Talk:2009 Lecture 13

Background Reading

• Cytomegalovirus induces abnormal chondrogenesis and osteogenesis during embryonic mandibular development

Tina Jaskoll, George Abichaker, Parish P Sedghizadeh, Pablo Bringas, Jr, and Michael Melnick BMC Dev Biol. 2008; 8: 33. Published online 2008 March 27. doi: 10.1186/1471-213X-8-33. PMCID: PMC2330031

• Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis

Amitabha Bandyopadhyay, Kunikazu Tsuji, Karen Cox, Brian D Harfe, Vicki Rosen, and Clifford J Tabin PLoS Genet. 2006 December; 2(12): e216. Prepublished online 2006 November 6. Published online 2006 December 22. doi: 10.1371/journal.pgen.0020216. PMCID: PMC1713256

• Isolation and characterization of side population stem cells in articular synovial tissue

Takeshi Teramura, Kanji Fukuda, Shinji Kurashimo, Yoshihiko Hosoi, Yoshihisa Miki, Shigeki Asada, and Chiaki Hamanishi BMC Musculoskelet Disord. 2008; 9: 86. Published online 2008 June 12. doi: 10.1186/1471-2474-9-86. PMCID: PMC2440379

• http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2579486&tool=pmcentrez The sclera and the corneal stroma that are anatomically continuous have common characteristics such as mechanical rigidity, and share a common origin, i.e., the neural crest.

• http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2714097&tool=pmcentrez

• Heart and craniofacial muscle development: a new developmental theme of distinct myogenic fields.

Tzahor E. Dev Biol. 2009 Mar 15;327(2):273-9. Epub 2009 Jan 6. Review. PMID: 19162003

• The genetics of vertebrate myogenesis.

Bryson-Richardson RJ, Currie PD. Nat Rev Genet. 2008 Aug;9(8):632-46. Review. PMID: 18636072 http://www.nature.com/nrg/journal/v9/n8/pdf/nrg2369.pdf

• Cell fusions in mammals.

Larsson LI, Bjerregaard B, Talts JF. Histochem Cell Biol. 2008 May;129(5):551-61. Epub 2008 Mar 20. Review. PMID: 18351375

• Context matters: in vivo and in vitro influences on muscle satellite cell activity.

Cornelison DD. J Cell Biochem. 2008 Oct 15;105(3):663-9. Review. PMID: 18759329

Summary

The following text is extracted and modified from previous lecture slides and should be used as a "trigger" to remind you of some key concepts.

• Bone DevelopmentSkeleton patterningshape and location of each specific skeletal element2 ossification typesEndochondrial, IntramembranousCell differentiation in skeletonchondrocyte in cartilageosteoblast in boneosteoclast in bonemolecular control of major function of skeletonskeleton growth (fetal, neonatal, puberty)bone remodelling, mineralizationAbnormalities

• Background - Mesoderm Development Somite, somatic Vertebra Development Somite, sclerotome Limb and Axial Development Endochondrial ossification Head Development Intramembranous ossification

