Untersuchung von Gli Transkriptionsfaktoren während der Chondrozytendifferenzierung
Final Report Abstract
During endochondral ossification chondrocytes of the early cartilage anlagen undergo distinct steps of differentiation, from proliferating into hypertrophic chondrocytes, which are subsequently replaced by bone an bone marrow. One of the key regulators of endochondral ossification is the secreted growth factor Indian hedgehog (Ihh), which regulates at least three distinct steps of this process: (1) together with a second secreted factor, Parathyroid hormone related protein (PTHrP), Ihh regulates the onset of hypertrophic differentiation in a negative feed back mechanism. Independent of PTHrP Ihh regulates the (2) proliferation of chondrocytes and (3) the onset of osteoblast differentiation in the perichondrium. In this project we have analyzed the role of the zinc finger transcription factors Gli2 and Gli3 in translating the Ihh signal in the developing bone. Both transcription factors can act as proteolytically processed repressors in absence of hedgehog signals and as unprocessed activators upon hedgehog signaling in other tissues. Analyzing mouse mutants carrying a loss of function mutation of Gli3 in an Ihh deficient background revealed that loss of Gli3 rescues the Ihh phonotype in chondrocytes to a significant degree demonstrating that Gli3 acts mainly as a repressor of Ihh target genes. In contrast loss of Gli2 does not rescue the Ihh phenotype nor do Gli2;Gli3 double mutants (no Gli activator; no repressor) show a more severe phenotype than Gli3;Ihh mutants (Gli2 activator and repressor). Thus, Gli2 does not act as a strong repressor in chondrocytes. As Gli2 mutant mice show slightly disturbed chondrocyte proliferation and the Ihh phenotype is not completely rescued in Gli3;Ihh mutants, Gli2 seems to act mainly as a activator of Ihh target genes. Nevertheless as neither loss of Gli2 nor Gli3 leads to severe defects in chondrocyte differentiation both transcription factors can substitute for the other if Ihh signals are present. During osteoblast differentiation loss of neither Gli2 nor Gli3 does induce the ossification process in Ihh mutants, indicating that an activator form of either factor is required to mediate Ihh signaling in osteoblasts. Investigation of mice expressing Gli2 in part of the perichondrium in a Gli3 deficient background revealed that activation of Ihh target genes by Gli2 is sufficient to induce the differentiation of osteoblasts. By detailed molecular analysis of chondrocyte differentiation in Gli3 mutants we identified the determination of the region of round, low proliferating chondrocytes at the distal ends of the cartilage anlagen as a new function of Ihh/Gli3 signaling. Two newly identified markers of this cell type, UCMA1 and Fgfr1, demonstrate that loss of Gli3 leads to a reduced pool of distal chondrocytes. As a similar effect can be observed in mice overexpressing Ihh close to the joint region the repressor function of Gli3 seems to be required to expand this pool of chondrocytes. Furthermore we found that Gli3 physically interacts with the transcription factor Trps1, another regulator of distal chondrocytes These studies provided first insight into molecular mechanisms that regulate the differentiation of this specific type of chondrocytes, which will later give rise to the secondary ossification center and the articular cartilage. To identify genes that mediate the Ihh signal in chondrocytes, we have started to identify Gli3 target genes by Chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-Seq). During the course of this project a (ChIP-Seq) protocoll fhas been established, which allows enrichment of Gli3 target genes over input DNA and unspecific binding of ß-actin. The precipitated DNA has been amplified and sequenced. First results revealed enrichment of promoter regions of the previously identified target genes Gli1 and Ptch1.
Publications
- (2005). Gli3 has repressing and activating functions downstream of Ihh in regulating two distinct steps of chondrocyte differentiation. Development 132, 5249-60
Koziel, L., Wuelling, M., Schneider, S., Vortkamp, A.
- (2008). Ucma - A novel secreted factor represents a highly specific marker for distal chondrocytes. Matrix Biol. 27, 3-11. [Epub 2007 Jul 13]
Tagariello A, Luther J, Streiter M, Didt-Koziel L, Wuelling M, Surmann-Schmitt C, Stock M, Adam N, Vortkamp A, Winterpacht A
- (2009). Trps1, a regulator of chondrocyte proliferation and differentiation, interacts with the activator form of Gli3. Dev. Biol 328, 40-53. [Epub 2009 Jan 14]
Wuelling, M., Kaiser, F. J., Buelens, L. A., Braunholz, D., Shivdasani, R. A., Depping, R. and Vortkamp, A.
- (2010). Gli2 activator function in preosteoblasts is sufficient to mediate Ihh-dependent osteoblast differentiation, whereas the repressor function of Gli2 is dispensable for endochondral ossification. Dev Dyn. 239,1818-26
Kesper DA, Didt-Koziel L, Vortkamp A
- (2011). Sonic hedgehog signaling during adrenal development. Mol Cell Endocrinol. Oct. 2011 [Epub ahead of print]
Laufer, E., Kesper, D., Vortkamp, A. King, P.
