial markers was reduced and expression of the sm22 LacZ transgene, a smooth-muscle marker, was attenuated in BAAs and conotruncus of Nkx2.5 cre ; Bmp4 conditional mutants. Moreover, we found tissue-specific functions for Bmp4 in the regulation of cellular proliferation and apoptosis. We also demonstrate a strong genetic interaction between Bmp4 and Bmp7 in OFT development. Our findings uncover a previously uncharacterized function for Bmp4 in vascular remodeling of the BAAs, and they show definitively that Bmp4, in cooperation with Bmp7, has a central role in OFT septation. T he vertebrate heart can be subdivided into inflow, outflow, and primitive-ventricular regions (1). The cardiac outflow tract (OFT), which develops from the anterior part of the linear heart tube, forms the right-sided conotruncal region after heart looping. Initially unseptated, the OFT divides into the pulmonary trunk (PT) and aorta, and it is critical for separation of postnatal pulmonary and systemic circulation. Congenital OFT malformations are common, making an understanding of the genetic pathways regulating OFT development an important goal in developmental biology and clinical medicine.At defined areas of the OFT, endocardial cells undergo an epithelial to mesenchymal transformation (perhaps in response to a signal from overlying myocardium) and invade the intervening space to form the endocardial cushions. The cardiac neural crest also invades the forming aorto-pulmonary (AP) septum and OFT cushions (2). The OFT myocardium receives an additional input from splanchnic and branchial-arch mesoderm, the anterior or secondary heart field (SHF), which may be important for OFT lengthening and morphogenesis (3).Bmp4 is a member of the Bone morphogenetic protein (Bmp) subclass of transforming growth factor type ␤ (TGF-␤)-signaling molecules (4). Bmp4 expression in splanchnic and branchial-arch mesoderm (which contributes to OFT myocardium) and within the OFT myocardium itself suggests a role in OFT morphogenesis (ref.5 and see below). Investigation of Bmp4 function in cardiac development has been hampered by the early embryonic lethality of Bmp4 null mutant embryos (6). Recent work analyzing an allele of the ubiquitously expressed Bmp type 2 receptor (Bmpr2), containing a partial ectodomain deletion, revealed defective proximal OFT septation in mouse embryos, providing insight into Bmp function in the OFT (7). However, because Bmpr2 is broadly expressed, the developmental mechanisms responsible for the cushion defects remain unclear. Overexpression of noggin in chick embry...

Pitx2, a paired-related homeobox gene that encodes multiple isoforms, is the gene mutated in the haploinsufficient Rieger Syndrome type 1 that includes dental, ocular and abdominal wall anomalies as cardinal features. Previous analysis of the craniofacial phenotype of Pitx2-null mice revealed that Pitx2 was both a positive regulator of Fgf8 and a repressor of Bmp4-signaling,suggesting that Pitx2 may function as a coordinator of craniofacial signaling pathways. We show that Pitx2 isoforms have interchangeable functions in branchial arches and that Pitx2 target pathways respond to small changes in total Pitx2 dose. Analysis of Pitx2allelic combinations that encode varying levels of Pitx2 showed that repression of Bmp signaling requires high Pitx2 while maintenance of Fgf8 signaling requires only low Pitx2. Fate-mapping studies with a Pitx2 cre recombinase knock in allele revealed that Pitx2 daughter cells are migratory and move aberrantly in the craniofacial region of Pitx2 mutant embryos. Our data reveal that Pitx2 function depends on total Pitx2 dose and rule out the possibility that the differential sensitivity of target pathways was a consequence of isoform target specificity. Moreover, our results uncover a new function of Pitx2 in regulation of cell motility in craniofacial development.

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