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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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Endothelial cell morphology is affected in the placental labyrinth of Hoxa13 homozygous mutants.(A, B) Transmission electron microscopy (TEM) reveals the initial elongation of the wild-type EC in the developing labyrinth vessels, whereas homozygous mutant littermates (B) exhibit rounded endothelia with attenuated cell bodies. Asterisks denote the endothelial cells; arrows depict the EC bodies in wild-type and mutant vessels. (C, D) Wild-type controls exhibit a mature elongated endothelial cell morphology by E13.5 (arrows), whereas homozygous mutant littermates (D) exhibit a severe loss in the elongated morphology (arrows), causing edema in the surrounding placental tissues (arrowheads). F = fetal vessel lumen; M = maternal space. (E, F) No differences in basement membrane (arrows) ultrastructure were detected in the labyrinth vessels between wild-type and homozygous mutant embryos, confirming that the loss of HOXA13 function is directly affecting endothelial morphology and function. F = fetal vessel lumen; N = endothelial cell nucleus. (G) Quantitative analysis of multiple labyrinth vessel TEM micrographs revealed that nearly 50% of the homozygous mutant endothelia exhibited an intermediate or severely abnormal morphology compared to wild-type or heterozygous mutant controls.
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pgen-1000073-g004: Endothelial cell morphology is affected in the placental labyrinth of Hoxa13 homozygous mutants.(A, B) Transmission electron microscopy (TEM) reveals the initial elongation of the wild-type EC in the developing labyrinth vessels, whereas homozygous mutant littermates (B) exhibit rounded endothelia with attenuated cell bodies. Asterisks denote the endothelial cells; arrows depict the EC bodies in wild-type and mutant vessels. (C, D) Wild-type controls exhibit a mature elongated endothelial cell morphology by E13.5 (arrows), whereas homozygous mutant littermates (D) exhibit a severe loss in the elongated morphology (arrows), causing edema in the surrounding placental tissues (arrowheads). F = fetal vessel lumen; M = maternal space. (E, F) No differences in basement membrane (arrows) ultrastructure were detected in the labyrinth vessels between wild-type and homozygous mutant embryos, confirming that the loss of HOXA13 function is directly affecting endothelial morphology and function. F = fetal vessel lumen; N = endothelial cell nucleus. (G) Quantitative analysis of multiple labyrinth vessel TEM micrographs revealed that nearly 50% of the homozygous mutant endothelia exhibited an intermediate or severely abnormal morphology compared to wild-type or heterozygous mutant controls.

Mentions: To determine the identity of the cells expressing HOXA13 in developing labyrinth, we examined whether these cells co-express the endothelial marker PECAM-1 (Figure 3) [35]–[37]. Characterization of HOXA13 and PECAM-1 expression confirmed that only the cells expressing PECAM-1 (cell surface) also express HOXA13 (nucleus), suggesting that HOXA13 is functioning in the labyrinth vascular endothelial cells (EC) (Figure 3A–3F). HOXA13 expression was not detected in the placental trophoblasts (data not shown). Interestingly, the elongated morphology normally attributed to the labyrinth vascular endothelia was also affected in homozygous mutants, which appeared rounded compared to controls in the E12.5 labyrinths (Figure 3C and 3D). To determine the onset of the EC phenotype, we characterized the labyrinth vessels at E10.5 (Figure 3E and 3F). At E10.5, the EC in both the heterozygous control and homozygous mutant labyrinths exhibited only the rounded EC morphology (Figure 3E and 3F). This result suggests that the vascular specification of the EC, as indicated by their elongated morphology, occurs between E10.5 and E12.5 and denotes when the loss of HOXA13 function phenotype first manifests in the developing labyrinth EC (Figure 3C–3F). Close examination of the affected EC using transmission electron microscopy confirmed the timing of the onset of this phenotype as wild type EC exhibited lengthening of the cell body as early at E11.5 whereas homozygous mutant EC exhibited shortened cell bodies that lacked uniform contact with the underlying vessel walls (Figure 4A–4D). Finally, consistent with the reduction in the EC cell body was the loss of vessel wall integrity in the E11.5 and E13.5 mutant vessels, resulting in extracellular edema between mutant labyrinth vessels and the underlying syncytiotrophoblasts, while edema was not detected in the wild type labyrinths (Figure 4A–4D).


