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HOXA13 Is essential for placental vascular patterning and labyrinth endothelial specification.

Shaut CA, Keene DR, Sorensen LK, Li DY, Stadler HS - PLoS Genet. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon, United States of America.

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.

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TIE-2 and LYVE-1 are co-expressed with HOXA13 in the labyrinth vascular endothelia and exhibit altered expression in E13.5 homozygous mutants.(A, B) Immunohistochemical analysis of TIE2 expression (red signal) in the placental labyrinth reveals reduced levels in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (C, D) Higher magnification image of the placental labyrinth confirms that TIE2 (red signal) is co-expressed with HOXA13 (green nuclear signal) in the labyrinth vascular endothelia. (E, F) Immunohistochemical analysis of LYVE-1 expression (red signal) in the placental labyrinths reveals elevated levels of LYVE-1 in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (G, H) Analysis of LYVE-1 expression at higher magnification (red signal) confirms that the PECAM-1–positive endothelial cells (green signal) co-express LYVE-1 and that LYVE-1 expression is elevated in the labyrinth vasculature of Hoxa13 homozygous mutants.
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pgen-1000073-g009: TIE-2 and LYVE-1 are co-expressed with HOXA13 in the labyrinth vascular endothelia and exhibit altered expression in E13.5 homozygous mutants.(A, B) Immunohistochemical analysis of TIE2 expression (red signal) in the placental labyrinth reveals reduced levels in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (C, D) Higher magnification image of the placental labyrinth confirms that TIE2 (red signal) is co-expressed with HOXA13 (green nuclear signal) in the labyrinth vascular endothelia. (E, F) Immunohistochemical analysis of LYVE-1 expression (red signal) in the placental labyrinths reveals elevated levels of LYVE-1 in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (G, H) Analysis of LYVE-1 expression at higher magnification (red signal) confirms that the PECAM-1–positive endothelial cells (green signal) co-express LYVE-1 and that LYVE-1 expression is elevated in the labyrinth vasculature of Hoxa13 homozygous mutants.

Mentions: Next, to validate that the genes detected by the microarray analysis were mis-expressed in the affected EC, we performed quantitative real-time PCR (qRT-PCR) using total RNA derived from affinity purified vascular labyrinth EC. In all cases, the mis-expression trend determined by the microarray analysis (increased or decreased in mutant placentas) was conserved (Table 1). Moreover, the enrichment of the EC also caused an increase in the detected fold-change differences between wild type and homozygous mutant EC, a finding consistent with an endothelial-specific expression pattern or function for the affected genes. Immunohistochemical analysis of TIE2, LYVE-1, NEUROPILIN-1, and ENPP2 confirmed the altered EC-specific expression levels detected by microarray and qRTPCR (Figures 9 and S1, Table 1, and unpublished data). In particular, the pro-vascular receptor tyrosine kinase, TIE2, was noticeably reduced in the mutant labyrinth EC which also express HOXA13 at E10.5–13.5 (Figures 9A–9D and S1).


HOXA13 Is essential for placental vascular patterning and labyrinth endothelial specification.

Shaut CA, Keene DR, Sorensen LK, Li DY, Stadler HS - PLoS Genet. (2008)

TIE-2 and LYVE-1 are co-expressed with HOXA13 in the labyrinth vascular endothelia and exhibit altered expression in E13.5 homozygous mutants.(A, B) Immunohistochemical analysis of TIE2 expression (red signal) in the placental labyrinth reveals reduced levels in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (C, D) Higher magnification image of the placental labyrinth confirms that TIE2 (red signal) is co-expressed with HOXA13 (green nuclear signal) in the labyrinth vascular endothelia. (E, F) Immunohistochemical analysis of LYVE-1 expression (red signal) in the placental labyrinths reveals elevated levels of LYVE-1 in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (G, H) Analysis of LYVE-1 expression at higher magnification (red signal) confirms that the PECAM-1–positive endothelial cells (green signal) co-express LYVE-1 and that LYVE-1 expression is elevated in the labyrinth vasculature of Hoxa13 homozygous mutants.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2367452&req=5

pgen-1000073-g009: TIE-2 and LYVE-1 are co-expressed with HOXA13 in the labyrinth vascular endothelia and exhibit altered expression in E13.5 homozygous mutants.(A, B) Immunohistochemical analysis of TIE2 expression (red signal) in the placental labyrinth reveals reduced levels in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (C, D) Higher magnification image of the placental labyrinth confirms that TIE2 (red signal) is co-expressed with HOXA13 (green nuclear signal) in the labyrinth vascular endothelia. (E, F) Immunohistochemical analysis of LYVE-1 expression (red signal) in the placental labyrinths reveals elevated levels of LYVE-1 in E13.5 Hoxa13 homozygous mutants compared to heterozygous controls. (G, H) Analysis of LYVE-1 expression at higher magnification (red signal) confirms that the PECAM-1–positive endothelial cells (green signal) co-express LYVE-1 and that LYVE-1 expression is elevated in the labyrinth vasculature of Hoxa13 homozygous mutants.
Mentions: Next, to validate that the genes detected by the microarray analysis were mis-expressed in the affected EC, we performed quantitative real-time PCR (qRT-PCR) using total RNA derived from affinity purified vascular labyrinth EC. In all cases, the mis-expression trend determined by the microarray analysis (increased or decreased in mutant placentas) was conserved (Table 1). Moreover, the enrichment of the EC also caused an increase in the detected fold-change differences between wild type and homozygous mutant EC, a finding consistent with an endothelial-specific expression pattern or function for the affected genes. Immunohistochemical analysis of TIE2, LYVE-1, NEUROPILIN-1, and ENPP2 confirmed the altered EC-specific expression levels detected by microarray and qRTPCR (Figures 9 and S1, Table 1, and unpublished data). In particular, the pro-vascular receptor tyrosine kinase, TIE2, was noticeably reduced in the mutant labyrinth EC which also express HOXA13 at E10.5–13.5 (Figures 9A–9D and S1).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon, United States of America.

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.

Show MeSH
Related in: MedlinePlus