Modulation of cell adhesion is essential to the cell rearrangements that characterize Xenopus gastrulation. The spatial and temporal regulation of cell movement requires a highly coordinated cross-talk between cadherin and integrin adhesion receptors. Beta-parvin is an integrin associated scaffolding protein consisting of two calponin homology (CH) domains. Xenopus beta-parvin is highly conserved being ~95% similar to mammalian orthologs. Beta-parvin is expressed in the blastocoel roof and dorsal marginal zone of the embryo during gastrulation, suggesting a potential role in morphogenesis. Over-expression of full-length beta-parvin has no effect on embryogenesis, however, over-expression of either CH domain causes a failure in gastrulation. When over-expressed the CH1 domain causes a failure in fibronectin (FN) matrix assembly, epiboly and convergent extension in vivo. CH1 domain over-expression also inhibits tissue separation (TS) and Brachet’s cleft formation. The CH1 domain of beta-parvin localizes to sites of cell-cell adhesion, and down-regulates C-cadherin adhesion through activation of Rac1, independent of receptor expression. Significantly, the CH1 domain can rescue convergent extension downstream of integrin ex vivo suggesting a role for beta-parvin in the integrin mediated control of cell intercalation. Over-expression of the CH2 domain also inhibits morphogenesis in a similar fashion as CH1. However, the CH2 domain localizes to sites of integrin adhesion and inhibits integrin function resulting in a loss of FN assembly. The CH2 domain binds ILK and inhibits integrin function. When over-expressed the CH2 domain promotes TS in the pre-involution mesoderm through the activation of Rho. While the CH1 domain inhibits TS through Rac and the CH2 domain promotes TS through Rho, full-length beta-parvin over-expression has no embryonic phenotype and its signaling properties appear to be intermediate between expression of either isolated CH domain. At the dorsal lip full-length beta-parvin shuttles between integrin in the pre-involution mesoderm and cell-cell adhesion sites in the post-involution mesoderm indicating it plays significant roles in the previously characterized integrin-cadherin cross talk. My research has defined novel roles for beta-parvin as a key player in the regulation of integrin-cadherin cross-talk during tissue morphogenesis.

No description available.

During vertebrate gastrulation, a well-orchestrated series of cell movements leads to the formation of the three germ layers: ectoderm, mesoderm and endoderm. In zebrafish, a model organism for vertebrate development, the mesendodermal progenitor cells separate from the ectodermal cells and migrate towards the animal pole. To identify proteins controlling these processes, I used a comparative proteomic approach following two alternative strategies: (1) Based on the notion that Wnt11 regulates cell movement and morphology during gastrulation independent of transcriptional regulation, I performed a screen aimed at the identification of proteins phosphorylated upon Wnt11 signalling. To regulate Wnt11 expression tightly, I engineered a transgenic slb/wnt11-/- fish line expressing wnt11 under the control of a heat shock promoter. Using this line, I performed a quantitative comparison of protein phosphorylation with or without Wnt11 pathway activation by analysing 32P-labelled embryo extracts on 2D gels. (2) Since these experiments did not reveal any Wnt11 targets, I addressed, in the second approach, proteomic differences causal for the changes in cell adhesion and motility observed in mesendodermal cells upon involution. Quantitative 2D gel analysis comparing ectodermal and mesendodermal cells revealed 37 significantly regulated spots, 36 of which I identified by mass spectrometry. Interestingly, the majority of these proteins were not regulated on a transcriptional level as determined by an accompanying microarray analysis confirming the complementary nature of proteomics and transcriptomics. Among the identified targets, several proteins, including Ezrin2, had previously been assigned a cytoskeleton-related function. I characterised Ezrin2 in more detail showing that Ezrin2 is specifically activated by phosphorylation in mesendodermal cells and that it is required for proper gastrulation movements. In the course of this study, I developed techniques for proteomic analysis of early zebrafish embryos, including a protocol to remove the yolk. I identified several cytoskeleton-related proteins in a comparative proteomic screen for regulators of gastrulation movements. The subsequent characterisation of Ezrin2 confirmed the power of proteomics for the analysis of developmental processes. In conclusion, this work provides a foundation to study developmental and cell biological questions in early zebrafish embryos using proteomics.

