UNSW Embryology

Musculoskeletal Development - Bone

© Dr Mark Hill (2008)

Acknowledgements

Introduction

Bone is formed through a lengthy process involving ossification of a cartilage formed from mesenchyme.

Early ossification in long bones (More? movie developing mouse)	Bone and cartilage in the developing knee (cartilage blue, bone red)

Two main forms of ossification occur in different bones, intramembranous (eg skull) and endochondral (eg vertebra) ossification. Ossification continues postnatally, through puberty until mid 20s.

Endochondral Ossification in the Limb

Bones within the limb form by endochondrial ossification (begins Carnegie stage 18) throughout embryo. This process is the replacement of cartilage with bone (week 5-12).(More? limb development).

Osteoblasts manufacture bone and are derived from mesodermal in origin, arising from multipotential mesenchymal cells and further differentiate into bone-lining cells and osteocytes. (More? lineages | Osteoblast to Osteocyte)

Osteoclasts resorb bone and are derived from hematopoietic precursor cells formed by the fusion of monocytic cells at the bone sites to be resorbed. (More? Osteoclasts | Cardiovascular System - Blood)

The marrow of bones is the site of haematopoiesis, the generation of blood cells. At birth nearly all bones are a source of blood cells this is restricted with postnatal development to a few specific bones. Pluripotential stem cells reside in the marrow and are a self renewing source of stem cells or commitment to a progenitor cell.

The transcription factor Core Binding Factor 1 (Cbfa1) plays an essential role in osteoblast differentiation, bone formation, matrix production and mineralization. (More? molecular development)

Page Links: Introduction | Some Recent Findings | Reading | Computer Activities | Development Overview | Bone Matrix | Bone Cell Lineages | Osteoblast to Osteocyte | Osteoclasts | Ossification Stages | Glossary | Bone Development Terms | References

Some Recent Findings

Zhang C, Cho K, Huang Y, Lyons JP, Zhou X, Sinha K, McCrea PD, de Crombrugghe B. Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix. Proc Natl Acad Sci U S A. 2008 May 5.

"the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. ...we speculate that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/beta-catenin activity"

Maeda Y, Nakamura E, Nguyen MT, Suva LJ, Swain FL, Razzaque MS, Mackem S, Lanske B. Indian Hedgehog produced by postnatal chondrocytes is essential for maintaining a growth plate and trabecular bone. Proc Natl Acad Sci U S A. 2007 Apr 4;

"Indian hedgehog (Ihh) is essential for chondrocyte and osteoblast proliferation/differentiation during prenatal endochondral bone formation. ...These results demonstrate, for the first time, that postnatal chondrocyte-derived Ihh is essential for maintaining the growth plate and articular surface and is required for sustaining trabecular bone and skeletal growth."

Agoston H, Khan S, James CG, Gillespie JR, Serra R, Stanton LA, Beier F. C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and -independent pathways. BMC Dev Biol. 2007 Mar 20;7(1):18

"C-type natriuretic peptide (CNP) has recently been identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanisms mediating its effects are not completely understood. ...Our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral bone growth"

Liu X, Bruxvoort KJ, Zylstra CR, Liu J, Cichowski R, Faugere MC, Bouxsein ML, Wan C, Williams BO, Clemens TL. Lifelong accumulation of bone in mice lacking Pten in osteoblasts. Proc Natl Acad Sci U S A. 2007 Feb 7; (More? OMIM - Pten)

"Bone formation is carried out by the osteoblast, a mesenchymal cell whose lifespan and activity are regulated by growth factor signaling networks. Growth factors activation.... is negatively regulated by the Pten phosphatase. ...Mice deficient in Pten in osteoblasts were of normal size but demonstrated a dramatic and progressively increasing bone mineral density throughout life."

(More? Bone Development References)

Reading

• Human Embryology (3rd ed.) Larson Ch11 p311-339
• The Developing Human: Clinically Oriented Embryology (6th ed.) Moore and Persaud Ch15,16: p405-423, 426-430
• Before We Are Born (5th ed.) Moore and Persaud Ch16,17: p379-397, 399-405
• Essentials of Human Embryology Larson Ch11 p207-228
• Human Embryology, Fitzgerald and Fitzgerald
• Human Embryology and Developmental Biology, (3rd ed.) Carlson Ch9,10: p173-193, 209-222

Computer Activities

Relevant Webpages

The following webpages pages should be looked initially at in relation to bone development: Somite | Limb | Axial Skeleton | Human Bone | Skull | UNSW Cell Biology Medicine Practical: Bone Development, Structure | Developmental Biology (6th ed.) Gilbert Osteogenesis: The Development of Bones

Lectures/Laboratories: These Lecture links are to current and historic courses and may also link directly to PDF version of Lecture slides (educational use only).

• ANAT2341 (2008) Lecture Bone Development
• UNSW Cell Biology Medicine Practical: Bone Development, Structure
• ANAT2300 (2004) Lecture 6 Mesoderm
• ANAM1006 (2003) Musculoskel Development Lab
• PDF Documents ANAT2300 (2004) Lecture 7 Bone (view) | ANAT2300 (2004) Lecture 7 Bone (print)

Human Embryology Movies: Fate of the Somite (315Kb) | Vertebrae (366Kb)

Embryo Images Unit: (External links require Internet connection) Body Cavities, Musculoskeletal &Limb Development | Embryo Images Online | Musculoskeletal Development | Musculoskeletal (Early Week 4) | Musculoskeletal (Late Week 4)

Developmental Biology (6th ed.) Gilbert: (External links require Internet connection) Paraxial Mesoderm: The Somites and Their Derivatives | Osteogenesis: The Development of Bones

Bone Matrix

Bone matrix and marrow

The organic matrix of bone consists of:

• 95% Type I collagen
• 5% proteoglycans and noncollagenous proteins (osteopontin and osteocalcin).

Bone Cell Lineages

• Paraxial Mesoderm
  • Somite
    • sclerotome
      • osteoprogenitor cells
        • osteoblasts
          • osteocytes
        • stem cells (bone lining cells)
• Bone Marrow (pluripotential stem cell)
  • monocytes
    • osteoclasts

Development Overview

Below is a very brief overview using simple figures of 3 aspects of early musculoskeletal development covering : Mesoderm then Somite and Limb development

More detailed overviews are shown on other notes pages (Mesoderm and Somite, Vertebral Column, Limb) in combination with serial sections and Carnegie images.

In mammals the clavicle is one of the first bones to ossify by a mix of intramembranous and endochondral ossification from two centres of ossification within a single condensation. The suggested bird equivilant, the furculae (wishbone), ossifies only by intramembranous within a single center in each condensation.

 

Mesoderm Development

Cells migrate through the primitive streak to form mesodermal layer. Extraembryonic mesoderm lies adjacent to the trilaminar embryo totally enclosing the amnion, yolk sac and forming the connecting stalk.

Paraxial mesoderm accumulates under the neural plate with thinner mesoderm laterally. This forms 2 thickened streaks running the length of the embryonic disc along the rostrocaudal axis. In humans, during the 3rd week, this mesoderm begins to segment. The neural plate folds to form a neural groove and folds.

Segmentation of the paraxial mesoderm into somites continues caudally at 1 somite/90minutes and a cavity (intraembryonic coelom) forms in the lateral plate mesoderm separating somatic and splanchnic mesoderm.

Note intraembryonic coelomic cavity communicates with extraembryonic coelom through portals (holes) initially on lateral margin of embryonic disc.

Somites continue to form. The neural groove fuses dorsally to form a tube at the level of the 4th somite and "zips up cranially and caudally and the neural crest migrates into the mesoderm.

Next Somite Development

Somite Development

Mesoderm beside the notochord (axial mesoderm) thickens, forming the paraxial mesoderm as a pair of strips along the rostro-caudal axis.

Paraxial mesoderm towards the rostral end, begins to segment forming the first somite. Somites are then sequentially added caudally. The somitocoel, is a cavity forming in early somites, which is lost as the somite matures.

Cells in the somite differentiate medially to form the sclerotome (forms vertebral column) and laterally to form the dermomyotome.

The dermomyotome then forms the dermotome (forms dermis) and myotome (forms muscle).

Neural crest cells migrate beside and through somite.

The myotome differentiates to form 2 components dorsally the epimere and ventrally the hypomere, which in turn form epaxial and hypaxial muscles respectively. The bulk of the trunk and limb muscle coming from the Hypaxial mesoderm. Different structures will be contributed depending upon the somite level.

Osteoblast to Osteocyte

Several models exist for the conversion or maturation of an osteoblast to osteocyte, a recent review suggests that slowing osteoblasts gradually get buried by their more rapid neighbours leading to their differentiation into osteocytes.

Franz-Odendaal TA, Hall BK, Witten PE. Buried alive: how osteoblasts become osteocytes. Dev Dyn. 2006 Jan;235(1):176-90. Review.

"...We investigate how these data support five schemes that describe how an osteoblast could become entrapped in the bone matrix (in mammals) and suggest one of the five scenarios that best fits as a model. Those osteoblasts on the bone surface that are destined for burial and destined to become osteocytes slow down matrix production compared to neighbouring osteoblasts, which continue to produce bone matrix. That is, cells that continue to produce matrix actively bury cells producing less or no new bone matrix (passive burial)."

Osteoclasts

Osteoclasts resorb bone and are derived from hematopoietic precursor cells formed by the fusion of monocytic cells at the bone sites to be resorbed.

References: Bruzzaniti A, Baron R. Molecular regulation of osteoclast activity. Rev Endocr Metab Disord. 2006 Sep 2. | Li Z, Kong K, Qi W. Osteoclast and its roles in calcium metabolism and bone development and remodeling. Biochem Biophys Res Commun. 2006 May 5;343(2):345-50. Epub 2006 Mar 6. | Roodman GD. Regulation of osteoclast differentiation. Ann N Y Acad Sci. 2006 Apr;1068:100-9.

Links: Dev Biology - Pathway for osteoclast differentiation | Dev Biology - Osteoclast Commitment, Differentiation, and Function

Ossification Stages

The process of ossification of bones, as determined postnatally clinically, include a series of stages.

1. Stage 1 - non-ossified epiphysis
2. Stage 2 - discernible ossification centre
3. Stage 3 - partial fusion
4. Stage 4 - total fusion

An additional stage has been used recently (Stage 5), which is the disappearance of the epiphyseal scar after total fusion.

Reference: Schmeling A, Schulz R, Reisinger W, Mühler M, Wernecke KD, Geserick G.Studies on the time frame for ossification of the medial clavicular epiphyseal cartilage in conventional radiography. Int J Legal Med. 2004 Feb;118(1):5-8.

References

Links: Journals | Online Textbooks | Search Textbooks | PubMed | Search PubMed | Glossary

Journals

Journal of Bone and Mineral Research JBMR "provides a forum for papers of the highest quality pertaining to all areas of the biology and physiology of bone, the hormones that regulate bone and mineral metabolism, and the pathophysiology and treatment of disorders of bone and mineral metabolism."

Bone official Journal of the International Bone and Mineral Society "The Journal is an interdisciplinary forum for the rapid publication of original, experimental or clinical studies, and review articles dealing with both normal and pathological processes which occur in bone or in other tissues affecting bone metabolism."

Online Textbooks

Developmental Biology (6th ed.) Gilbert Osteogenesis: The Development of Bones

Search NLM Online Textbooks- "bone development" : Endocrinology | Molecular Biology of the Cell | The Cell- A molecular Approach

PubMed

Reviews

Pogue R, Lyons K. BMP signaling in the cartilage growth plate. Curr Top Dev Biol. 2006;76:1-48.

Mundy GR, Elefteriou F. Boning up on ephrin signaling. Cell. 2006 Aug 11;126(3):441-3.

Franz-Odendaal TA, Hall BK, Witten PE. Buried alive: how osteoblasts become osteocytes. Dev Dyn. 2006 Jan;235(1):176-90. Review.

Li Z, Kong K, Qi W. Osteoclast and its roles in calcium metabolism and bone development and remodeling. Biochem Biophys Res Commun. 2006 May 5;343(2):345-50. Epub 2006 Mar 6.

Roodman GD. Regulation of osteoclast differentiation. Ann N Y Acad Sci. 2006 Apr;1068:100-9.

Mackie EJ. Osteoblasts: novel roles in orchestration of skeletal architecture. Int J Biochem Cell Biol. 2003 Sep;35(9):1301-5.

Ducy P. Cbfa1: a molecular switch in osteoblast biology. Dev Dyn. 2000 Dec;219(4):461-71. Review.

Articles

Prefumo F, Canini S, Crovo A, Pastorino D, Venturini PL, De Biasio P. Correlation between first trimester fetal bone length and maternal serum pregnancy-associated plasma protein-A (PAPP-A). Hum Reprod. 2006 Nov;21(11):3019-21.

Nakaoka R, Hsiong SX, Mooney DJ. Regulation of chondrocyte differentiation level via co-culture with osteoblasts. Tissue Eng. 2006 Sep;12(9):2425-33.

Iqbal J, Sun L, Kumar TR, Blair HC, Zaidi M. Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation. Proc Natl Acad Sci U S A. 2006 Sep 26

Sun L, Peng Y, Sharrow AC, Iqbal J, Zhang Z, Papachristou DJ, Zaidi S, Zhu LL, Yaroslavskiy BB, Zhou H, Zallone A, Sairam MR, Kumar TR, Bo W, Braun J, Cardoso-Landa L, Schaffler MB, Moonga BS, Blair HC, Zaidi M. FSH directly regulates bone mass. Cell. 2006 Apr 21;125(2):247-60. "Postmenopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels. Although FSH levels rise sharply in parallel, a direct effect of FSH on the skeleton has never been explored. We show that FSH is required for hypogonadal bone loss"

Bruzzaniti A, Baron R. Molecular regulation of osteoclast activity. Rev Endocr Metab Disord. 2006 Sep 2;

Hutchison C, Pilote M, Roy S. The axolotl limb: A model for bone development, regeneration and fracture healing. Bone. 2006 Aug 17;

Search PubMed

Search Oct2006 "bone development" 30,188 reference articles of which 3,029 were reviews.

Search PubMed: term= bone+development | endochondrial ossification | intramembranous ossification | osteoblast | cartilage+development

(More? PubMed- Medline)

Selected Lists of References from PubMed March 1999 search results are available for Department of Anatomy computers without internet access: Somite Reviews | Somitogenesis Abstracts | Mesoderm Review List

Computers with internet access can search from either Below or directly from PubMed Internet Access

Bone Development Terms

• annulus fibrosus- the circularly arranged fibers (derived from sclerotome)that together with the nucleus pulposus (derived from notochord) form the intervertebral disc (IVD) of the vertebral column.
• axial mesoderm- (=notochord)
• cartilage- connective tissue from mesoderm in the embryo forms initial skeleton replaced by bone. In adult, found on surface of bone joints.
• centrum- the primordium of the vertebral body formed initially by the sclerotome.
• clavicle- (L. little key) bone which locks sholder to body.
• cloacal membrane- at caudal (anal) end of gastrointestinal tract (GIT) where surface ectoderm and GIT endoderm meet forms the openings for GIT, urinary, reproductive tracts. (see also buccopharyngeal membrane)
• connective tissue-
• costotransverse-
• costovertebral-
• dermatome-
• dermomyotome- dorsolateral half of each somite that forms the dermis and muscle.
• ectoderm- the layer (of the 3 germ cell layers) which form the nervous system from the neural tube and neural crest and also generates the epithelia covering the embryo.
• endochondrial ossification- the process of replacement of the cartilagenous framework by osteoblasts with bone.
• epaxial myotome- the dorsal portion of the myotome that generates dorsal skeletal muscles (epaxial muscles).
• erector spinae-
• extracellular matrix- material secreted by and surrounding cells. Consists if fibers and ground substance.
• fibroblast growth factors- (FGF) a family of at least 10 secreted proteins that bind membrane tyrosine kinase receptors. A patterning switch with many different roles in different tissues. (FGF8 = androgen-induced growth factor (AIGF)
• fibroblast growth factor receptor- receptors comprise a family of at least 4 related but individually distinct tyrosine kinase receptors (FGFR1- 4). They have a similar protein structure, with 3 immunoglobulin-like domains in the extracellular region, a single membrane spanning segment, and a cytoplasmic tyrosine kinase domain.
• growth factor- usually a protein or peptide that will bind a cell membrane receptor and then activates an intracellular signaling pathway. The function of the pathway will be to alter the cell directly or indirectly by changing gene expression. (eg shh)
• hox- (=homeobox) family of transcription factors that bind DNA and activate gene expression. Expression of different Hox genes along neural tube defines rostral-caudal axis and segmental levels.
• hypaxial myotome- the ventral portion of the myotome that generates ventral skeletal muscles (hypaxial muscles).
• intercostal- the region between adjacent ribs, usually comprising intercostal muscles and connective tissue.
• intervertebral disc- (IVD) the annulus fibrosus+nucleus pulposus together form the intervertebral disc (IVD) of the vertebral column. This is the flexible region between each bony vertebra that allows the column to be bent.
• intervertebral foramina-
• lumbar plexus- mixed spinal nerves innervating the lower limb form a complex meshwork (crossing).
• mesoderm- the middle layer of the 3 germ cell layers of the embryo. Mesoderm outside the embryo and covering the amnion, yolk and chorion sacs is extraembryonic mesoderm.
• myoblast- the undifferentiated mononucleated muscle cells that will fuse together to form a multinucleated myotube, then mature into a muscle fibre.
• MyoD- transcription factor involved in the determination of muscle cells in the somite. A basic helix-loop-helix factor which binds DNA.
• myotome- the portion of the dermamyotome that generates skeletal muscle. Has 2 components epaxial (dorsal muscles ) hypaxial (ventral muscles).
• neural crest- cell region at edge of neural plate, then atop the neural folds, that remains outside and initially dorsal to the neural tube when it forms. These paired dorsal lateral streaks of cells migrate throughout the embryo and can differentiate into many different cell types(=pluripotential). Those that remain on the dorsal neural tube form the sensory spinal ganglia (DRG). Neural crest cells migrate into the somites.
• Pax- name derived from Drosophila gene 'paired' (prd) the 'paired box' is a amino end 124 amino-acid conserved domain (signature aa 35-51: P-C-x(11)-C-V-S). Transcription factor of the helix-turn-helix structural family, DNA binding, and activating gene expression. In human, nine member proteins from Pax-1 to Pax-9. Regulate differentiation of many different tissues. Some members of the family (PAX3, PAX4, PAX6, PAX7) also contain a functional homeobox domain.
• pedicle-
• sacrum-
• sclerotome- ventromedial half of each somite that forms the vertebral body and intervertebral disc.
• segmentation- to break a solid structure into a number of usually equal size pieces.
• somatic mesoderm- derived from lateral mesoderm closest to the ectoderm and separated from other component of lateral mesoderm (splanchnic, near endoderm) by the intraembryonic coelom.
• somite- segmental block (ball) of mesoderm formed from paraxial mesoderm adjacent to notochord (axial mesoderm). Differentiates to form initially sclerotome and dermamyotome (then dermotome and myotome).
• somitic mesoderm-
• somitocoel- a transient cavity that appears within each of the the early forming somites then is lost.
• somitogenesis- the process of segmentation of the paraxial mesoderm to form "mesoderm balls" beginning cranially (humans day20) and extending caudally at 1 somite/90 minutes until approx. 44 pairs have been formed.
• sonic hedgehog- (=shh) secreted growth factor that binds patched (ptc) receptor on cell membrane. SHH function is different for different tissues in the embryo. In the nervous system, it is secreted by the notochord, ventralizes the neural tube, inducing the floor plate and motor neurons. In the Limb it is secreted by the zone of polarizing activity (ZPA) organizing limb axis formation.
• tarsal-
• Tbx- T-box genes (transcription factor) involved in mouse forelimb (Tbx4) and hindlimb (Tbx5) specification.
• transcription factor- a factor (protein or protein with steroid) that binds to DNA to alter gene expression, usually to activate. (eg steroid hormone+receptor, Retinoic acid+Receptor, Hox, Pax, Lim, Nkx-2.2).
• transverse abdominal m.-
• trochanter-
• vertebral body- formed by centrum, vertebral arch, facets for ribs. It is the mature vertebral structure formed by the 5 secondary ossification centers after puberty.
• vertebral column- name given to the complete structure formed from the alternating segments of vertebra and intervertebral discs which support the spinal cord.
• vertebral foramen- the dorsal cavity within each vertebra, generated by the vertebral arch that surrounds the spinal cord.
• vertebral canal-
• Wnt7a- The designation 'Wnt' was derived from 'wingless' and 'int'. The Wnt gene was first defined as a protooncogene, int1. Humans have at least 4 Wnt genes: Wnt7a gene is at 3p25 encoding a 349aa secreted glycoprotein. A patterning switch with different roles in different tissues. The mechanism of Wnt distribution (free diffusion, restricted diffusion and active transport) and all its possible cell receptors are still being determined. At least one WNT receptor is Frizzled (FZD). The Frizzled gene family encodes a seven-transmembrane receptor.

Glossary of Terms

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Quick Links

Development Notes ----------------- Menstrual Cycle Week 1 Week 2 Week 3 Week 4 Placenta Serial Images Genes/DNA Molecular Dev Abnormal Dev Postnatal Dev Notes Index

System Notes ----------------- Heart/Cardiovascular Respiratory Coelomic cavity Gastrointestinal Tract Head and Neck Neural Neural Crest Eye Ear Nose Endocrine Urogenital Musculoskeletal Integumentary

Movies ----------------- Embryo Growth Ultrasound Week 1 Week 2 Week 3 Week 4 Stage 13/14 Stage 22 Neural Head and Neck Heart Vasculature Gastrointestinal Urogenital Other Embryos Larsen Internet

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