PMC

(A) Representative Western blots of phosphorylated FAK (Tyr397) in untreated WT and Raldh2−/− yolk sacs and in RA-treated (in vivo) WT and Raldh2−/− yolk sacs at 9.5 dpc demonstrate lower levels of FAK phosphorylation (Tyr397) in Raldh2−/− mutants compared to WT which is increased back to WT levels after in vivo rescue with RA. Actin was used as the loading control. (B) Paxillin was immunoprecipitated from protein lysates from 9.5 dpc WT and Raldh2−/− yolk sacs and representative Western blots show levels of FAK and paxillin. Complex formation between FAK and paxillin occurs in WT yolk sacs during normal vascular remodeling (B, left panel). In contrast, the association between FAK and paxillin was diminished in Raldh2−/− yolk sacs that fail to undergo vascular remodeling (B, left panel). However, after in vivo RA-rescue of yolk sac vascular remodeling in Raldh2−/− mutants, complex formation between FAK and paxillin was observed at a level similar to that detected in WT yolk sacs (B, right panel).

(A) mRNA expression of vinculin, paxillin, FAK, and Tensin1 was assessed by qRT-PCR in yolk sacs harvested at 9.0 dpc (15-20 somite stage) versus 9.5 dpc (25-30 somite stage). Relative to corresponding WT yolk sacs isolated from littermates at either 9.0 dpc or 9.5 dpc, mRNA expression of these genes is increased in Raldh2−/− yolk sacs specifically at 9.5 dpc and not prior at 9.0 dpc. Transcript levels were normalized by using β-actin as the internal control. (B) Protein levels of FAK, paxillin, and vinculin were determined by Western blot in 8.0 dpc WT, Raldh2 heterozygote (+/−), and Raldh2 null (−/−) yolk sacs isolated at the 3-6 somite stage prior to cardiac function and establishment of blood flow. At this stage, protein levels in Raldh2 −/− yolk sacs were not found to be different from WT or Raldh2 +/− yolk sacs. Actin was used as the loading control.

(A) For normal vascular remodeling to proceed, RA is required for maintenance of the yolk sac endoderm, the site of production of multiple growth factors such as VEGF-A, IHH and bFGF. These factors act on the underlying mesoderm to promote endothelial cell migration via association of FAK with paxillin, FAK phosphorylation (Tyr397), and activation of Rac1 and its downsteam effector WAVE2. In the absence of RA signaling (B), decreased production of VEGF-A, IHH, and bFGF results due to loss of endoderm integrity. As a consequence, the association of FAK and paxillin is disrupted, activation of FAK and Rac1 is diminished, there is loss of WAVE2 expression, and increased activation of RhoA. The altered activation states of these signaling proteins result in increased vinculin-containing focal adhesion sites throughout the yolk sac mesoderm and ultimately lead to a defect in endothelial cell migration and lack of vascular remodeling.

(A-C) Representative images are shown of 9.5 dpc yolk sacs from WT (A) and Raldh2 −/− (B) embryos illustrating morphologic rescue (C) of the yolk sac vascular remodeling defect observed in Raldh2−/− yolk sacs. (D) Representative Western blots demonstrate that increased protein expression of vinculin in untreated Raldh2−/− yolk sacs is reduced to levels similar to WT yolk sacs after in vivo RA-rescue, however increased protein levels of paxillin and FAK are not reverted to WT levels after RA-rescue. (E-F) After RA-rescue, Raldh2−/− yolk sacs (F) at 9.5 dpc express minimal levels of vinculin (green) in the underlying mesodermal layer that is similar to that seen in the mesodermal layer of WT yolk sacs (E). Nuclei are stained with DAPI (blue). VE = visceral endoderm; Mes = mesoderm; L = blood vessel lumen; RA = retinoic acid; scale bars = 1000 μm (A, B, C); 10 μm (E, F).

(A) A WT control embryo cultured for 48 hrs. from 7.5-9.5 dpc in rat serum exhibits yolk sac vascular remodeling in contrast to (B) a WT embryo cultured in the presence of the Rac1-specific inhibitor NSC23766 (50 μM). (C—F) Whole-mounts of yolk sacs from WT untreated and NSC23766-treated embryos are shown. (C) PECAM-1 (red) delineates a large arborizing vitelline blood vessel and smaller blood vessels in an untreated WT embryo. (D) Diminished PECAM-1 expression correlates with the absence of large, medium, and small vessel formation in the yolk sac of a NSC23766-treated embryo. To verify Rac1 inhibition, WAVE2 (green) expression was compared in untreated and NSC23766-treated embryos. (G) WAVE2 (green) is expressed in the developing yolk sac blood vessels of untreated WT embryos. (H) In contrast, loss of WAVE2 expression was observed in the yolk sac from NSC23766-treated embryos. Nuclei are stained with DAPI (blue). Scale bars = 1000 μm (A, B); 90 μm (C, D, E); 30 μm (F).