During vertebrate gastrulation, a well-orchestrated series of cell movements leads to the formation of the three germ layers: ectoderm, mesoderm and endoderm. In zebrafish, a model organism for vertebrate development, the mesendodermal progenitor cells separate from the ectodermal cells and migrate towards the animal pole. To identify proteins controlling these processes, I used a comparative proteomic approach following two alternative strategies: (1) Based on the notion that Wnt11 regulates cell movement and morphology during gastrulation independent of transcriptional regulation, I performed a screen aimed at the identification of proteins phosphorylated upon Wnt11 signalling. To regulate Wnt11 expression tightly, I engineered a transgenic slb/wnt11-/- fish line expressing wnt11 under the control of a heat shock promoter. Using this line, I performed a quantitative comparison of protein phosphorylation with or without Wnt11 pathway activation by analysing 32P-labelled embryo extracts on 2D gels. (2) Since these experiments did not reveal any Wnt11 targets, I addressed, in the second approach, proteomic differences causal for the changes in cell adhesion and motility observed in mesendodermal cells upon involution. Quantitative 2D gel analysis comparing ectodermal and mesendodermal cells revealed 37 significantly regulated spots, 36 of which I identified by mass spectrometry. Interestingly, the majority of these proteins were not regulated on a transcriptional level as determined by an accompanying microarray analysis confirming the complementary nature of proteomics and transcriptomics. Among the identified targets, several proteins, including Ezrin2, had previously been assigned a cytoskeleton-related function. I characterised Ezrin2 in more detail showing that Ezrin2 is specifically activated by phosphorylation in mesendodermal cells and that it is required for proper gastrulation movements. In the course of this study, I developed techniques for proteomic analysis of early zebrafish embryos, including a protocol to remove the yolk. I identified several cytoskeleton-related proteins in a comparative proteomic screen for regulators of gastrulation movements. The subsequent characterisation of Ezrin2 confirmed the power of proteomics for the analysis of developmental processes. In conclusion, this work provides a foundation to study developmental and cell biological questions in early zebrafish embryos using proteomics.

Isolierung des Sp1-verwandten Transkriptionsfaktors Knopfkopf mittels eines PCR-basierten Homologie-Screens in der Maus. Das Gen Knopfkopf wurde anschließend hinsichtlich der evolutiven Verwandtschaftsbeziehungen zum Drosophila-Gen buttonhead eingeordnet. Eine funktionelle Charakterisierung erfolgte mit Hilfe einer gezielten Geninaktivierung durch homologe Rekombination (knock out). Es konnte gezeigt werden, dass das Gen in der Embryogenese der Maus essentiell ist für die Entwicklung der Extremitäten, der Nase und des Zentralen Nervensystems sowie der sekundären Gastrulation. / Isolation of the Sp1-related transkription factor Knopfkopf by a PCR-based homology screen in the mouse. The Knopfkopf gene was analysed regarding its evolutionary relationship with the Drosophila gene buttonhead. The functional characterization was done via a targeted gene inactivation by a homologous recombination (knock out). It was shown that the gene is necessary during the mouse embryogenesis for the development of limbs, nose, central nervous system, as well as the secondary gastrulation.

Bone Morphogenetic Proteins (BMPs) sind extrazellulär vorkommende Wachstumsfaktoren und werden der Superfamilie der Transforming Growth Factors β (TGF-β) zugeordnet. Entgegen ihrem Namen spielen sie nicht nur eine Rolle bei der Ausbildung und Regeneration der Knochenmatrix, sondern regulieren bereits während der Embryonalentwicklung zahlreiche Abläufe. Unter anderem sind sie an der Festlegung der Körperachsen und Entwicklung der Organanlagen beteiligt. Später steuern sie das Wachstum von Organen und Geweben und sind schließlich im adulten Organismus für deren Homöostase und Regeneration verantwortlich. Bei fast allen Mitgliedern der TGF-β Superfamilie erfolgt die Signalbildung nach derzeitigem Kenntnisstand durch die Bindung an transmembrane Serin/Threonin-Kinaserezeptoren, die in zwei Untergruppen unterteilt werden können. Dabei werden von einem Liganden jeweils zwei Typ I- und zwei Typ II-Rezeptoren gebunden, wodurch ein aktiver Komplex entsteht, der im Inneren der Zelle eine Signalkaskade auslöst. Um die vielseitigen Aufgaben der BMPs spezifisch vermitteln zu können, gibt es zahlreiche Mechanismen, die Signalbildung stringent neben der Ligand-Rezeptor-Interaktion zu regulieren. Die Small Mothers Against Decapentaplegic (Smad)-Signalkaskade im Zellinneren wird beispielsweise durch die Interaktion der inhibitorischen Smads mit rezeptor-regulierten Smads oder durch den proteasomalen Abbau der rezeptor-regulierten Smads durch die Bindung von Ubiquitin-Ligasen der Smurf-Familie beeinflusst. Auch auf Membranebene besteht die Möglichkeit der negativen Signalmodulation durch Pseudorezeptoren oder der Verstärkung der Signalbildung durch positive Effektoren wie beispielsweise aktivitätssteigernde Co-Rezeptoren. Ein Charakteristikum der TGF-β Superfamilie stellt jedoch die Vielzahl an sekretierten, löslichen Modulatorproteinen dar. Die meist glykosylierten Proteine üben, bis auf wenige Ausnahmen, einen antagonistischen Effekt auf die BMPs aus. Bei dem BMP-spezifischen Modulatorprotein Twisted gastrulation handelt es sich um ein extrazelluläres Glykoprotein, das im Gegensatz zu den meisten anderen BMP-Modulatoren jedoch eine duale Funktion als Besonderheit aufweist. Es zeigt zum einen eine anti-BMP-Wirkung, indem es den BMP-inhibierenden Einfluss von Chordin durch Bildung eines stabilen ternären Komplexes verstärkt; andererseits kann Twisted gastrulation in Gegenwart spezifischer Metalloproteasen eine proteolytische Spaltung von Chordin und die anschließende Freisetzung von aktivem BMP fördern und so eine BMP-Aktivität vermittelnde Wirkung aufweisen. Twisted gastrulation hat keine beziehungsweise nur eine äußerst geringe Homologie zu anderen (Modulator)Proteinen. Um daher den komplexen Wirkmechanismus detailliert molekular beschreiben zu können, ist die Aufklärung der Struktur /Funktions-beziehungen essentiell. Im Rahmen dieser Arbeit konnten unterschiedliche Expressionsstrategien für die rekombinante Herstellung von Twisted gastrulation etabliert werden, welche eine umfassende Charakterisierung des Proteins in vitro ermöglichen. Erste Kristallisationsversuche von isoliertem Twisted gastrulation für die Aufklärung der dreidimensionalen Struktur mittels Röntgenbeugung verliefen ohne Erfolg, allerdings gelang die Präparation stabiler ternärer Proteinkomplexe für weiterführende Kristallisationsansätze. Hochdurchsatzverfahren für die Expression und Interaktionsanalyse erlauben zudem die Untersuchung einer Vielzahl von Twisted gastrulation-Proteinvarianten. Auf diese Weise konnten Aminosäuren identifiziert werden, die an der Wechselwirkung von Twisted gastrulation mit seinem Interaktionspartner BMP 2 beteiligt sind. Dies ermöglichte eine detaillierte Lokalisation des Bindeepitops im N-terminalen Bereich von Twisted gastrulation. Dabei konnte auch gezeigt werden, dass die Glykosylierung von Twisted gastrulation für die Wechselwirkung mit BMP-2 von Bedeutung ist. Eine experimentelle Strukturanalyse von Twisted gastrulation für die detaillierte Aufklärung des Mechanismus der Interaktion mit BMP-2 und anderen Modulatorproteinen bleibt allerdings weiterhin aufgrund der Einzigartigkeit dieses Modulatorproteins zwingend erforderlich. Für eine Fortsetzung der Untersuchungen bietet der stabile ternäre Komplex eine gute Voraussetzung in Hinblick auf weitere Kristallisationsansätze. / Bone Morphogenetic Proteins (BMPs) are secreted growth factors belonging to the superfamily of Transforming Growth Factors β (TGF-β). In contrast to their name they are not only involved in bone morphogenesis and regeneration, but also regulate numerous events during embryonic development. Furthermore, they play a key role in body axis determination, embryonic patterning as well as organogenesis, later in the adult organism these factors control organ and tissue homeostasis and regeneration. At the current state of research all classical members of the TGF-β family signal through transmembrane serine/threonine kinase receptors, which can be subdivided into type I- and type II-receptors. For receptor activation the ligand induces receptor heteromerisation forming an active complex containing type I- and type II-receptors which subsequently triggers intracellular signaling cascades. To ensure highly specific signaling a complex network of regulatory mechanisms has evolved. The Small Mothers Against Decapentaplegic (Smad) signaling cascade for example is modulated by inhibitory Smads interacting with the receptor-regulated Smads or through proteasomal degradation of the receptor-regulated Smads by binding to ubiquitin-ligases of the Smurf-family. In the membrane, pseudoreceptors such as BAMBI lacking a kinase domain or GPI-anchored co-receptors such as the RGM-receptor family or Cripto can positively or negatively regulate BMP/TGF-β signaling. A highlight of the TGF-β superfamily is the multitude of different secreted soluble modulator proteins. Most present glycoproteins, which act as BMP-antagonists by interfering with BMP-signaling. The BMP-specific modulator Twisted gastrulation is such an extracellular glycoprotein, which however features an unique dual function. On the one hand, by forming a stable ternary complex it strongly enhances the BMP-antagonizing (anti-BMP) activity of another BMP-modulator Chordin. On the other hand, upon proteolytic processing of Chordin in this ternary complex in the presence of specific metalloproteases such as Tolloid, Twisted gastrulation facilitates the dissociation of the ternary complex releasing active BMP an thereby exerts a pro (moting) BMP-activity. Twisted gastrulation exhibits very low if at all any homology to other proteins and seems also unique among the large variety of BMP-modulator proteins. To understand its dual mode of action on a molecular level analysis of the structure/function-relationship is a pre-requisite. In this project we have established different strategies for recombinant production of Twisted gastrulation for a comprehensive in vitro characterization. Although crystallization of Twisted gastrulation for X-ray diffraction analysis failed, we could successfully prepare stable ternary complexes consisting BMP-2, a type II-receptor and Twisted gastrulation which are ideally suited for protein crystallization. High-throughput expression and interaction-analysis schemes allowed us to study a multitude of single amino acid variants of Twisted gastrulation. This resulted in the identification of several amino acid residues in Twisted gastrulation, which form the binding epitope for BMP-2 confirming its supposed location in the N-terminal half of Twisted gastrulation. We could also show that the N-glycosylation of Twisted gastrulation is involved in high-affinity BMP binding and required for its BMP-modulatory activity in vivo. For a full analysis of its molecular mechanism of BMP-2 interaction and its interplay with other modulator proteins, a structure analysis of Twisted gastrulation is required in the near future. Providing efficient recombinant sources and the preparation of stable ternary complexes will likely facilitate this.

Thèse de doctorat : Sciences du vivant. Biologie du développement : Strasbourg 1 : 2007. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 20 p.

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 106-112). Also available for download via the World Wide Web; free to University of Oregon users.

published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 180-186).