DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Eleonora Minina :
Interaction of Ihh/Pthlh, BMP and FGF signaling in regulating chondrocyte proliferation and differentiation
Ihh/Pthlh, BMP and FGF signaling in chondrogenesis
Interaktion der Ihh/Pthlh, BMP und FGF Signalwege in der Regulierung der Chondrozytenproliferation und Differenzierung

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Abstract

Bones of vertebrate limbs are formed by endochondral ossification. During this process a cartilage model of the future skeletal elements initially forms. Chondrocytes in the cartilage model undergo various steps of differentiation from proliferating into hypertrophic chondrocytes, which are subsequently replaced by bone. One of the critical steps during this process is the regulation of chondrocyte proliferation and differentiation in the developing cartilage anlagen. Previous studies have demonstrated that Indian hedgehog (Ihh) and Parathyroid hormone-like peptide (Pthlh) interact in a negative feedback loop to regulate chondrocyte differentiation. Ihh, which is expressed in prehypertrophic chondrocytes, activates the expression of Pthlh in the periarticular region of the developing skeletal elements. Pthlh in turn signals back to its receptor (Pthr) in proliferating and hypertrophic chondrocytes, and prevents the onset of hypertrophic differentiation of chondrocytes, thereby inhibiting Ihh expression. In addition, Ihh was shown to positively regulate chondrocyte proliferation. Several lines of evidence suggest an interaction of the Ihh/Pthlh pathway with other signaling factors, including Bone morphogenetic proteins (BMPs) and Fibroblast growth factors (FGFs). Previous studies on chicken limbs have shown that ectopically expressed activated BMP receptor 1A upregulates the expression of Pthlh and blocks chondrocyte differentiation. Therefore, it was hypothesized that BMPs act downstream of Ihh and mediate the Ihh signals to induce Pthlh expression. In this study, a potential interaction of the Ihh/Pthlh and BMP signaling pathways was analyzed using a culture system for embryonic mouse and chick limbs, which was supplemented with activators and inhibitors of both signaling pathways. The results have shown that BMPs are not acting as secondary signals of Ihh in mediating the induction of Pthlh expression. Instead BMP signaling acts at various steps of chondrocyte differentiation and interacts with Ihh signaling in different ways. First, both BMP and Ihh signals are essential for chondrocyte proliferation and act in parallel. Second, BMP signaling regulates the expression level of Ihh thereby coordinating the rate of chondrocyte proliferation and the onset of hypertrophic differentiation. Third, a negative role for BMP signaling was identified in regulating the differentiation of terminal hypertrophic cells. A third signaling pathway critical for proliferation and differentiation of chondrocytes is the FGF signaling pathway. Constitutive activation of the FGF receptor 3 (Fgfr3) gene results in specific dwarfism syndromes in human and mice. On the molecular level activated FGF signaling results in reduced proliferation and differentiation of chondrocytes. In this study the epistatic relationship of FGF signaling with the Ihh/Pthlh and BMP signaling pathways was analyzed. Activation of FGF signaling in the limb culture system showed decreased chondrocyte proliferation and reduced Ihh expression similar to that in a mouse model for human dwarfism (FGFach mouse). Moreover FGF signaling acts upstream of Ihh/Pthlh signaling and regulates the onset of hypertrophic differentiation indirectly by regulating Ihh expression. Furthermore, FGF signaling advances the hypertrophic differentiation process instead of inhibiting it, as it was proposed in previous studies. Additionally, as activation of FGF signaling resembles a loss of BMP signals the interaction of FGF and BMP signaling was analyzed. Both pathways were found to have antagonistic functions during chondrocyte development. Moreover, activation of BMP signaling in FGFach mice in the limb culture system can increase the reduced rate of chondrocyte proliferation and Ihh expression thus rescuing the dwarfism phenotype. Summarizing, the results of this study allowed to integrate three signaling pathways, Ihh/Pthlh, BMP and FGF, into a common control network regulating chondrocyte development.

