Funktionelle Charakterisierung des Tbx18-Gens der Maus in der Somitogenese der Maus
Final Report Abstract
Compartmentalization of somites along their anterior-posterior (AP) axis is crucial for the development of the metameric organization of the vertebrate axial skeleton as well as the peripheral nervous system because anterior and posterior somite halves differentially contribute to the developing vertebrae and provide distinct substrates for the migration of neural crest and spinal nerves. AP somite patterning is established in the anterior presomitic mesoderm (PSM) by a complex feed-back regulatory loop that restricts Notch signaling to a posterior and expression of the transcription factor Mesp2 to an anterior half of a prospective somite. Maintenance of this polarization within epithelial somites and the lateral sclerotome involves the antagonistic activity of two transcription factors, the paired-type homeobox protein Uncx4.1 and the T-box protein, Tbx18. Uncx4.1 is required downstream of Dll1/Notch to maintain the posterior somite half whereas Tbx18 is required and sufficient to confer anterior somite half identity. Given the crucial nature of Tbx18 in maintaining AP-somite polarity and vertebral column integrity, we proposed to further elucidate the molecular function of Tbx18 in this process, by studying its molecular nature as transcriptional modulator in vitro and in vivo, by analyzing its molecular regulation, and by defining the molecular circuits in which this factor acts to activate distinct effector gene programs. Here, we show that Tbx18 and the closely related Tbx15 and Tbx22 genes encode structurally highly related transcriptional repressors. The proteins harbor a classical nuclear localization signal in the N-terminus, they bind to similar combinations of DNA-binding sites, and possess a critical eh1-motif that confers transcriptional repression by recruitment of groucho corepressors in vitro. Using conditional misexpression of a Tbx18-VP16 fusion protein in vivo, we confirm that Tbx18 acts in the somitic mesoderm, i.e. in vivo, as transcriptional repressor as well. We found that Tbx18 homodimerizes and heterodimerizes with other T-box proteins. Using a yeast-two hybrid screen we identified a number of proteins that may form complexes with Tbx18 in vivo. We validated interaction with Pax3, and showed its functional relevance for AP somite patterning in Tbx18/Pax3 compound mutants. Tbx18 and Tbx22 are coexpressed and co-regulated in anterior stripes in the anterior PSM and in early epithelial somites. We generated Tbx18/Tbx22 double mutants and showed that the combined loss of both genes enhanced the phenotypic defects in vertebral column development compared to Tbx18 single mutants. Posteriorization of somites occurred earlier and was accompanied by strong upregulation of Uncx4.1 and unexpectedly Dll1 in early epithelial somites. Using conditional overexpression approaches we provide evidence that Tbx18 is sufficient to repress Dll1, a regulation that may even be of direct nature as supported by transactivation assays in vitro. Using transcriptome comparison of anteriorized and posteriorized somites as well as data base mining with subsequent in situ hybridization analysis of wildtype and mutant embryos, we identify a number of genes that are differentially expressed in anterior and posterior compartments of somites and the sclerotome, respectively, dependent on Tbx18. These genes provide a pool of candidates for effectors of AP somite compartmentalization the dependence of which on Notch, Mesp2, Uncx4.1 and Tbx18/Tbx22 activities in the somitic mesoderm needs to be clarified in future studies. Finally, we employed misexpression approaches to show that Notch signaling and Uncx4.1 are sufficient to repress Tbx18 in vivo. The molecular nature of this regulation remains to be deciphered since no genomic elements have been found ithat are sufficient to recapitulate endogenous expression of Tbx18 in somites. Together, our experimental efforts further dissect the genetic programs that maintain AP- subdivision of somites. We define Tbx18/Tbx22 as crucial regulators of anterior somite half identity, and identify upstream regulators, and candidates for interacting proteins as well as effector genes, the analysis of which may open avenues to unravel the underling molecular pathways.
Publications
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(2007). TBX22 missense mutations found in X-linked cleft palate (CPX) patients affect DNA binding, sumoylation and transcriptional repression. Am. J. Hum. Gen. 81, 700-712
Andreou, A.M., Pauws, E., Jones, M.C., Singh, M.K., Bussen, M., Doudney, K., Moore, G.E., Kispert, A., Brosens, J.J. and Stanier, S.
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(2007). Transcriptional repression by the T-box proteins Tbx18 and Tbx15 depends on Groucho corepressors. J. Biol. Chem. 282, 25748-25759
Farin, H.F., Bussen, M., Schmidt, M.K., Singh, M.K., Schuster-Gossler, K. and Kispert, A.
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2007). Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signaling and Tbx6. EMBO Reports 8, 784-789
Wittler, L., Shin, E.-h., Grote, P., Kispert, A., Beckers, A., Gossler, A., Werber, M., and Herrmann, B.G.
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(2008). T-box Protein Tbx18 Interacts with the Paired Box Protein Pax3 in the Development of the Paraxial Mesoderm. J. Biol. Chem. 283, 25372-25380
Farin, H.F., Mansouri, A., Petry, M., Kispert, A.
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2008). TBX15 mutations cause craniofacial dysmorphism, hypoplasia of scapula and pelvis, and short stature in Cousin syndrome. Am. J. Hum. Genet. 83, 649-655
Lausch, E., Hermanns, P., Farin, H.F., Alanay, Y., Unger, S., Nikkel, S., Steinwender, C., Scherer, G., Spranger, J., Zabel, B., Kispert, A., Superti-Furga, A.
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(2009). Tbx20 interacts with Smads to confine Tbx2 expression to the atrioventricular canal. Circ. Res. 105, 442-452
Singh, R., Horsthuis, T., Farin, H.F., Grieskamp, T., Norden, J., Petry, M., Wakker, V., Moorman, A.F., Christoffels, V.M., Kispert, A.
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(2010). Hydroureternephrosis due to loss of Sox9 regulated smooth muscle cell differentiation of the ureteric mesenchyme. Hum. Mol. Genet 19, 4918-4929
Airik, R., Trowe, M.O., Foik, A., Farin, H.F., Petry, M., Schuster-Gossler, K., Schweizer, M., Scherer, G., Kist, R., Kispert, A.
