Unbegrentzte Selbsterneuerungkapazität und Pluripotenz sind charakteristische Merkmale von embryonalen Stammzellen (ES-Zellen). Dennoch sind die molekularen und zellulären Mechanismen, die für das Schicksal der ES-Zellen zuständig sind, noch nicht genau definiert. Um regulierende Faktoren des undifferenzierten Zustands von ES-Zellen zu identifizieren, wurden undifferenzierte ES Zellen, "Embryoid Bodies", spontan differenzierte und mit Retinsäure differenzierte ES Zellen mittels Microarray-Analysen verglichen. Neben bereits etablierten Pluripotenz-Markern, wurde Sprouty4 als eines der am stärksten degerulierten Transkripte unter diesen Bedingungen identifiziert. Sprouty4 ist als Inhibitor des ERK (Extracellular signal-regulated protein kinase)-Signalweges bekannt, aber seine Rolle in ES-Zellen wurde noch nicht definiert. Mittels Genexpression und Western BlotAnalysen konnte gezeigt werden, dass Sprouty4 in undifferenzierten ES-Zellen stark exprimiert ist und im Verlauf der Differenzierung schnell herunterreguliert wird. In vivo war Sprouty4 auf die innere Zellmasse (ICM) der Mausblastozyste beschränkt. Außerdem wurde gezeigt, dass der Sprouty4 Promotor durch direkte Bindung der PluripotenzMarkern Nanog, Klf4 und Stat3 reguliert wird. ES-Zellen, die Sprouty4 konstitutiv exprimieren, waren resistent gegen Differenzierung durch Zugabe von Retinsäure oder Bildung von Embryoid Bodies. Hingegen führte die Expression einer dominant-negativen Mutante von Sprouty4 zu einer erhörten Sensitivierung von ES-Zellen gegenüber der Differenzierung und zur Bildung extraembryonaler Gewebe begleitet von Endoreduplikation. Zusammenfassend konnten unsere Ergebnisse zeigen, dass die enge Regulation des ERK-Signalweges und warscheinlich anderer Signalwege durch Sprouty4 notwendig ist, um die Balance zwichen Pluripotenz und Differenzierung embryonaler Stammzellen zu kontrollieren. / A hallmark feature of embryonic stem (ES) cells is the ability to self-renew indefinitely while maintaining pluripotency. However, the molecular and cellular mechanisms underlying ES cell fate are poorly understood. To identify signaling pathway molecules that maintain the uncommitted state of ES cells, a microarray analysis was performed comparing undifferentiated ES cells, mature embryoid bodies, spontaneously differentiated and retinoic acid-induced differentiated ES cells. Among several well-validated pluripotency markers, Sprouty4 was identified as one of the most highly deregulated transcripts under these conditions. Sprouty4 is known as an inhibitor of the extracellular signal-regulated protein kinase (ERK/MAPK) pathway however its role in ES cells has not yet been defined. Gene expression and western-blot analyses have shown that Sprouty4 is highly expressed in ES cells and strongly downregulated upon differentiation whilst in vivo, Sprouty4 is confined to the founder population of ES cells, the inner cell mass of mouse blastocysts. Moreover, the Sprouty4 promoter was found to be regulated via the direct binding of the intrinsic pluripotency-associated factors Nanog, Klf4 and Stat3. ES cells engineered to constitutively express a wild-type version of Sprouty4 were found to be resistant to differentiation induced by retinoic acid or embryoid bodies formation. Conversely, expression of a dominant negative Sprouty4 mutant activating the ERK/MAPK pathway in a sustained manner sensitized ES cells to differentiation and triggered endoreduplication leading to the formation of extraembryonic tissue. Taken together, these results highlight the essential role of Sprouty4 in the tight regulation of the ERK/MAPK pathway- and probably others- for the balance between pluripotency and lineage commitment in mouse embryonic stem cells.