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

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

Endothelial cell morphology is affected in the placental labyrinth of Hoxa13 homozygous mutants.(A, B) Transmission electron microscopy (TEM) reveals the initial elongation of the wild-type EC in the developing labyrinth vessels, whereas homozygous mutant littermates (B) exhibit rounded endothelia with attenuated cell bodies. Asterisks denote the endothelial cells; arrows depict the EC bodies in wild-type and mutant vessels. (C, D) Wild-type controls exhibit a mature elongated endothelial cell morphology by E13.5 (arrows), whereas homozygous mutant littermates (D) exhibit a severe loss in the elongated morphology (arrows), causing edema in the surrounding placental tissues (arrowheads). F = fetal vessel lumen; M = maternal space. (E, F) No differences in basement membrane (arrows) ultrastructure were detected in the labyrinth vessels between wild-type and homozygous mutant embryos, confirming that the loss of HOXA13 function is directly affecting endothelial morphology and function. F = fetal vessel lumen; N = endothelial cell nucleus. (G) Quantitative analysis of multiple labyrinth vessel TEM micrographs revealed that nearly 50% of the homozygous mutant endothelia exhibited an intermediate or severely abnormal morphology compared to wild-type or heterozygous mutant controls.
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pgen-1000073-g004: Endothelial cell morphology is affected in the placental labyrinth of Hoxa13 homozygous mutants.(A, B) Transmission electron microscopy (TEM) reveals the initial elongation of the wild-type EC in the developing labyrinth vessels, whereas homozygous mutant littermates (B) exhibit rounded endothelia with attenuated cell bodies. Asterisks denote the endothelial cells; arrows depict the EC bodies in wild-type and mutant vessels. (C, D) Wild-type controls exhibit a mature elongated endothelial cell morphology by E13.5 (arrows), whereas homozygous mutant littermates (D) exhibit a severe loss in the elongated morphology (arrows), causing edema in the surrounding placental tissues (arrowheads). F = fetal vessel lumen; M = maternal space. (E, F) No differences in basement membrane (arrows) ultrastructure were detected in the labyrinth vessels between wild-type and homozygous mutant embryos, confirming that the loss of HOXA13 function is directly affecting endothelial morphology and function. F = fetal vessel lumen; N = endothelial cell nucleus. (G) Quantitative analysis of multiple labyrinth vessel TEM micrographs revealed that nearly 50% of the homozygous mutant endothelia exhibited an intermediate or severely abnormal morphology compared to wild-type or heterozygous mutant controls.
Mentions: To determine the identity of the cells expressing HOXA13 in developing labyrinth, we examined whether these cells co-express the endothelial marker PECAM-1 (Figure 3) [35]–[37]. Characterization of HOXA13 and PECAM-1 expression confirmed that only the cells expressing PECAM-1 (cell surface) also express HOXA13 (nucleus), suggesting that HOXA13 is functioning in the labyrinth vascular endothelial cells (EC) (Figure 3A–3F). HOXA13 expression was not detected in the placental trophoblasts (data not shown). Interestingly, the elongated morphology normally attributed to the labyrinth vascular endothelia was also affected in homozygous mutants, which appeared rounded compared to controls in the E12.5 labyrinths (Figure 3C and 3D). To determine the onset of the EC phenotype, we characterized the labyrinth vessels at E10.5 (Figure 3E and 3F). At E10.5, the EC in both the heterozygous control and homozygous mutant labyrinths exhibited only the rounded EC morphology (Figure 3E and 3F). This result suggests that the vascular specification of the EC, as indicated by their elongated morphology, occurs between E10.5 and E12.5 and denotes when the loss of HOXA13 function phenotype first manifests in the developing labyrinth EC (Figure 3C–3F). Close examination of the affected EC using transmission electron microscopy confirmed the timing of the onset of this phenotype as wild type EC exhibited lengthening of the cell body as early at E11.5 whereas homozygous mutant EC exhibited shortened cell bodies that lacked uniform contact with the underlying vessel walls (Figure 4A–4D). Finally, consistent with the reduction in the EC cell body was the loss of vessel wall integrity in the E11.5 and E13.5 mutant vessels, resulting in extracellular edema between mutant labyrinth vessels and the underlying syncytiotrophoblasts, while edema was not detected in the wild type labyrinths (Figure 4A–4D).

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