• Textbook ReferencesHuman Embryology (3rd ed.) LarsonChapter 11 Limb Dev (bone not well covered in this textbook)The Developing Human (6th ed.) Moore & PersaudChapter 15 the skeletal systemBefore we Are Born (5th ed.) Moore and PersaudCh16,17: p379-397, 399-405Essentials of Human Embryology LarsonCh11 p207-228Human Embryology Fitzgerald and FitzgeraldMolecular biology of the Cell (4th edn)
• Introductionossification of a cartilage formed from mesenchyme2 main forms of ossification IntramembranousskullEndochondrialVertebra, limb long bonesOssification continues postnatallythrough puberty until mid 20sEarly ossification occurs at ends of long bones
• Movie: Ossification SitesEarly Fetal SkeletonEarly Fetal SkeletonVertebra- Sclerotomal cellsMovie: Segmental VertebraEndochondrial OssificationVertebra Cartilage to BoneVertebra Cartilage to BoneMature BoneMature BoneDeveloping JointSkeleton Cell Lineage
• Bone Components
• CellsOsteoblasts, osteocytes, osteroclastsOrganic Matrix95% Type I collagen5% proteoglycans and noncollagenous proteinsOsteopontinOsteocalcin
• Bone Cell TypesChondrocytemesenchymal originOsteoblastmesenchymal origin Osteoclastmonocytic originGenes controlling skeleton patterning and cell differentiation are differentPatterning- Limb BonesBone Mandible Development1st arch prechondrogenic condensation in mouse embryoseveral skeletal elementsarise from a single condensation and persist for varying timeBoneBluecore component gives rise to Meckel‚Äôs cartilage (duration 12h)Brownrostral component forms symphyseal cartilage between two lower jaws (lasts 28h)Greencaudal component that lies across the mandibularhyoid arch boundary (dotted line), (lasts 30h)forms malleusRedmost caudal component (duration of 30h)forms incusPhases of DevelopmentCell Condensationkey stage in skeletal and mesenchymal tissue development Initially dispersed population of cellsgathers together to differentiate into a single cell/tissue typecartilage, bone, muscle, tendon, kidney, lung earliest stage during tissue formationwhen tissue specific genes are up-regulatedCell CondensationExtracellular matrix molecules, cell surface receptors and cell adhesion moleculesinitiate condensation formationset condensation boundariesfibronectin, tenascin, syndecan, and N-CAMHox genes (Hoxd-11-13) and other transcription factors (CFKH-1, MFH-1, osf-2)modulate the proliferation of cells within condensationsCell adhesion indirectly through Hox genes (Hoxa-2, Hoxd-13)directly via cell adhesion molecules (N-CAM and N-cadherin)Cell Condensation Growthregulated by BMPs which activatePax-2, Hoxa-2, Hoxd-11 and other genesceases when Noggin inhibits BMP signallingNext stage of skeletal developmentovert cell differentiationCondensation Formation GenesMesenchyme Condensationcondensation formation (blue) transition from condensation to overt differentiation (green arrow) condensation shown using peanut agglutinin lectinelevated levels of cyclic-AMP and major genes expressed at condensation stage (Pax-1, Pax-9, Sox-9)differentiating cartilage is visualised with Alcian bluemajor genes associated with five stages in condensationInitiate; Set Boundary; Proliferate; Adhere, GrowTwo pathways that stop condensation growth shown in yellow and redCessation of condensation leads to differentiation (green arrow), which involves both upregulation of genes to initiate differentiation and downregulation of genes to terminate condensation.Control of chondrocyte and osteoblast differentiationTranscription FactorsEarly transcription factorsSox9 Late transcription factors Cbfa1 and other factors promote differentiation of type II collagen expressing proliferating chondrocytes into pre-hypertrophic (pre-HC) cells expressing Indian hedgehog (Ihh) and PTH/PTHrP receptorIndian HedgehogIHH acts on Cells in perichondriumfavours production of PTHrPwhich inhibits chondrocyte hypertrophywhereas FGFs/FGFR3 antagonizes Ihh expressionBone collarinduces expression of Cbfa1triggering osteoblast differentiation chondrocytesOver-expression of Cbfa1 induces Ihh expressionHypertrophic Chondrocytes(HCs) secrete VEGFpromoting vascular invasionthen hypertrophic calcified cartilage becomes resorbed by recruited chondroclasts/osteoclasts via MMP9
• OsteoblastsOsteoblasts derived from the bone collarreplace cartilage matrix with a matrix rich in type I collagen leading to bone formationknown molecules affecting differentiation of pre-osteoblasts into osteoblastsHoxa2LeptinCuboidal osteoblastson newly formed bone as well as some osteocytes embedded in the bone matrixOsteoblast Development Cbfa1Yellow - activation domains Green - repression domainsGrey - Runt DNA-binding domain Q/A - glutamine/alanine repeatsPST - proline/serine/threonine-rich regionNLS - nuclear localization signalVWRPY - C-terminal repressor motifRegulation of Cbfa1 expressionRed- Transcription factorsBlue- secreted moleculesBrown- cofactorsGreen- ‚Äúmodifier complexes‚Äù Expression Level RegulationRed Arrows- (activation)blunted lines (repression)Functional Regulation green arrows (activation)blunted lines (repression)Cofactor binding is indicated by a black line.
• Osteoblast DevelopmentCbfa1expressionIn situ hybridization10.5 day mouse embryoArrows - primordia of shoulder bonetrachea (T) - low levels present
• Osteoblast DevelopmentCbfa1 and type II collagen expression14.5 day mouse embryoossifying ribs Cbfa1expressioncartilaginous structuresdo not express Cbfa1type II collagen expression
• Osteoblast Differentiationgenetic control of osteoblast differentiation and functionRed - Transcription factorsBlue - secreted moleculesBlack arrows - stimulatory or inhibitory roles of these moleculesGrey arrows - possible regulatory pathwaysMechanisms of Osteoclast Differentiation
• Leptin Role?bone remodelling thought to be mainly an autocrine-paracrine resorption mechanisms are under control of hormonessuggested this also may regulate bone formationmolecular endocrinology suggests a common central regulation via leptinbone formationbody weightreproduction
• Bone Morphogenic ProteinsFirst BMPs identified by ability to induce ectopic bone formation when implanted under skin of rodentsrecapitulation of all events occurring during skeletogenesisMore than 30 BMPs have been identifiedexpression pattern and analysis of spontaneously mutated or genetically depleted mice show a much broader range of functionactivities localized at sites of epithelial-mesenchymal interactionsincluding but not restricted to the skeleton
• Bone Morphogenetic Proteins BMPslarge family of cytokines related to members of transforming growth factor-beta superfamilyMouse - required for mesoderm formation and for development and patterning of many different organ systemsXenopus - role in gastrulation and neurulation in XenopusExtracellular modifiers of BMP activity may constitute an opposing morphogenetic system
• Endochondrial BoneIntramembranous BoneFgf and receptor expression
• Intramembranous BoneFgf and receptor expression