Abstract The embryonic urogenital system (UGS) generates the metanephric kidney, gonad and the adrenal gland. It is well known that the development of the UGS is regulated by sequential and reciprocal epithelial and mesenchymal tissue interactions but the secreted mediators involved are still poorly known. The action of such inductive signals is typically regulated by specific antagonists. The Sprouty (Spry) proteins compose one family of cytoplasmic regulators that typically repress the function of the receptor tyrosine kinase (RTK) signal transduction pathways. The DAN/Cerberus (Cer) family that encodes secreted proteins bind and antagonize the Bmp, Wnt and Nodal signals. In this study the roles of Spry and Cer1 was addressed during mouse UGS development by targeted expression of SPROUTY2 (SPRY2) and Cer1 in the ureteric bud and Wolffian duct under the Pax2 promoter. Changes induced in the UGS assembly process were analyzed in detail to reveal the normal developmental roles of these proteins. SPRY2 expression led to either complete lack of the kidney, reduction in the kidney size or formation of unilateral kidney with reduced size. The SPRY2 mediated reduction in kidney size was accompanied by inhibition of expression of genes that are known to regulate kidney development. The results indicated that the Spry may take part in kidney development by coordinating the reciprocal cell signaling between the ureteric bud, the mesenchymal cells and stromal cells. In addition to the kidney, the gain of SPRY2 function revealed an important role in the control of male gonadogenesis. SPRY2 over expression in the Wolffian duct malformed the Wolffian duct derivatives, diminished the number of seminiferous tubules and the amount of the interstitial tissue associated with reduced mesonephric cell migration to the testis. Exogenous FGF9 rescued mesonephric cell migration inhibited by SPRY2. It was concluded that Spry protein contribute to male sexual organogenesis by antagonizing Fgf9 signaling. When the Cer1 gene was over expressed in the ureteric bud this lead unexpectedly to increased kidney size. The Cer1 mediated promotion of kidney size was demonstrated to involve enhanced ureteric bud morphogenesis. At the molecular level Cer1 protein function lead to inhibition of Bmp4 gene expression and concurrent upregulation of Gdnf and Wnt11 expression. Notably, excess BMP4 reduced the Cer1 stimulated ureteric bud branching and downregulated normally expression of Gdnf and Wnt11 in the embryonic kidney. Based on the presented data it is proposed that the establishment of mammalian organ size is under the control of both systemic and the intrinsic factors. Together the work demonstrates significant roles for the proteins that typically inhibit growth factor signaling or signal transduction. Hence organogenesis is controlled by coordination between positive and negative growth factor regulator signals.

The present thesis project focused on the preclinical study of Neurotensin receptor 1 (Ntsr1), Endoglin/CD105 (Eng), Glypican 6 (Gpc6) and Sprouty homolog 4 (Spry4) as potential markers or molecular targets for future therapeutic interventions of gastrointestinal stromal tumors (GIST). Ntsr1 expression was characterized was reported in a paper that I co-authored entitled “Neurotensin receptor 1 is expressed in gastrointestinal stromal tumors but not in interstitial cells of Cajal.” by Gromova et al. PLOS ONE, 2009. As radio-labeled NTSR1 ligand analogues have already been reported for whole-body imaging and therapeutic interventions, prompting us to investigate NTSR1 as a target for in vivo imaging of GIST.Next, expression of Eng was characterized in the KitK641E murine GIST model, human GIST, GIST882 and BaF3 cells. This study has been reported in “Endoglin/CD105 is expressed in KIT positive cells in the gut and gastrointestinal stromal tumors” by Gromova et al. JCMM, 2011, a paper I co-authored. As result, an American start-up approached us to assess their proprietary compound targeting ENG on GIST882 cells. However, concerns were raised about possible non-selective action and the project was stalled by the company.Subsequently, GIST tissue microarrays were examined by immunohistochemistry using the sole commercially available GPC6 antibody. No statistical correlation could be found between GPC6-ir and GIST clinic-pathological features and concerns were raised about the reliability of the GPC6 antibody used. Ultimately, Spry4 was investigated in the last part of my thesis. In vitro, we have demonstrated that Spry4 is specifically upregulated by the ERK pathway in GIST882 cells. In vivo, Spry4 deficient mice showed an ICC hyperplasia in antrum and colon, using a new ICC quantification method developed in the lab, which was reminiscent of the oncogenic GIST murine model KitK641E. Similarities between Spry4 KO and KitK641E heterozygous animals were even further emphasized by functional studies, as both genotypes showed a delay in transit time. This study lead to the publication “Hyperplasia of interstitial cells of Cajal in Sprouty homolog 4 deficient mice” by Thys et al. 2015, PLOS ONE. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished