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Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis <em>In vivo</em>

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Newcastle University 

Endoglin is an auxiliary receptor for TGFbeta signalling. Heterozygous germline Endoglin mutations have been identified in patients with the vascular abnormality, Hereditary Haemorrhagic Telangiectasia. Endoglin is upregulated in endothelial cells during angiogenesis and loss of Endoglin in the mouse results in embryonic lethality at mid-gestation. This phenotype points to an important role of Endoglin in new blood vessel formation but precludes analysis at later stages in development and in postnatal life. To bypass this limitation and allow further investigations of the function of Endoglin we have generated a floxed Endoglin allele in which loxP sites flank exons 5 and 6. Mice homozygous for this allele are normal and in the presence of appropriate Cre lines will allow time and cell specific Endoglin deletion for in vivo analysis of function in cardiovascular development and disease.

Generation of a floxed allele of the mouse Endoglin gene.

Endogline est un r&eacute;cepteur auxiliaire de signalisation TGF. Lign&eacute;e germinale h&eacute;t&eacute;rozygotes endogline mutations ont &eacute;t&eacute; identifi&eacute;es chez des patients avec l&#39;anomalie vasculaire, t&eacute;langiectasie h&eacute;morragique h&eacute;r&eacute;ditaire. Endogline est augment&eacute;e dans les cellules endoth&eacute;liales au cours de l&#39;angiogen&egrave;se et la perte de l&#39;endogline dans les r&eacute;sultats de la souris dans la d&eacute;termination de la l&eacute;talit&eacute; embryonnaire au milieu. Ce ph&eacute;notype pointe vers un r&ocirc;le important de l&#39;endogline dans la formation de vaisseaux sanguins, mais s&#39;oppose &agrave; l&#39;analyse &agrave; un stade ult&eacute;rieur, au d&eacute;veloppement et &agrave; la vie postnatale. Pour contourner cette limitation et permettre de nouvelles recherches de la fonction de l&#39;endogline, nous avons g&eacute;n&eacute;r&eacute; un all&egrave;le d&#39;endogline floxed quel loxP sites flanquent exons 5 et 6. Souris homozygotes pour cet all&egrave;le sont normaux et en pr&eacute;sence de Cre appropri&eacute; lignes permettra de temps et de cellules sp&eacute;cifique endogline suppression pour l&#39;analyse in vivo de la fonction en d&eacute;veloppement cardiovasculaire et la maladie.

G&eacute;n&eacute;ration d&#39;un all&egrave;le du g&egrave;ne endogline souris de floxed.

Endoglin &egrave; un recettore ausiliario per la segnalazione del TGFbeta. Linea germinale eterozigote Endoglin sono state identificate mutazioni in pazienti con anormalit&agrave; vascolari, teleangiectasia emorragica ereditaria. Endoglin &egrave; sovraregolato nelle cellule endoteliali durante l&#39;angiogenesi e la perdita di Endoglin nei risultati del mouse di letalit&agrave; embrionale a mid-gestation. Questo fenotipo punta a un ruolo importante di Endoglin nella formazione di sangue nuovo vaso ma esclude analisi nelle fasi successive nello sviluppo e nella vita postnatale. Per aggirare questa limitazione e consentire ulteriori indagini della funzione di Endoglin noi abbiamo generato un allele Endoglin floxed in cui loxP siti fiancheggiano esoni 5 e 6. Topi omozigoti per questo allele sono normali e in presenza di adeguata Cre linee permetter&agrave; tempo e cellule specifiche eliminazione di Endoglin per l&#39;analisi in vivo della funzione sviluppo cardiovascolare e malattia.

Generazione di un allele floxed del gene della Endoglin del mouse.

Arteriovenous malformations (AVMs) result in anomalous direct blood flow between arteries and veins, bypassing the normal capillary bed. Depending on size and location, AVMs may lead to severe clinical effects including systemic cyanosis (pulmonary AVMs), hemorrhagic stroke (cerebral AVMs) and high output cardiac failure (hepatic AVMs). The factors leading to AVM formation are poorly understood, but patients with the familial disease hereditary hemorrhagic telangiectasia (HHT) develop AVMs at high frequency. As most HHT patients have mutations in ENG (endoglin) or ACVRL1 (activin receptor-like kinase 1), a better understanding of the role of these genes in vascular development is likely to reveal the etiology of AVM formation.

Pathogenesis of arteriovenous malformations in the absence of endoglin.

Malformations art&eacute;rioveineuses (MAV) r&eacute;sultent du d&eacute;bit sanguin directe anormale entre les art&egrave;res et les veines, sans passer par le lit capillaire normal. Selon la taille et l&#39;emplacement, SMAV peut entra&icirc;ner des effets cliniques graves y compris syst&eacute;mique cyanose (MAV pulmonaires), accident vasculaire c&eacute;r&eacute;bral h&eacute;morragique (MAV c&eacute;r&eacute;bral) et l&#39;insuffisance cardiaque de haut rendement (MAV h&eacute;patiques). Les facteurs conduisant &agrave; AVM formation sont mal compris, mais les patients atteints de la t&eacute;langiectasie h&eacute;morragique h&eacute;r&eacute;ditaire maladie familiale (VRD) d&eacute;veloppent MAV &agrave; haute fr&eacute;quence. Comme la plupart des patients de VRD pr&eacute;sentent des mutations dans ENG (endogline) ou ACVRL1 (activin r&eacute;cepteur-like kinase 1), une meilleure compr&eacute;hension du r&ocirc;le de ces g&egrave;nes dans le d&eacute;veloppement vasculaire est susceptible de r&eacute;v&eacute;ler l&#39;&eacute;tiologie de la formation de l&#39;AVM.

Pathogen&egrave;se des malformations art&eacute;rio-veineuses en l&#39;absence de l&#39;endogline.

Malformazioni artero-venose (MAV) risultano nel flusso di sangue diretto anomale tra arterie e vene, ignorando il letto capillare normale. A seconda della dimensione e posizione, SMA pu&ograve; portare a gravi effetti clinici tra cui cianosi sistemica (MAV polmonari), ictus emorragico (MAV cerebrale) e guasto cardiaco ad alto rendimento (MAV epatiche). I fattori che portano alla formazione di AVM sono poco comprensibili, ma i pazienti con teleangiectasia emorragica ereditaria malattia familiare (HHT) sviluppano MAV ad alta frequenza. Come la maggior parte dei pazienti HHT hanno mutazioni in ITA (endoglin) o ACVRL1 (activin receptor-like chinasi 1), una migliore comprensione del ruolo di questi geni nello sviluppo vascolare rischia di rivelare l&#39;eziologia della formazione di AVM.

Patogenesi delle malformazioni artero-venose in assenza di endoglin.

TGFÎ² signalling is required for normal cardiac development. To investigate which cell types are involved, we used mice carrying a floxed Type II TGFÎ² receptor (Tgfbr2fl) allele and Cre-lox genetics to deplete this receptor in different regions of the heart. The three target tissues and corresponding Cre transgenic lines were atrioventricular myocardium (using cGata6-Cre), ventricular myocardium (using Mlc2v-Cre), and vascular endothelium (using tamoxifen-activated Cdh5(PAC)-CreERT2). Spatio-temporal Cre activity in each case was tracked via lacZ activation from the Rosa26R locus. Atrioventricular-myocardial-specific Tgfbr2 knockout (KO) embryos had short septal leaflets of the tricuspid valve, whereas ventricular myocardial-specific KO embryos mainly exhibited a normal cardiac phenotype. Inactivation of Tgfbr2 in endothelial cells from E11.5 resulted in deficient ventricular septation, accompanied by haemorrhage from cerebral blood vessels. We conclude that TGFÎ² signalling through the Tgfbr2 receptor, in endothelial cells, plays an important role in cardiac development, and is essential for cerebral vascular integrity.

The TGFÎ² type II receptor plays a critical role in the endothelial cells during cardiac development.

TGF&beta; signalisation est n&eacute;cessaire pour le d&eacute;veloppement cardiaque normal. Afin d&#39;&eacute;tudier quels types de cellules sont impliqu&eacute;s, nous avons utilis&eacute; des souris portant un all&egrave;le de r&eacute;cepteurs (Tgfbr2fl) floxed Type II TGF&beta; et g&eacute;n&eacute;tique de Cre-lox &agrave; &eacute;puiser ce r&eacute;cepteur dans diff&eacute;rentes r&eacute;gions du c&oelig;ur. Les trois ciblent tissus et correspondantes Cre lign&eacute;es transg&eacute;niques &eacute;taient auriculo-ventriculaire myocarde (&agrave; l&#39;aide de la cGata6-Cre), myocarde ventriculaire (&agrave; l&#39;aide de la Mlc2v-Cre) et endoth&eacute;lium vasculaire (&agrave; l&#39;aide de Cdh5(PAC)-CreERT2) activ&eacute;s par le tamoxif&egrave;ne. Spatio-temporelle Cre activit&eacute; dans chaque cas a &eacute;t&eacute; suivie par l&#39;activation du g&egrave;ne lacZ du locus Rosa26R. Les embryons de knockout (KO) de Tgfbr2 auriculo-ventriculaire-myocardique sp&eacute;cifiques ont abr&eacute;g&eacute; folioles septales de la valve tricuspide, alors que les embryons de KO sp&eacute;cifique du myocarde ventriculaires ont principalement un ph&eacute;notype cardiaque normal. Inactivation de Tgfbr2 dans les cellules endoth&eacute;liales de E11.5 a entra&icirc;n&eacute; d&eacute;ficient cloisonnement ventriculaire, accompagn&eacute;e d&#39;h&eacute;morragie des vaisseaux sanguins c&eacute;r&eacute;braux. Nous concluons que le TGF&beta; signalisation via le r&eacute;cepteur Tgfbr2, dans les cellules endoth&eacute;liales, joue un r&ocirc;le important dans le d&eacute;veloppement cardiaque et est essentiel pour l&#39;int&eacute;grit&eacute; vasculaire c&eacute;r&eacute;brale.

Le r&eacute;cepteur II de TGF&beta; type joue un r&ocirc;le essentiel dans les cellules endoth&eacute;liales au cours du d&eacute;veloppement cardiaque.

Segnalazione di TGF&beta; &egrave; necessaria per lo sviluppo cardiaco normale. Per indagare su quali tipi di cellule sono coinvolte, abbiamo utilizzato topi portatori di un allele di recettore (Tgfbr2fl) tipo II TGF&beta; floxed e Cre-lox genetica per esaurire questo recettore in diverse regioni del cuore. I tre tessuti di destinazione e corrispondenti linee transgeniche Cre erano miocardio atrioventricolare (utilizzando cGata6-Cre), miocardio ventricolare (utilizzando Mlc2v-Cre) e dell&#39;endotelio vascolare (utilizzando tamoxifene-attivato Cdh5(PAC)-CreERT2). Spazio-temporale Cre attivit&agrave; in ogni caso &egrave; stato monitorato attraverso lacZ attivazione dal locus Rosa26R. Embrioni di knockout (KO) Tgfbr2 atrioventricolare del miocardio specifico ha avuto breve volantini settale della valvola tricuspide, mentre gli embrioni di KO specifiche del miocardio ventricolari esposta principalmente un fenotipo cardiaco normale. Inattivazione di Tgfbr2 in cellule endoteliali da E11.5 ha portato nel setto ventricolare carenza, accompagnata da emorragia da vasi sanguigni cerebrali. Concludiamo TGF&beta; segnalazione attraverso il recettore Tgfbr2, nelle cellule endoteliali, gioca un ruolo importante nello sviluppo cardiaco che &egrave; essenziale per l&#39;integrit&agrave; vascolare cerebrale.

Il TGF&beta; type II receptor gioca un ruolo critico in cellule endoteliali durante lo sviluppo cardiaco.

TGFÎ² signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life. To address this we used a tamoxifen inducible Cre-LoxP strategy in neonatal mice to deplete the TypeII TGFÎ² receptor (Tgfbr2) specifically in endothelial cells. This resulted in multiple micro-haemorrhages, and glomeruloid-like vascular tufts throughout the cerebral cortices and hypothalamus of the brain as well as in retinal tissues. A detailed examination of the retinal defects in these mutants revealed that endothelial adherens and tight junctions were in place, pericytes were recruited and there was no failure of vascular smooth muscle differentiation. However, the deeper retinal plexus failed to form in these mutants and the angiogenic sprouts stalled in their progress towards the inner nuclear layer. Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells. This evidence suggests that TGFÎ² signalling is not required for vessel maturation, but is essential for the organised migration of endothelial cells as they begin to enter the deeper layers of the retina. Thus, TGFÎ² signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life.

Endothelial expression of TGFÎ² type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system.

TGF&beta; dans les cellules endoth&eacute;liales est important pour l&#39;angiogen&egrave;se dans le d&eacute;veloppement embryonnaire pr&eacute;coce, mais on conna&icirc;t mal son r&ocirc;le dans la vie postnatale pr&eacute;coce. Pour rem&eacute;dier &agrave; cette nous avons utilis&eacute; une strat&eacute;gie de Cre-LoxP inductible de tamoxif&egrave;ne souris n&eacute;onatales pour &eacute;puiser le r&eacute;cepteur de TypeII TGF&beta; (Tgfbr2) sp&eacute;cifiquement dans les cellules endoth&eacute;liales. Il en est r&eacute;sult&eacute; plusieurs micro-h&eacute;morragies et touffes vasculaires glomeruloid-comme dans tout le cortex c&eacute;r&eacute;braux et l&#39;hypothalamus du cerveau ainsi que dans les tissus r&eacute;tiniens. Un examen d&eacute;taill&eacute; des anomalies r&eacute;tiniennes chez ces mutants a r&eacute;v&eacute;l&eacute; qu&#39;adherens endoth&eacute;liale et des jonctions serr&eacute;es &eacute;taient en place, ont &eacute;t&eacute; recrut&eacute;s des p&eacute;ricytes et il n&#39;y n&#39;avait aucune d&eacute;faillance de la diff&eacute;renciation du muscle lisse vasculaire. Cependant, le plexus r&eacute;tiniens plus profond n&#39;a pas pu former dans ces mutants et les choux angiog&eacute;nique bloqu&eacute;s dans leur progression vers la couche nucl&eacute;aire interne. Au contraire les cellules endoth&eacute;liales leaders forment touffes glom&eacute;rulaires avec les cellules des muscles lisses associ&eacute;es. Cette &eacute;vidence sugg&egrave;re que le TGF&beta; signalisation n&#39;est pas n&eacute;cessaire pour la maturation du navire, mais est essentiel pour la migration organis&eacute;e des cellules endoth&eacute;liales lorsqu&#39;elles commenceront &agrave; entrer dans les couches profondes de la r&eacute;tine. Signalisation de TGF&beta; est donc essentiel dans les cellules endoth&eacute;liales vasculaires pour le maintien de l&#39;int&eacute;grit&eacute; vasculaire &agrave; l&#39;angiog&eacute;nique avant qu&#39;il migre dans le d&eacute;veloppement des tissus nerveux en d&eacute;but de vie postnatal.

Expression endoth&eacute;liale de r&eacute;cepteur de TGF&beta; type II est n&eacute;cessaire pour maintenir l&#39;int&eacute;grit&eacute; vasculaire au cours du d&eacute;veloppement postnatal du syst&egrave;me nerveux central.

TGF&beta; segnalazione in cellule endoteliali &egrave; importante per l&#39;angiogenesi precoce dello sviluppo embrionale, ma poco si sa circa il suo ruolo nei primi anni di vita postnatale. Per risolvere questo problema abbiamo usato una strategia Cre-LoxP inducibile tamoxifene nei topi neonatali per esaurire il recettore TypeII TGF&beta; (Tgfbr2) in particolare nelle cellule endoteliali. Ci&ograve; ha provocato molteplici micro-emorragie e ciuffi vascolare glomeruloid-come in tutto le cortecce cerebrale e ipotalamo del cervello cos&igrave; come nei tessuti della retina. Un esame dettagliato dei difetti della retina in questi mutanti ha rivelato che adherens endoteliale e giunzioni strette erano a posto, sono stati reclutati pericytes e non non c&#39;era nessun guasto della muscolatura liscia vascolare differenziazione. Tuttavia, il plesso pi&ugrave; profondo della retina non &egrave; riuscito a formare in questi mutanti e i germogli di angiogenic in stallo nei loro progressi verso lo strato nucleare interno. Invece le cellule endoteliali leader formano glomerulare ciuffi con cellule muscolari lisce associato. Questa evidenza suggerisce che il TGF&beta; segnalazione non &egrave; necessario per la maturazione del vaso, ma &egrave; essenziale per la migrazione delle cellule endoteliali organizzata come cominciano a entrare in strati pi&ugrave; profondi della retina. Cos&igrave;, segnalazione di TGF&beta; &egrave; essenziale nelle cellule endoteliali vascolari per mantenere l&#39;integrit&agrave; vascolare angiogenic davanti come migra nello sviluppo dei tessuti neurali nei primi anni di vita postnatale.

Endoteliali espressione del recettore II tipo TGF&beta; &egrave; necessaria per mantenere l&#39;integrit&agrave; vascolare durante lo sviluppo postnatale del sistema nervoso centrale.

Article	Year
Generation of a floxed allele of the mouse Endoglin gene. Genesis (New York, N.Y. : 2000)\| PubMed ID: 17506087	2007
Pathogenesis of arteriovenous malformations in the absence of endoglin. Circulation research\| PubMed ID: 20224041	2010
The TGFÎ² type II receptor plays a critical role in the endothelial cells during cardiac development. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 20652948	2010
Endothelial expression of TGFÎ² type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system. PloS one\| PubMed ID: 22745736	2012

Kathleen R. Allinson

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