BMP2- Clinical StudiesImplantation of recombinant human BMP-2 (rhBMP-2) can augment alveolar ridge in animal models when placed in a periodontal environmentrestore new bone and attachment tissuesClinical studies support ability of rhBMP-2 implants to induce physiologic boneThe bone morphogenetic protein family: multifunctional cellular regulators in the embryo and adult. Wozney JM. Eur J Oral Sci. 1998 Jan;106 Suppl 1:160-6. ReviewPotential applications and delivery strategies for bone morphogenetic proteins. Kirker-Head CA. Adv Drug Deliv Rev. 2000 Sep 15;43(1):65-92. Review

Bone AbnormalitiesAbnormalitiesCongenital Hip DislocationInstability: 1:60 at birth; 1:240 at 1 wk: Dislocation untreated; 1:700congenital instability of hiplater dislocates by muscle pulls or gravityfamilial predisposition female predominanceGrowth of femoral head, acetabulum and innominate bone are delayed until the femoral head fits firmly into the acetabulumAbnormalities- Scoliosisassymetric growth impairment of vertebral bodieslateral deviation of spineLateral flexionForward flexionRotation of vertebral column on long axiscompensated by movement of vertebral column above and below affected region producing a primary and two secondary curvesprogresses rapidly in adolescencebecomes fixed once bone growth is completedHox2a KnockoutSynergistic Interactionsmouse skeleton (a)sternum (b)regions affected by short ear (se) and brachypodism (bp) mutationsSynergistic Interactionsse and bp mutationsaffect different parts of mouse skeletoncoloured red and blue, respectivelyse/se; bp/bp double mutants show the sum of individual phenotypes and also the sternum is affected (green)se and bp gene products, BMP5 and GDF5, respectively, have a synergistic effect in sternal developmentAbnormalities FGF receptors