HOXA13 Is Essential for Placental Vascular Patterning and Labyrinth Endothelial Specification

Abstract

In eutherian mammals, embryonic growth and survival is dependent on the formation of the placenta, an organ that facilitates the efficient exchange of oxygen, nutrients, and metabolic waste between the maternal and fetal blood supplies. Key to the placenta's function is the formation of its vascular labyrinth, a series of finely branched vessels whose molecular ontogeny remains largely undefined. In this report, we demonstrate that HOXA13 plays an essential role in labyrinth vessel formation. In the absence of HOXA13 function, placental endothelial cell morphology is altered, causing a loss in vessel wall integrity, edema of the embryonic blood vessels, and mid-gestational lethality. Microarray analysis of wild-type and mutant placentas revealed significant changes in endothelial gene expression profiles. Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia. ChIP analysis of HOXA13–DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo. In vitro, HOXA13 binds sequences present in the Tie2 and Foxf1 promoters with high affinity (K _d = 27–42 nM) and HOXA13 can use these bound promoter regions to direct gene expression. Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.

Author Summary

Defects in placental development are a common cause of mid-gestational lethality. Key to the placenta's function is its vascular labyrinth, a series of finely branched vessels that facilitate the efficient exchange of gases, nutrients, and metabolic waste between the maternal and fetal blood supplies. In this study, we identify a novel role for the transcription factor HOXA13 in formation of the placental vascular labyrinth. In the absence of HOXA13 function, labyrinth vessel branching and endothelial specification is compromised, causing mid-gestational lethality due to placental insufficiency. Analysis of the genes affected by the loss of HOXA13 function revealed significant reductions in the expression of several pro-vascular genes, including Tie2 and Foxf1. Analysis of the Tie2 and Foxf1 promoters confirmed that HOXA13 binds sites present in each promoter with high affinity in the placenta, and in vitro, HOXA13 can use these bound sequences to regulate gene expression. These results suggest that Tie2 and Foxf1 are direct transcriptional targets of HOXA13 in the developing placental labyrinth, providing a novel transcriptional pathway to consider when examining pathologies of the placenta and placental insufficiency, as well as the evolutionary mechanisms required for the emergence of the vascular placenta in eutherian mammals.