(A-B) Representative images of WT (A) and Raldh2−/− (B) yolk sacs harvested from embryos cultured in the presence of exogenous rh-VEGF-A, human/mouse recombinant IHH, and rh-bFGF demonstate restoration of yolk sac vascular remodeling in Raldh2−/− mutants (B). (C) Levels of phosphorylated FAK (Tyr397) in WT vs Raldh2−/− yolk sacs from embryos cultured in the presence of exogenous VEGF-A, IHH, and bFGF were determined by Western blot. Increased levels of phosphorylated FAK (Tyr397) similar to WT levels were observed in Raldh2−/− mutants after VEGF-A, IHH, and bFGF mediated rescue. Actin was used as the loading control. (D-F) Immunofluorescence for phospho-FAK (Tyr397) (green) was performed on yolk sac sections from WT (D), Raldh2−/− (E), and VEGF-A+IHH+bFGF-rescued Raldh2−/− (F) yolk sacs. Phospho-FAK (Tyr397) (green) was expressed in endothelial cells within the mesodermal layer of WT yolk sacs (inset, D) and was diminished in Raldh2−/− mutants (inset, E). Endothelial expression of phospho-FAK (Tyr397) increased to WT levels in VEGF-A + IHH + bFGF-rescued Raldh2−/− mutants (inset, F). Nuclei are stained with DAPI (blue). VE = visceral endoderm; Mes = mesoderm; L = blood vessel lumen; scale bars = 1000 μm (A, B); 20 μm (D, E, F).

(A) Representative Western blots are shown that demonstrate increased protein levels of the focal adhesion proteins vinculin, paxillin, and FAK in Raldh2−/− yolk sacs versus WT yolk sacs at 9.5 dpc. Actin was used as the loading control. (B) Quantitation of Western blots for vinculin, paxillin, and FAK is represented by bar graphs indicating the mean band density +/− SEM measured for Raldh2−/− mutants relative to WT for each focal adhesion protein. Vinculin (p = 0.0027), paxillin (p = 0.032), and FAK (p = 0.0036) were significantly increased in Raldh2−/− yolk sacs as determined by a two-tailed student t-test (*p ≤ 0.05 as denoted by an asterisk; n = number of independent Western blot experiments repeated for each focal adhesion protein). Representative fluorescence confocal images are shown which demostrate the localization of vinculin (green) in WT (C) and Raldh2−/− yolk sacs (D). Vinculin expression is increased in the cytoplasm of yolk sac mesodermal cells and endoderm cell-cell borders at 9.5 dpc in Raldh2−/− mutants compared to WT. Nuclei are stained with DAPI (blue). VE = visceral endoderm; Mes = mesoderm; L = blood vessel lumen; scale bars = 10 μm.

(A) Activated and total Rac1 and (C) activated and total RhoA from pulldowns performed on lysates derived from WT vs. Raldh2−/− yolk sacs were determined at 9.5 dpc. RacGTP from WT and Raldh2−/− whole yolk sacs isolated at 9.5 dpc was pulled down utilizing the PAK-1 binding domain bound to glutathione-agarose beads. Similarly, RhoGTP was pulled down with the Rhotekin Rho binding domain bound to glutathione-agarose beads. Levels of activated Rac1 were found to be decreased in Raldh2−/− mutants relative to WT (A). Conversely, RhoA activation was increased in Raldh2−/− mutants relative to WT (C). (B, D) Quantitation of RacGTP (B) and RhoGTP (D) by band densitometry represented by bar graphs denoting fold differences in Rac1 and RhoA activation in 9.5 dpc Raldh2−/− yolk sacs (represented as the mean ± SEM) relative to WT. Data representing the activation for each RhoGTPase were averaged from 6 independent experiments (n = 6) and analyzed by Student’s two-tailed t-test; *p values ≤ 0.05 were considered significant and are denoted by an asterisk (Rac GTP: p = 0.025; RhoGTP: p = 0.016). (E-J) Representative yolk sac sections from WT and Raldh2−/− embryos double-labeled with PECAM-1 (red) and WAVE2 (green) are shown. PECAM-1 (red) marks the endothelial cell layer of yolk sac blood vessels from both WT (E) and Raldh2−/− (H) embryos. (F) WAVE2 (green) is present in the endothelium of the WT yolk sac blood vessel and (G) colocalizes with PECAM-1 (yellow). (I) In contrast, endothelial WAVE2 expression is lost in the Raldh2−/− yolk sac blood vessel and relocalizes to the cell-cell borders of the endoderm layer. Nuclei are stained with DAPI (blue). VE = visceral endoderm; Mes = mesoderm; L = blood vessel lumen; scale bars = 20 μm.