University of Groningen Hirschsprung Disease
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University of Groningen Hirschsprung disease - genetics and development Burzynski, Grzegorz Maciej IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2006 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Burzynski, G. M. (2006). Hirschsprung disease - genetics and development. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 26-09-2021 Appendix 4 ________________________________________________________________________________________________________________________________________________ Abstract Signalling of the RET receptor triggers one of the most important signal transduction route, crucial for the migration, proliferation and differentiation of the neural crest stem cells (NCSC’s), which are responsible for the formation of the enteric nervous system (ENS). We isolated NCSC’s from E.12-E.14 mouse embryo intestines and cultured them in selection media with or without GDNF, the ligand of RET. To identify genes involved in/or triggered by the RET pathway, we performed gene expression profiling using Agilent Whole Mouse Genome Arrays. Not only would this give information on GDNF-induced RET signalling in NCSC’s, it might also be helpful in identifying genes involved in the development of Hirschsprung disease (HSCR). Several, important signalling pathways for ENS and, more generally, embryonic development were found to be significantly, differentially regulated by Gdnf stimulation. Genes belonging to the Notch and Wnt routes were found to be downregulated upon Gdnf stimulation. Furthermore, Gdnf seemed to negatively regulate routes promoting differentiation of the ENS progenitors like the retinoic acid (RA) pathway and semaphorins signalling. Conversely, genes and pathways that have been previously shown to stimulate proliferation and/or migration of NCSC’s were found to be upregulated in cells treated with Gdnf. Among them the FGF pathway, forkhead transcription factors and the BMP signalling route. From the previously identified genes in HSCR, only the Ret co-receptor Gfr α1 was found to be down-regulated by Gdnf. We also identified 3 genes, differentially regulated upon Gdnf stimulation, which are located in regions previously shown to harbour HSCR susceptibility genes (3p21 and 9q31). In sum, we provide evidence that Gdnf stimulation support the maintenance of pluripotency, proliferation and migration of the NCSC’s. Further, we provide a profile of genes’ expression in the ENS precursors, regulated by the RET signalling and identified possible candidate HSCR predisposing genes. 120 Gene expression profile of ENS precursors ________________________________________________________________________________________________________________________________________________ Introduction The neural crest (NC) is formed at the time of neural tube closure, at the border of the neural plate and non-neural ectoderm. Shortly after the neural tube closure is complete, neural crest cells undergo an epithelial to mesenchymal transition, they delaminate from the neuroepithelium and migrate along various pathways in the embryo to form distinct neural crest tissue derivatives (Barembaum and Fraser, 2005). A predominant region for ENS formation is the vagal region, corresponding to somites 1-7, giving rise to the vast majority of parasympathetic neurons and glia cells of the ENS. Vagal crest cells upon delamination (in mouse E.8.5-9.0) migrate ventrally to the region posterior to the branchial arches, close to the dorsal aorta. Subsequently cells enter the foregut mesenchyme, migrate caudally and eventually colonize the whole length of the GI tract (Durbec et al., 1996, Kapur et al., 1992). In mouse, vagal crest cells enter the foregut at E.9.0- E9.5, and by E11.5 the whole small intestine is colonized. The entire GI tract is colonized by E13.5 (Durbec et al., 1996; Kapur et al., 1992; Young et al., 1998). Respectively in humans, ENS precursors invade the foregut at embryonic week 4 and reach the terminal hindgut at week 7 (Wallace and Burns, 2005). ENS precursors form the myenteric and submucosal plexuses, of which the myenteric plexus arises earlier than the submucosal plexus. Ganglia develop gradually from the foregut to the hindgut region, what resembles the vagal cells migration wave. Submucosal ganglia are formed later by cells which migrate transversely from the myenteric region towards the gut lumen (Gershon et al., 1994). This process of proliferation and migration of NCSC’s from the dorsal neural tube towards the foregut and later colonization of the whole GI tract, followed by differentiation into ENS neurons and glia cells is a very complex procedure. Timing and harmonious action of growth factors, their receptors, signalling effectors and transcription factors is essential for each stage of ENS 121 Appendix 4 ________________________________________________________________________________________________________________________________________________ development (Taraviras and Pachnis, 1999, Young et al., 2001). Important during the first steps in the induction and delamination of neural crest is signalling of the WNT, NOTCH, BMP, FGF and Retinoic Acid (RA) signalling routes (La Bonne and Bronner-Fraser, 1998; Cornell and Eisen et al., 2005; Villanueva et al., 2002; Garcia-Castro et al., 2002). Subsequently, neural crest cells acquire distinct fate, depending on the region of origin and upon migration to different embryonic regions where surrounding tissue express appropriate instructive signals. The RET protein with GFR α’s as co-receptors, and GDNF as its ligand are the major components of the most important signalling cascade in ENS development (Durbec et al., 1996; Taraviras et al., 1999). GDNF serves as a chemoattractant for the migrating vagal cells that express RET towards and within the gut. High levels of GDNF expression are observed in the stomach mesenchyme ahead of the migrating NCSC’s and later in the cecum region assuring proper course and successful colonization of the small intestine (Natarajan et al. 2002, Young et al., 2001). However, in the GI tract regions posterior to the cecum, no GDNF expression ahead of NCSC’s cells was detected. Instead, GDNF is expressed alongside the streams of the migrating cells and most likely exert proliferative and survival rather than having a migratory effect (Natarajan et al., 2002). Defects in proper migration, proliferation or differentiation of the ENS progenitors can lead in humans to the most common disorder affecting enteric neurons – Hirschsprung disease (HSCR) [MIM 142623]. It is a congenital disorder characterized by intestinal obstruction due to an absence of enteric ganglia among variable lengths of the intestinal tract. HSCR is genetically a complex, both heterogenic and non-Mendelian disease, and to date 10 genes have been shown to be involved in the disease etiology (see first chapter). Nevertheless, the RET gene is a major disease risk factor as the majority of the mutations are being found either in the RET coding region or in its regulatory sequences (Chakravarti and Lyonnet, 2001; Emison 122 Gene expression profile of ENS precursors ________________________________________________________________________________________________________________________________________________ et al., 2005). Other genes and susceptibility loci, which were found to be involved in HSCR, are believed to belong to the RET signalling cascades or are believed to interact with the RET route. As mentioned, RET signalling is extremely important in NCSC migration proliferation and differentiation, nevertheless the network that transmits RET signals within the cell and more importantly the actual downstream effectors are largely unknown. Similarly, understanding the interconnections between different signalling cascades is poorly understood. In this study we tried to get more insight into the RET network and downstream effectors by the isolation of migratory vagal neural crest cells from mice embryo intestines. We have compared gene expression profiles of cells where RET signalling was triggered by Gdnf to those where RET signalling was inactive. By this approach we hoped to identify genes belonging to or interfering with RET signalling pathway, also those important in HSCR. We make an overview of the genes, whose expression in ENS progenitors was significantly influenced by Gdnf stimulation. 123 Appendix 4 ________________________________________________________________________________________________________________________________________________ Material and Methods Isolation of NCSC’s and culture conditions Anesthetized pregnant female C57BL/6 mice were sacrificed by cervical dislocation; embryos at E11-14 were removed