title: Analyses of small Rho-GTPases signaling molecules during vertebrate development and angiogenesis
creator: Dietz, Christian Tobias
subject: ddc-570
subject: 570 Life sciences
description: The members of the Rho family of small GTPases were originally identified to be involved in a variety of cellular processes, including regulation of F-actin cytoskeleton and microtubule dynamics, cell polarity, vesicle transport and gene expression. In recent studies G-proteins have been implicated to function as key modulators of angiogenesis. G-protein signaling is thereby strongly associated with angiogenic processes, such as the regulation of vascular permeability, remodeling of the extracellular matrix, endothelial migration, proliferation, morphogenesis, and survival. Much of our knowledge regarding G-protein signaling was hitherto predominantly obtained by in vitro experiments or by in vivo studies performed in invertebrates. However, little is known about their regulation and function in vertebrates. Thus, this thesis was aimed at uncovering the role of selected G-proteins during vertebrate development with the focus on angiogenesis. Recently, the guanine nucleotide exchange factor (GEF) Ect-2 interacting BTB-kelch protein KLEIP (KLHL20) was implicated in angiogenesis as a novel and essential regulator of endothelial function that controls the VEGF-induced activation of the small GTPase RhoA. In order to unravel Kleip’s function during angiogenesis in vivo, as well as during development, the Kleip gene was disrupted in mice. Constitutive inactivation of Kleip accomplished by gene-trapping led to a partially lethal phenotype. Some Kleip-deficient embryos died between midgestation and birth due to cranial hemorrhages. This dysfunction in maintaining vascular integrity was furthermore supported by studies with splice-blocking morpholino-induced downregulation of klhl20 in zebrafish embryos as well as by an in vitro transwell permeability assay. Whole-mount immunostainings of the embryonic vasculature in Kleip-mutants exhibited in comparison to their wild-type littermates significantly extended cranial vessels suggesting as well as for the bleedings a failure in the recruitment of mural cells to the nascent vessels. Interestingly, specific NG2 immunostainings displayed no alterations in pericyte coverage-mediated vessel stabilization thereby rather indicating defects in endothelial adhesion. Moreover, Kleip was identified to be indispensable for neonatal development. Homozygous offspring exhibited a high neonatal mortality from around 50% due to respiratory failure. Histological analysis of newborn mutant lungs exhibited reduced airspace, and marked thickening of alveolar septae, which represent the characteristic features of maturation defects. Thus, our studies demonstrate the importance of Kleip for lung morphogenesis and suggest that it could possibly be involved in the pathogenesis of respiratory distress syndroms (RDS). In additional subprojects selected proteins of the G-protein signaling cascade were characterized with regard to angiogenesis. On the one hand it could be shown for the first time that the dock180 and elmo1 formed GEF for the small GTPase Rac1 is expressed in the zebrafish endothelium. Furthermore, the spatially and temporarily restricted photoactivatable morpholino-based expression-silencing of elmo1 in the ventral mesoderm severely impaired the formation of the fish vasculature, suggesting an endothelial cell autonomous function of elmo1. Next to the analysis of GEFs during angiogenesis the downstream mediators of RhoA signaling were analyzed. In previous studies the pharmacological inhibition of both serine/threonine kinases ROCK I/II with the relative unspecific inhibitor Y-27632 revealed contrary results. Here it could be demonstrated that inhibition of ROCK I/II activity with the inhibitor H-1152 enhanced endothelial signaling implicating that both kinases function as negative regulators of sprouting angiogenesis. In conclusion, all analyzed G-protein signaling molecules were identified as essential regulators involved in the formation of a functional vasculature.
date: 2011
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/11797/1/Doktorarbeit_Christian_Dietz.pdf
identifier: DOI:10.11588/heidok.00011797
identifier: urn:nbn:de:bsz:16-opus-117973
identifier:   Dietz, Christian Tobias  (2011) Analyses of small Rho-GTPases signaling molecules during vertebrate development and angiogenesis.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/11797/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng