Talk:Musculoskeletal System - Bone Development

From Embryology
About Discussion Pages  
Mark Hill.jpg
On this website the Discussion Tab or "talk pages" for a topic has been used for several purposes:
  1. References - recent and historic that relates to the topic
  2. Additional topic information - currently prepared in draft format
  3. Links - to related webpages
  4. Topic page - an edit history as used on other Wiki sites
  5. Lecture/Practical - student feedback
  6. Student Projects - online project discussions.
Links: Pubmed Most Recent | Reference Tutorial | Journal Searches

Glossary Links

Glossary: A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | Numbers | Symbols | Term Link

Cite this page: Hill, M.A. (2021, November 30) Embryology Musculoskeletal System - Bone Development. Retrieved from

Bone Development - Intramembranous Ossification

Zone Acronym Cells Properties
Resting or
RZ small uniform compactly located chondrocytes rich in lipid and cytoplasmic vacuoles low rates of chondrocytes replication, proteoglycan and collagen type IIB synthesis.
Proliferative PZ chondrocyte are tightly bound in columns parallel to the axis of the length of the bone can proliferate and differentiate.
Hypertrophic HZ upper zone of maturation and the lower zone of degeneration layer where blood vessels invade, along with chondroclasts that degrade and remodel the cartilage extracellular matrix
Calcification CZ Osteoblast precursor cells that adhere to the remnants of the cartilage ECM form bone tissue in primary ossification centres to assemble the provisional calcification zone

Old Histology Slides: developing Vertebra | Vertebra medium

Endochondral ossification slides: Developing bone | Bone, Developing (LS, Femur) Cat H&E


Funato N. (2020). New Insights Into Cranial Synchondrosis Development: A Mini Review. Front Cell Dev Biol , 8, 706. PMID: 32850826 DOI.

Review - New Insights Into Cranial Synchondrosis Development

The synchondroses formed via endochondral ossification in the cranial base are an important growth center for the neurocranium. Abnormalities in the synchondroses affect cranial base elongation and the development of adjacent regions, including the craniofacial bones. In the central region of the cranial base, there are two synchondroses present-the intersphenoid synchondrosis and the spheno-occipital synchondrosis. These synchondroses consist of mirror image bipolar growth plates. The cross-talk of several signaling pathways, including the parathyroid hormone-like hormone (PTHLH)/parathyroid hormone-related protein (PTHrP), Indian hedgehog (Ihh), Wnt/β-catenin, and fibroblast growth factor (FGF) pathways, as well as regulation by cilium assembly and the transcription factors encoded by the RUNX2, SIX1, SIX2, SIX4, and TBX1 genes, play critical roles in synchondrosis development. Deletions or activation of these gene products in mice causes the abnormal ossification of cranial synchondrosis and skeletal elements. Gene disruption leads to both similar and markedly different abnormalities in the development of intersphenoid synchondrosis and spheno-occipital synchondrosis, as well as in the phenotypes of synchondroses and skeletal bones. This paper reviews the development of cranial synchondroses, along with its regulation by the signaling pathways and transcription factors, highlighting the differences between intersphenoid synchondrosis and spheno-occipital synchondrosis.

Kegelman CD, Nijsure MP, Moharrer Y, Pearson HB, Dawahare JH, Jordan KM, Qin L & Boerckel JD. (2020). YAP and TAZ promote periosteal osteoblast precursor expansion and differentiation for fracture repair. J. Bone Miner. Res. , , . PMID: 32835424 DOI.

