Novel Genes in Mesodermal Development

Glial-cell-line-derived neurotrophic factor (GDNF) signaling through the receptor tyrosine kinase Ret and its co-receptor Gfrα1 is indispensable for the ureteric bud (UB) outgrowth from Wolffian ducts during kidney differentiation. To search for novel genes regulated by GDNF in Wolffian ducts and early UB epithelium, we performed a genome wide analysis of Wolffian ducts exposed by GDNF in vitro, and identified 69 up-regulated genes. Plakophilin2 gene encoding for the armadillo protein- Pkp2 and Visinin like 1 gene, encoding for a neuronal calcium sensor protein- VSNL1 were among the highly up-regulated genes and their expression was missing in the Gdnf- null kidneys. Analysis of the Pkp2- null allele kidneys revealed a defect in ureteric branching. The Pkp2-/- kidney explants did not undergo UB formation and branching in vitro, but responded to GDNF by producing supernumerary buds, suggesting that Pkp2 is not essential for UB formation from the Wolffian duct. VSNL1 characterized the tip domain of the UB epithelium regardless of the inducer. In the UB tips, VSNL1 displayed a unique mosaic expression pattern that demonstrated the heterogeneity of cells in the tip. VSNL1 expression was mutually exclusive with β-catenin activity as shown in Bat-gal Wnt-reporter mice. VSNL1 was downregulated in both β-catenin stabilized and β-catenin deficient kidneys. Moreover, VSNL1 transduction compromised β-catenin stability in cultured cells. The results suggest an antagonistic effect between VSNL1 and β-catenin. The spatial and temporal expression of VSNL1 and its mRNA was further addressed during mouse embryogenesis. Vsnl1 was specifically confined to the cardiac anlagen. All myocardial cells that contributed to the remodeling of the venous pole of the heart were positive for Vsnl1. An expansion of the VSNL1 expression domain to the ventricles was seen soon after the first week of postnatal life, and this pattern was maintained to adulthood. The function of PlexinB2 during renal development was finally addressed by analyzing the kidney phenotype in Plxnb2- null mice. These embryos displayed hypoplastic kidneys, and occasional unilateral double kidneys and ureters. The defect in branching was intrinsic to the ureteric epithelium. By co-immunoprecipitation experiments, PlexinB2 was shown to interact with Ret suggesting that PlexinB2, activated by Sema4C ligand, may modulate ureteric branching by a direct regulation of Ret activity.Gliaperäisen neurotrofisen tekijän (GDNF) signalointi Ret tyrosiinikinaasin ja coreseptori GFRa1:n kautta on välttämätöntä uretersilmun (UB) kasvulle sikiön munuaisen erilaistumisen kuluessa. Löytääk-semme uusia, GDNF:n säätelemiä geenejä teimme koko genomin kattavan analyysin niistä geeneistä Wolffin tiehyeessä, jotka ovat in vitro GDNF:n säätelemiä. Löysimme 69 geeniä, joiden tasot nousivat GDNF:n vaikutuksesta. Plakofilin 2 geeni, joka koodaa armadillo proteiinia Pkp2, ja Visinin-like 1 geeni, joka koodaa neuro-naalista kalsium-sensori proteiinia Vsnl1, ilmentyivät voimakkaasti GDNF:n vaikutuksesta ja ne puut-tuivat GDNF-poistogeenisistä munuaisista. Pkp2- poistogeenisten munuaisten analyysi osoitti häiriön ureterin haarautumisessa, koska kudosviljel-mässä Pkp2 poistogeenisten hiirten Wolffin tiehyeet eivät muodostaneet UB:tä. Sen sijaan ne reagoivat GDNF:lle muodostamalla lukuisia uretersilmuja. Tämän perusteella Pkp2 ei vaikuta välttämättömältä UB:n muodostumiselle Wolffin tiehyeestä. Vsnl1 ilmentyi UB:n kärjessä riippumatta siitä millä menetelmällä UB:n muodostus oli saatu aikaan. Vsnl1:n ilmentyminen UB:n kärjessä oli mosaiikkinen, minkä perusteella kärjen solut ovat heterogeenisiä. Vsnl1:tä ilmentävät solut olivat peilikuva b-kateniinin aktiivisuutta heijastelevan Bat-gal histokemian kanssa. Vsnl1:n tasot UB:n kärjissä olivat voimakkaasti laskeneet geneettisissä malleissa, joissa β-kateniinin tasot olivat kohonneet tai sen ilmentyminen oli estetty. Lisäksi osoitimme, että Vsnl1:n il-mentäminen viljellyissä soluissa laski aktivoituneen β-kateniinin tasoa. Tulokset viittaavat siihen, että Vsnl1 antagonisoi β-kateniinin signalointia. Seuraavaksi tutkin Vsnl1:n ilmentymistä hiiren sikiön kehityksen aikana. Vsnl1 ilmentyi varhain sydä-men aiheessa. Kaikki sydänlihaksen solut, joista myöhemmin kehittyivät laskimorakenteet, ilmensivät Vsnl1:tä. Syntymän jälkeisen viikon kuluessa Vsnl:n ilmentyminen laajeni sydämen kammioihin ja tämä ilmentymiskuvio säilyi aikuisuuteen. Lopuksi selvitin PleksiiniB2:n tehtäviä munuaisen erilaistumisessa tutkimalla PleksiiniB2-poistogeenisiä hiiriä. Niissä ovat hypoplastiset munuaiset ja satunnaisesti myös kaksoisuretereja ja munuaisia. UB:n haarautumishäiriö johtui häiriöstä epiteelissä mutta munuaismesenkyymi oli normaali. Koimmunopre-sipitaatio osoitti, että PleksiiniB2 on vuorovaikutuksessa Ret:n kanssa ja PleksiiniB2:n aktivoituu munuaisessa todennäköisesti Sema4C:n vaikutuksesta. Sema4C säätelee ureterin haarautumista todennäköisesti vaikuttamalla suoraan Ret:n aktivaatioon

Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons

The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation

Development of the urogenital system is regulated via the 3 ' UTR of GDNF

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cellline derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3' untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf(hyper) mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3' UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.Peer reviewe

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

Development of the urogenital system is regulated via the 3 ' UTR of GDNF

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cellline derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3' untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf(hyper) mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3' UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

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PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia

Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

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