Functional redundancy of Tbx15 and Tbx18 in mouse limb development
Final Report Abstract
Vertebrate limbs are body appendages with a stereotyped pattern of skeletal elements that arise by growth and patterning processes coordinated by specialized regions of the developing limb bud. While the molecular nature of the signaling systems establishing the main axes of the limb bud have been identified, much less is known about the mechanisms controlling differentiation and regionalization along the proximal-distal (PD) axis. Tbx15 and Tbx18, two closely related T-box genes, are expressed in a largely overlapping pattern in the proximal limb bud mesenchyme. Null alleles of Tbx15 and Tbx18 cause minor and no defects, respectively, in limb development. Thus, we hypothesized that Tbx15 and Tbx18 redundantly regulate the development of proximal limb skeletal elements. We therefore wished to analyze expression and regulation of the two genes during limb development, characterize Tbx15/Tbx18 compound mutant embryos, and investigate the biochemical equivalence of the encoded proteins. Here, we showed in the mouse, that Tbx15 and Tbx18 are redundantly required at early stages of limb development for the normal formation of girdle, stylopod and zeugopod skeletal elements in both forelimbs and hindlimbs. Since distal condensations are relatively unaffected, hand and feet become directly attached to the body in Tbx15-/-;Tbx18-/- embryos, mimicking the situation in extreme cases of congenital and induced (X-ray or thalidomide) forms of phocomelia. Reduction of precartilagenous anlagen of stylopod and zeugopod occurs in absence of changes of PD marker gene expression but correlates with cell dispersal, decreased proliferation and increased apoptosis. In vivo misexpression and in vitro cell mixing experiments indicate that Tbx15/Tbx18 confer specific adhesive properties to mesenchymal cells that promote recruitment into the proximal limb bud region and subsequent chondrogenesis. Proximal restriction of Tbx18 expression is achieved by locally repressing signals from adjacent tissues including the apical ectodermal ridge (AER), suggesting the dynamic subdivision in a proximal region that differentiates early and a distal region from which the autopod differentiates later. Our findings corroborate the differentiation front model and provide a cellular mechanism to explain the selective loss of proximal skeletal elements as observed in phocomelia. Tbx15 and Tbx18 encode structurally highly related transcriptional repressors. Both proteins harbor a classical nuclear localizing signal in the N-terminus. They bind to similar combinations of DNA-(T) binding sites, and possess a critical eh1-motif that confers transcriptional repression by recruitment of groucho corepressors. Both protein bind to a number of transcription factors including Pax3, an interaction that was found to be of functional relevance in girdle formation. Protein destabilizing mutations in TBX15 were found in patients suffering from Cousin- Syndrome, a complex disease entity with a skeletal malformation spectrum including scapula dysplasia highly similar to that of mice homozygous for a null mutation in the Tbx15 ortholog. We expanded our work program to (re-)analyze the role of Tbx2 in digit development. We characterized Tbx2 as a critical mediator for the termination of positive feedback loop involving Sonic hedgehog (Shh), Gremlin1 (Grem1), and Fibroblast growth factors (Fgfs) in the posterior limb bud mesenchyme. We showed that in Tbx2-deficient hindlimbs Shh/Fgf4 signaling is prolonged resulting in increased posterior limb size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. Tbx2 likely acts cell-autonomously to repress Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4 expression in the overlying epithelium. Together our experimental efforts identified novel regulatory modules important for the development of proximal skeletal elements (Tbx15/Tbx18) and the correct termination of distal limb outgrowth (Tbx2). Thus, we have provided insight into the etiology of rare human congenital diseases including phocomelia and polydactyly, and have opened avenues to further decipher the underling molecular pathways.
Publications
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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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(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). Expression and requirement of T-box transcription factors Tbx2 and Tbx3 during secondary palate development in the mouse. Dev. Biol 336, 145-155
Zirzow, S., Lüdtke, T.-H., Brons, J.F., Petry, M., Christoffels, V.M., Kispert, 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.
