Wnt4 is essential to normal mammalian lung development

AbstractWnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4−/− embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4−/− lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein–Induced Retinal AngiogenesisHighlights

OBJECTIVE: Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2, and Alk3 in mouse retinal vessels. APPROACH AND RESULTS: Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2. Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. CONCLUSIONS: Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels

Annexin A3 Regulates Early Blood Vessel Formation.

Annexins are a large family of calcium binding proteins that associate with cell membrane phospholipids and are involved in various cellular processes including endocytosis, exocytosis and membrane-cytoskeletal organization. Despite studies on numerous Annexin proteins, the function of Annexin A3 (Anxa3) is largely unknown. Our studies identify Anxa3 as a unique marker of the endothelial and myeloid cell lineages of Xenopus laevis during development. Anxa3 transcripts are also detected in endothelial cells (ECs) of zebrafish and mouse embryos, suggesting an important evolutionary function during formation of blood vessels. Indeed, Anxa3 loss-of-function experiments in frog embryos reveal its critical role during the morphogenesis of early blood vessels, as angioblasts in MO injected embryos fail to form vascular cords. Furthermore, in vitro experiments in mammalian cells identify a role for Anxa3 in EC migration. Our results are the first to reveal an in vivo function for Anxa3 during vascular development and represent a previously unexplored aspect of annexin biology

Anxa3 gain-of-function has no effect on developing blood vessels <i>in vivo</i>.

<p>(A,B) Whole-mount <i>in situ</i> hybridization for Aplnr expression in GFP and Anxa3 mRNA injected embryos (stage 34, lateral views). No changes in overall vessel morphology or levels of Aplnr expression were observed. (C) The percent (%) of GFP and Anxa3 mRNA injected embryos displaying normal vascular formation, as assessed by Aplnr expression, are shown. Aortic arches, aa; intersomitic vessel, isv; n, number of embryos assayed; posterior cardinal vein, pcv and vascular plexus, vp.</p

Expression of Anxa3 in the endothelial lineage is conserved across multiple species.

<p>(A,B) Whole-mount <i>in situ</i> hybridization analysis for vascular endothelial growth factor receptor 2 (VegfR2) and Anxa3b expression in <i>Danio rerio</i> embryos, 24 hours post fertilization (hpf). Close-up, lateral views of the tail regions are shown. Anxa3b transcripts are restricted to the developing dorsal aorta (DA), whereas VegfR2 is also expressed in the intersomitic vessels (isv). (C-F) Embryonic (E) day 8.25 mouse embryos were assayed for Anxa3 and the vascular marker PlexinD1 by whole-mount <i>in situ</i> hybridization (anterior views). Similar to PlexinD1, Anxa3 is observed in the paired dorsal aortae (da, arrows) and heart (h) region of the embryo. Anxa3 is absent in the extra-embryonic vessels of the yolk sac (ys) that are marked by PlexinD1 expression. This is highlighted in close-up views of ys from PlexinD1 and Anxa3 stained E8.25 embryos (D,F). (G,H) E9.25 heterozygous (^+/-) and homozygous (^-/-) VegfR2 embryos analyzed for Anxa3 expression by <i>in situ</i> hybridization (lateral views). Anxa3 is detected in the dorsal aorta (DA), intersomitic vessels (isv) and heart (h) region of VegfR2^+/- embryos, while VegfR2^-/- null embryos, which lack all blood vessels, displayed no observable vascular staining of Anxa3 (background staining is detected in the head and just outside the heart region).</p

Anxa3 is expressed in endothelial and myeloid cells of developing <i>Xenopus laevis embryos</i>.

<p>Whole-mount <i>in situ</i> hybridization analysis of Etv2, SpiB and Anxa3 transcripts in <i>Xenopus</i> embryos at stages (St) 20/21, 28/29 and 33/34 (A-M). Lateral (A,C,D,E,G,H,I,K,L) and ventral (B,F,J) views show a close association of Anxa3 with the developing endothelial (Etv2) and myeloid (SpiB) lineages. At early stages (I,J), Anxa3 is expressed in the anterior ventral blood island (aVBI), which consists of adjacent Etv2 (A,B) and SpiB (E,F) expressing cells. At St 28/29 and 33/34, Anxa3 (K,L) is detected in the major vascular structures highlighted by Etv2 expression (C,D): the posterior cardinal vein (pcv), vascular plexus flank (vp) and intersomitic vessels (isv). (M) High magnification view of an embryo showing strong expression of Anxa3 in the pcv and isv. Anxa3 is also observed in the myeloid cells (marked by SpiB) that migrate throughout the entire embryo (compare the G,H to K,L). Throughout development, Anxa3 is also present in the cement gland (cg).</p

Inhibition of ANXA3 function leads to vascular disruptions.

<p>Inhibition of ANXA3 function leads to vascular disruptions.</p

Resolution of Defective Dorsal Aortae Patterning in Sema3E-Deficient Mice Occurs Via Angiogenic Remodeling

10.1002/dvdy.23949DEVELOPMENTAL DYNAMICS2425580-59

<i>Lyve1^wt/Cre;Vegfr2^flox/flox</i> embryos exhibit lymphatic defects.

<p>(A,B) Immunofluorescence staining for Lyve-1 showing jugular lymph sacs in E14.5 <i>Vegfr2^flox/flox</i> and <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> embryos. (C) Graph showing that lymph sac area is not different between <i>Vegfr2^flox/flox</i> (380,110±76,279 pixels²; n = 8) and <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> embryos (287,350±59,931 pixels²; n = 5). (D,E) Images of back skin from E14.5 <i>Vegfr2^flox/flox</i> and <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> embryos stained with antibodies against podoplanin (green) and phospho-histone H3 (red). (F) At E14.5, there are significantly fewer lymphatic branch points in <i>Lyve-^wt/Cre;Vegfr2^flox/flox</i> embryos (11.63±0.5239; n = 3) than in <i>Vegfr2^flox/flox</i> embryos (19.37±0.5783; n = 3). (G) At E14.5, lymphatic vessel diameter is not significantly different between <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> (26.00±5.859 pixels; n = 3) and <i>Vegfr2^flox/flox</i> embryos (23.75±1.652 pixels; n = 4). (H) Graph showing that there are fewer proliferating lymphatic endothelial cells in <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> embryos than in <i>Vegfr2^flox/flox</i> embryos at E14.5 and E16.5. At E14.5, 19.45% of LECs in <i>Vegfr2^flox/flox</i> embryos (n = 4) were phospho-Histone H3-positive whereas 9.73% of LECs in <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> embryos (n = 4) were phospho-histone H3-positive. At E16.5, 26.94% of LECs in <i>Vegfr2^flox/flox</i> embryos (n = 5) were phospho-Histone H3-positive whereas 13.69% of LECs in <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> embryos (n = 7) were phospho-histone H3-positive. (I) Graph showing the percent of viable <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> mice at different developmental stages. (J-M) Images of non-edematous E14.5 (J,K), E16.5 (L) and E18.5 (M) <i>Vegfr2^flox/flox</i> and <i>Lyve-1^wt/Cre;Vegfr2^flox/flox</i> embryos. ** indicates P || 0.01; *** indicates P || 0.001.</p