YAP and TAZ promote periosteal osteoblast precursor expansion and differentiation for fracture repair

In response to bone fracture, periosteal progenitor cells proliferate, expand, and differentiate to form cartilage and bone in the fracture callus. These cellular functions require the coordinated activation of multiple transcriptional programs, and the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) regulate osteochondroprogenitor activation during endochondral bone development. However, recent observations raise important distinctions between the signaling mechanisms used to control bone morphogenesis and repair. Here, we tested the hypothesis that YAP and TAZ regulate osteochondroprogenitor activation during endochondral bone fracture healing in mice. Constitutive YAP and/or TAZ deletion from Osterix-expressing cells impaired both cartilage callus formation and subsequent mineralization. However, this could be explained either by direct defects in osteochondroprogenitor differentiation after fracture, or by developmental deficiencies in the progenitor cell pool prior to fracture. Consistent with the second possibility, we found that developmental YAP/TAZ deletion produced long bones with impaired periosteal thickness and cellularity. Therefore, to remove the contributions of developmental history, we next generated adult onset-inducible knockout mice (using Osx-CretetOff ) in which YAP and TAZ were deleted prior to fracture, but after normal development. Adult onset-induced YAP/TAZ deletion had no effect on cartilaginous callus formation, but impaired bone formation at 14 days post-fracture (dpf). Earlier, at 4 dpf, adult onset-induced YAP/TAZ deletion impaired the proliferation and expansion of osteoblast precursor cells located in the shoulder of the callus. Further, activated periosteal cells isolated from this region at 4 dpf exhibited impaired osteogenic differentiation in vitro upon YAP/TAZ deletion. Finally, confirming the effects on osteoblast function in vivo, adult onset-induced YAP/TAZ deletion impaired bone formation in the callus shoulder at 7 dpf, prior to the initiation of endochondral ossification. Together, these data show that YAP and TAZ promote the expansion and differentiation of periosteal osteoblast precursors to accelerate bone fracture healing.


Wang L, Huang J, Moore DC, Song Y, Ehrlich MG & Yang W. (2019). SHP2 regulates intramembranous ossification by modifying the TGFβ and BMP2 signaling pathway. Bone , 120, 327-335. PMID: 30471432 DOI.

SHP2 regulates intramembranous ossification by modifying the TGFβ and BMP2 signaling pathway

SHP2 is a ubiquitously expressed protein tyrosine phosphatase, which is involved in many signaling pathways to regulate the skeletal development. In endochondral ossification, SHP2 is known to modify the osteogenic fate of osteochondroprogenitors and to impair the osteoblastic transdifferentiation of hypertrophic chondrocytes. However, how SHP2 regulates osteoblast differentiation in intramembranous ossification remains incompletely understood. To address this question, we generated a mouse model to ablate SHP2 in the Prrx1-expressing mesenchymal progenitors by using "Cre-loxP"-mediated gene excision and examined the development of calvarial bone, in which the main process of bone formation is intramembranous ossification. Phenotypic characterization showed that SHP2 mutants have severe defects in calvarial bone formation. Cell lineage tracing and in situ hybridization data showed less osteoblast differentiation of mesenchymal cells and reduced osteogenic genes expression, respectively. Further mechanistic studies revealed enhanced TGFβ and suppressed BMP2 signaling in SHP2 ablated mesenchymal progenitors and their derivatives. Our study uncovered the critical role of SHP2 in osteoblast differentiation through intramembranous ossification and might provide a potential target to treat craniofacial skeleton disorders.


The emerging role of Hippo signaling pathway in regulating osteoclast formation

J Cell Physiol. 2018 Jun;233(6):4606-4617. doi: 10.1002/jcp.26372. Epub 2018 Jan 15.

Yang W1, Han W1, Qin A2, Wang Z3, Xu J3, Qian Y1.