Table of Contents

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TITLE PAGE AND SUMMARY
0. CONTENTS I
1. INTRODUCTION 1
1.1 Skeleton of vertebrates 1
1.2 Endochondral ossification 2
1.3 Structure of developing cartilage 4
1.4 Different signaling pathways regulating bone development 5
1.4.1 Human skeletal diseases 5
1.4.2 Ihh/Pthlh pathway directs endochondral ossification 7
1.4.3 BMP family 8
1.4.4 BMPs and Ihh may interact to regulate chondrocyte maturation 9
1.4.5 FGF signaling controls bone development 10
2. MATERIAL AND METHODS 13
2.1 Materials 13
2.1.1 Bacterial media 15
2.1.2 Limb-culture media 15
2.1.3 Reagents and buffers for in situ hybridization 16
2.1.4 Buffer for mice genotyping 17
2.1.5 Reagents and buffers for protein purification 17
2.1.6 Kits 19
2.1.7 Proteins 19
2.1.8 Table 1. Protein names and their synonyms 20
2.1.9 Table 2. Chicken and mouse DNA probes for in situ hybridization 21
2.1.10 Cell lines 22
2.1.11 Chicken and mouse lines 22
2.1.12 Bacterial strain 22
2.2 Methods 23
2.2.1 Genomic DNA preparation from mouse tail 23
2.2.2 Genotyping of mice 23
2.2.3 Limb culture 24
2.2.4 Harvesting and dehydration of limb tissue 25
2.2.5 BrdU labeling of limb explants 25
2.2.6 Silanization of slides 25
2.2.7 Preparation of DNA template for in vitro transcription 25
2.2.8 Labeling of antisense riboprobes 26
2.2.9 In situ hybridization 26
2.2.10 Washing of slides and dipping in photoemulsion 27
2.2.11 Production and purification of Noggin protein 27
2.2.12 Dot-blot hybridization for Noggin protein 28
3 RESULTS 29
3.1 Limb culture system 29
3.1.1 Concentration- and time-courses in the limb culture system 29
3.2 Ihh/Pthlh pathway 32
3.2.1 Ihh overexpression results in delayed chondrocyte differentiation 32
3.2.2 Modulation of Ihh signaling by Pthlh or cyclopamine in a limb culture 33
3.3 Interaction of BMP signaling and the Ihh/Pthlh pathway 34
3.3.1 BMP signaling regulates chondrocyte differentiation and proliferation 34
3.3.2 Bmp expression is upregulated in Ihh-overexpressing mice 36
3.3.3 BMPs are not mediators of Ihh signaling in regulating hypertrophic differentiation 36
3.3.4 Pthlh expression is not regulated by BMP signaling 38
3.3.5 BMPs and Ihh interact similarly in chick and in mouse 39
3.3.6 BMPs induces chondrocyte proliferation independent of Ihh 40
3.3.7 BMP signaling negatively regulates terminal hypertrophic differentiation 41
3.4 Interaction of FGF signaling with the Ihh/Pthlh pathway 43
3.4.1 FGF signaling regulates the chondrocyte differentiation and proliferation 43
3.4.2 FGFs advance hypertrophic differentiation by downregulating Ihh expression 45
3.4.3 FGF signaling regulates terminal hypertrophic differentiation independent of the Ihh/Pthlh system 47
3.4.4 FGF signaling reduces chondrocyte proliferation independent of Ihh/Pthlh 48
3.5 Interaction of FGF and BMP signaling 49
3.5.1 FGF signaling antagonizes BMP signal 49
3.5.2 BMPs rescue FGF-induced defects in achondroplasia mice 51
3.5.3 BMP and FGF signaling reciprocally regulate their expression 52
4 DISCUSSION 54
4.1 Analyzing of signaling systems using the limb culture system 54
4.2 Interaction of BMP signaling and the Ihh/Pthlh pathway 55
4.2.1 BMPs do not act as secondary signals of Ihh in regulating the onset of hypertrophic differentiation 55
4.2.2 BMP signaling regulates several aspects of chondrocyte development 57
4.2.3 The Bmp gene family 58
4.2.4 Interaction of BMP signaling with the Ihh/Pthlh pathway 59
4.3 Interaction of FGF and Ihh/Pthlh signaling 61
4.3.1 FGF signaling regulates bone development 61
4.3.2 FGF signaling accelerates hypertrophic differentiation 62
4.3.3 FGF and BMP signaling act as antagonists during cartilage development 64
4.3.4 BMP signaling rescues defects in achondroplasia mice 65
4.4 Conclusions 65
5 REFERENCES 67
6 APPENDIX 79
6.1 Abbreviations 79
6.2 Curriculum vitae 80
6.3 Publications and Meetings 81
6.4 Acknowledgments 82
6.5 Selbstständigkeitserklärung 83

More Information:

Online available: http://www.diss.fu-berlin.de/2002/256/indexe.html
Language of PhDThesis: english
Keywords: Ihh, Pthlh, BMP, FGF, chondrogenesis
DNB-Sachgruppe: 32 Biologie
Date of disputation: 14-Jun-2002
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Günter Korge
Second Referee: Prof. Dr. Roland Lauster
Third Referee: Prof. Dr. H. Kress, Prof. Dr. W. Schuster
Contact (Author): minina@molgen.mpg.de
Contact (Advisor): vortkamp@molgen.mpg.de
Date created:18-Nov-2002
Date available:27-Nov-2002

 

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