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Skeleton Development Patricia Ducy HHSC1616 x5-9299 pd2193@columbia
Skeleton Development Patricia Ducy HHSC1616 x
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Skeleton ≥ 200 elements Two tissues: cartilage, bone Three cell types:
chondrocytes, osteoblasts, osteoclasts Three “environments”: marrow, blood, SNS Growth Formation Resorption Necessity of in vivo analysis Blooming thanks to mouse genetics Advantage is that genes are conserved between muse and human
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Embryonic origin of the skeleton
Cranial neural crest cells Somitic mesoderm Lateral plate mesoderm Monocyte lineage Craniofacial skeleton Axial skeleton Appendicular Skeleton Complex origin however each and all bones will end up with the same strucutre Chondrocytes & Osteoblasts Osteoclasts
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Skeleton Biology Patterning Development Skeletogenesis Birth Life
Location and shape of skeletal elements Development Skeletogenesis Differentiated cells Bone structure Growth/Modeling Birth More spectacular biology and disorders during development But more dramatic biomedical concerns are post-natal Osteoporosis = 1/2 Arthritis =1/3 Life Homeostasis Fracture repair Remodeling Balance between Formation/Resorption
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Skeleton Pathologies Patterning Dysostoses Development Skeletogenesis
Dysplasia Mineralization defects Degenerative diseases Life Homeostasis
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Genetic defects associated with skeleton development
(RUNX2)
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Skeleton patterning Condensation of mesenchymal cells to form the scaffold of each future skeletal element Migration Adhesion Proliferation Early steps use signaling molecules and pathways generally involved in patterning other tissues (FGFs, Wnts, BMPs) Orchestrated by specific set of genes acting as territories organizers When not embryonic lethal disorders often localized Organizer genes often control not only skeleton but also skin, axis, muscles… Organizer genes often identified via comparative genetics Human genetics
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Hox transcription factors
First described in Drosophila where they control body plan organization Arranged in 4 genomic clusters in mammals Expression patterns follow the cluster arrangement
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Homeotic transformations in absence of Hox transcription factors
Wellik, Dev. Dynamics 236 (2007)
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Hox transcription factors control vertebrate limb patterning
Genomic organization Site of expression Initially identified in drosophila where they regulate antero-posterior axis Sequential expression in space and time according to the location within the cluster Discrete territories of influence ---->Defect limited to one area/element
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Mutations in HOXD13 cause synpolydactyly* in humans
*OMIM 18600, Patient with increased number of Ala repeat in HOXD13 Muragaki et al., Science 272 (1996)
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Skeletogenesis Cell differentiation Bone morphogenesis
Chondrocytes, osteoblasts, osteoclasts Bone morphogenesis Formation of growth plate cartilage, bone shaft and marrow cavity Vascular invasion and innervation Defects generalized
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Two skeletogenetic mechanisms
Endochondral ossification Differentiation of a cartilaginous scaffold (chondrocytes) later replaced by bone (osteoblasts) Most of the skeletal elements Intramembranous ossification Direct differentiation of the condensed mesenchymal cells into osteoblasts Many bones of the skull, clavicles
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Hypertrophy
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Sox9 Transcription factor of the HMG family
Regulates the expression of chondrocyte-specific genes Sox9 haploinsufficiency causes Campomelic dysplasia (OMIM ) Earliest known regulator of chondrocyte differentiation Mutation known for long time. Sex reversal understood. Meca in chondro not understood before col2 prom analysis and mouse studies
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Sox9-deficient cells cannot differentiate into chondrocytes
Bi et al., Nat. Genet 22 (1999)
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Sox9 Sox5, 6 Hypertrophy
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Karp et al., Development 127 (2000)
Accelerated chondrocyte hypertrophy in PTHrP-deficient mice +/+ PTHrP -/- PTHrP negative regulator of chondro maturation PTHrP receptor KO, same phenotype. PTHrP recept activting mutation, Jansen metaphyseal dysplasia ---> dwarfism due to lack of hypertrophic chondro Karp et al., Development 127 (2000)
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PTHrP Ubiquitously expressed growth factor
Shares the same receptor with PTH Mice “knockout” only phenotype is a generalized growth plate cartilage defect PTHrP protein signals to its receptor in the prehypertrophic chondrocytes and blocks their hypertrophic differentiation Mouse study pivotal
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Dwarfism in Ihh-deficient mice
+/+ Ihh -/- St-Jacques et al. , Genes Dev. 13 (1999)
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Indian hedgehog (Ihh) One of 3 members of the Hedgehog family of growth factors Widely expressed during development Expression positively regulated by the transcription factor Runx2
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Reduced chondrocyte proliferation and delayed chondrocyte hypertrophy in Ihh-deficient mice
+/+ Ihh -/- +/+ Ihh -/- St-Jacques et al. , Genes Dev. 13 (1999)
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Chondrocyte maturation is regulated by a PTHrP/Ihh feedback loop
Perichondrium Resting Proliferating Pre-hypertrophic PTHrP receptor Hypertrophic PTHrP No PTHrP No Ihh Ihh
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Schipani & Provost. Brith Defects Res. 69 (2003)
Mutations in the PTH/PTHrP receptor cause Jansen and Bloomstrand chondrodysplasia Activating mutations Loss-of-function mutations Jansen metaphyseal chondrodysplasia OMIM Blomstrand's lethal chondrodysplasia OMIM Schipani & Provost. Brith Defects Res. 69 (2003)
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Sox9 Sox5, 6 Ihh/PTHrP Runx2 Hypertrophy VEGF
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Endochondral ossification
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Arrest of osteoblast differentiation in Runx2-deficient mice
+/+ Runx2 -/- Otto et al., Cell 89 (1997)
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Runx2 One of three members of the runt family of transcription factors
Identified as a regulator of the Osteocalcin promoter Necessary and sufficient for osteoblast differentiation
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Lee et al. , Nat Genetics 16 (1997)
Cleidocranial dysplasia (CCD, OMIM ) is caused by Runx2 haploinsufficiency +/+ +/- Mundlos et al., Cell 89 (1997) Lee et al. , Nat Genetics 16 (1997)
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Intramembranous ossification
Suture formation Growth Mesenchymal cells from a layer and differentiate in situ Differentiation progress from the center of each bone Difficult to differentiate patterning disorders from differentiation defects Most disorders affect suture fusion ---> process well studied Suture morphogenesis controlled by epithelium-mesenchyme interactions Closure
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Disorders of suture fusion
Delay Msx2, Runx2 haploinsufficiency Acceleration = craniosynostosis Craniosynostoses, very frequent FGFR1, 2, 3 activating mutations Msx2 activating mutations Twist haploinsufficiency
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Osteoblast differentiation
Twist (Saethre-Chotzen Syndrome OMIM ) Runx2 Osx, ATF4 Osteoprogenitor Pre-osteoblast Osteoblast
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Delayed osteogenesis in absence of Atf4
WT Atf4-/- Many skeletal elements of WT embryo are mineralized which stained by Alizarin Red, such as Jaw, skull bones, ribs, and limbs. Mineralization of these bones are absent in mutant embryo As a result of delay in osteogenesis, these mice are born with open frontnelles also, similar to Rsk2-/- mice E14 Yang et al., Cell 117 (2004)
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Delayed osteogenesis in Atf4-deficient mice
WT Atf4 -/- P0 E16 E15 Histologically, we can easily observe a delay in bone formation in A-/- embryos, for example, at E15, there is bone collar in WT but not in -/-, E16, trabecular bones in WT but not in -/-, at birth, only few trabecular bones are seen in -/- while extensive in WT Moecularly, the markers for terminal differentiated Obs, such as Bsp and OC expression are decreased… Yang et al., Cell 117 (2004)
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ATF4 Divergent member of the ATF/CREB family of leucine-zipper transcription factors Required for amino-acid import Identified as a regulator of the Osteocalcin promoter Activated by the Rsk2 kinase If ATF4 is the substrate of RSK2, its deficiency may cause a skeletal phenotype, to understand its physiological role, we also analyzed Atf4-/- mice The function of ATF4 in amino acid import and synthesis is important, I will come back to this point later
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ATF4 Lack of ATF4 phosphorylation by inactivating mutations in Rsk2 causes the skeletal defects associated with Coffin-Lowry syndrome (OMIM ) Increased ATF4 phosphorylation by Rsk2 causes the skeletal defects associated with Neurofibromatosis Type I (OMIM ) If ATF4 is the substrate of RSK2, its deficiency may cause a skeletal phenotype, to understand its physiological role, we also analyzed Atf4-/- mice The function of ATF4 in amino acid import and synthesis is important, I will come back to this point later
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ATF4 Divergent member of the ATF/CREB family of leucine-zipper transcription factors Required for amino-acid import Identified as a regulator of the Osteocalcin promoter Activated by the Rsk2 kinase If ATF4 is the substrate of RSK2, its deficiency may cause a skeletal phenotype, to understand its physiological role, we also analyzed Atf4-/- mice The function of ATF4 in amino acid import and synthesis is important, I will come back to this point later
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Elefteriou et al., Cell Metab. 4 (2006)
A high protein diet normalizes bone formation in Atf4-/- and Rsk2-/- mice High protein diet BV/TV BFR Ob.S/BS Elefteriou et al., Cell Metab. 4 (2006)
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Elefteriou et al., Cell Metab. 4 (2006)
A low protein diet normalizes bone formation in a mouse model of Neurofibromatosis type I Normal diet Low protein diet wt Nf1ob-/- wt Nf1ob-/- These mice have been analyzed at 3 and 6 months of age and displayed an increase in bone mass visible in the right panel, This increase in bone mass originates from an increase in osteoblast activity and number, as measured by an increase in BFR and osteoblast surface and number. Therefore, this phenotype suggests that Neurofibromin is an inhibitor of bone formation, that may negatively regulate osteoblast function and proliferation. BV/TV 15.3±1 19.6±0.4* 14.8±0.7 15.3±0.6 BFR 153.3±11 313.9±7.0* 157.0±11 186.2±22 ObS/BS 19.1±0.8 31.8±1.6* 19.0±0.5 19.7±1.0 Elefteriou et al., Cell Metab. 4 (2006)
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Structure of a growing long bone
articular cartilage (chondrocytes) secondary ossification centre (osteoblasts/osteoclasts) reserve cartilage proliferating cells Growth plate Cartilage (chondrocytes) hypertrophic cells calcified cartilage trabecular bone (osteoblasts/osteoclasts) cortical bone (osteoblasts)
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Control of osteoclast differentiation and function
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Osteopenia in OPG-deficient mice
Bucay et al., Genes Dev. 12 (1998)
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Osteopetrosis in RANK-L deficient mice
Lacey et al., Cell 93 (1998)
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Osteoblast progenitor
OPG RANK-L Inactive complex A ctive complex RANK TRAF 6 TRAF 2/5 Osteoclast progenitor NF NF k k B B c-src JNK JNK Osteoclast Maturation AP1 activation
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Research directions Knowledge Diseases Patterning Development
Skeletogenesis Life Homeostasis
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