A delicate balance between osteoblastic bone formation and osteoclastic bone resorption is crucial for bone homeostasis. This process is regulated by the Hippo signaling pathway including key regulatory molecules RASSF2, NF2, MST1/2, SAV1, LATS1/2, MOB1, YAP, and TAZ. It is well established that the Hippo signaling pathway plays an important part in regulating osteoblast differentiation, but its role in osteoclast formation and activation remains poorly understood. In this review, we discuss the emerging role of Hippo-signaling pathway in osteoclast formation and bone homeostasis. It is revealed that specific molecules of the Hippo-signaling pathway take part in a stage specific regulation in pre-osteoclast proliferation, osteoclast differentiation and osteoclast apoptosis and survival. Upon activation, MST and LAST, transcriptional co-activators YAP and TAZ bind to the members of the TEA domain (TEAD) family transcription factors, and influence osteoclast differentiation via regulating the expression of downstream target genes such as connective tissue growth factor (CTGF/CCN2) and cysteine-rich protein 61 (CYR61/CCN1). In addition, through interacting or cross talking with RANKL-mediated signaling cascades including NF-κB, MAPKs, AP1, and NFATc1, Hippo-signaling molecules such as YAP/TAZ/TEAD complex, RASSF2, MST2, and Ajuba could also potentially modulate osteoclast differentiation and function. Elucidating the roles of the Hippo-signaling pathway in osteoclast development and specific molecules involved is important for understanding the mechanism of bone homeostasis and diseases. KEYWORDS: Hippo-signaling pathway; bone metabolism; bone resorption; osteoclast formation J Cell Physiol. 2018 Jun;233(6):4606-4617. doi: 10.1002/jcp.26372. Epub 2018 Jan 15. Review. PMID: 29219182

Stresses and strains on the human fetal skeleton during development

J R Soc Interface. 2018 Jan;15(138). pii: 20170593. doi: 10.1098/rsif.2017.0593.

Verbruggen SW1, Kainz B2, Shelmerdine SC3, Hajnal JV4, Rutherford MA5, Arthurs OJ6, Phillips ATM7, Nowlan NC8.


Mechanical forces generated by fetal kicks and movements result in stimulation of the fetal skeleton in the form of stress and strain. This stimulation is known to be critical for prenatal musculoskeletal development; indeed, abnormal or absent movements have been implicated in multiple congenital disorders. However, the mechanical stress and strain experienced by the developing human skeleton in utero have never before been characterized. Here, we quantify the biomechanics of fetal movements during the second half of gestation by modelling fetal movements captured using novel cine-magnetic resonance imaging technology. By tracking these movements, quantifying fetal kick and muscle forces, and applying them to three-dimensional geometries of the fetal skeleton, we test the hypothesis that stress and strain change over ontogeny. We find that fetal kick force increases significantly from 20 to 30 weeks' gestation, before decreasing towards term. However, stress and strain in the fetal skeleton rises significantly over the latter half of gestation. This increasing trend with gestational age is important because changes in fetal movement patterns in late pregnancy have been linked to poor fetal outcomes and musculoskeletal malformations. This research represents the first quantification of kick force and mechanical stress and strain due to fetal movements in the human skeleton in utero, thus advancing our understanding of the biomechanical environment of the uterus. Further, by revealing a potential link between fetal biomechanics and skeletal malformations, our work will stimulate future research in tissue engineering and mechanobiology. KEYWORDS: biomechanical stimuli; cine-MRI; finite element analysis; joint biomechanics; musculo-skeletal development

PMID: 29367236 DOI: 10.1098/rsif.2017.0593


Cell-matrix signals specify bone endothelial cells during developmental osteogenesis

Nat Cell Biol. 2017 Mar;19(3):189-201. doi: 10.1038/ncb3476. Epub 2017 Feb 20.

Langen UH1, Pitulescu ME1, Kim JM1, Enriquez-Gasca R2, Sivaraj KK1, Kusumbe AP1, Singh A1,2, Di Russo J3, Bixel MG1, Zhou B4, Sorokin L3, Vaquerizas JM2, Adams RH1. Abstract

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell-matrix signalling interactions. Loss of endothelial integrin β1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system. PMID 28218908 DOI: 10.1038/ncb3476


Disruption of Scube2 impairs endochondral bone formation

J Bone Miner Res. 2015 Jan 12. doi: 10.1002/jbmr.2451. [Epub ahead of print]

Lin YC1, Roffler SR, Yan YT, Yang RB.


SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2) belongs to a secreted and membrane-tethered multi-domain SCUBE protein family composed of 3 members found in vertebrates and mammals. Recent reports suggested that zebrafish scube2 could facilitate sonic hedgehog (Shh) signaling for proper development of slow muscle. However, whether SCUBE2 can regulate the signaling activity of two other hedgehog ligands (Ihh and Dhh), and the developmental relevance of the SCUBE2-induced hedgehog signaling in mammals remain poorly understood. In this study, we first showed that as compared with SCUBE1 or 3, SCUBE2 is the most potent modulator of IHH signaling in vitro. In addition, gain and loss-of-function studies demonstrated that SCUBE2 exerted an osteogenic function by enhancing Ihh-stimulated osteoblast differentiation in the mouse mesenchymal progenitor cells. Consistent with these in vitro studies and the prominent roles of Ihh in coordinating skeletogenesis, genetic ablation of Scube2 (-/-) caused defective endochondral bone formation and impaired Ihh-mediated chondrocyte differentiation and proliferation as well as osteoblast differentiation of -/- bone-marrow mesenchymal stromal-cell cultures. Our data demonstrate that Scube2 plays a key regulatory role in Ihh-dependent endochondral bone formation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. KEYWORDS: Bone µCT; Genetic animal models; Hedgehogs; Molecular pathways - development; osteoblast

PMID 25639508


A computational model of clavicle bone formation: a mechano-biochemical hypothesis

Bone. 2014 Apr;61:132-7. doi: 10.1016/j.bone.2014.01.007. Epub 2014 Jan 18.

Garzon-Alvarado DA1, Gutiérrez ML2, Calixto LF3.


Clavicle development arises from mesenchymal cells condensed as a cord extending from the acromion towards the sternal primordium. First two primary ossification centers form, extending to develop the body of the clavicle through intramembranous ossification. However, at its ends this same bone also displays endochondral ossification. So how can the clavicle be formed by both types of ossification? Developmental events associated with clavicle formation have mainly used histological studies as supporting evidence. Nonetheless, mechanisms of biological events such as molecular and mechanical effects remain to be determined. The objective of this work was to provide a mathematical explanation of embryological events based on two serial phases: first formation of an ossified matrix by intramembranous ossification based on three factors: systemic, local biochemical, and mechanical factors. After this initial phase expansion of the ossified matrix follows with mesenchymal cell differentiation into chondrocytes for posterior endochondral ossification. Our model provides strong evidence for clavicle formation integrating molecules and mechanical stimuli through partial differentiation equations using finite element analysis.

Copyright © 2014 Elsevier Inc. All rights reserved.

KEYWORDS: Clavicle formation; Finite elements analysis; Reaction diffusion; Turing pattern formation PMID 24444803

Vascularization of primary and secondary ossification centres in the human growth plate

BMC Dev Biol. 2014 Aug 28;14(1):36. [Epub ahead of print]

Walzer SM, Cetin E, Grübl-Barabas R, Sulzbacher I, Rueger B, Girsch W, Toegel S, Windhager R, Fischer MB.


BackgroundThe switch from cartilage template to bone during endochondral ossification of the growth plate requires a dynamic and close interaction between cartilage and the developing vasculature. Vascular invasion of the primarily avascular hypertrophic chondrocyte zone brings chondroclasts, osteoblast- and endothelial precursor cells into future centres of ossification.Vascularization of human growth plates of polydactylic digits was studied by immunohistochemistry, confocal-laser-scanning-microscopy and RT-qPCR using markers specific for endothelial cells CD34 and CD31, smooth muscle cells ¿-SMA, endothelial progenitor cells CD133, CXCR4, VEGFR-2 and mesenchymal progenitor cells CD90 and CD105. In addition, morphometric analysis was performed to quantify RUNX2+ and DLX5+ hypertrophic chondrocytes, RANK+ chondro- and osteoclasts, and CD133+ progenitors in different zones of the growth plate.ResultsNew vessels in ossification centres were formed by sprouting of CD34+ endothelial cells that did not co-express the mature endothelial cell marker CD31. These immature vessels in the growth plate showed no abluminal coverage with ¿-SMA+ smooth muscle cells, but in their close proximity single CD133+ precursor cells were found that did not express VEGFR-2, a marker for endothelial lineage commitment. In periosteum and in the perichondrial groove of Ranvier that harboured CD90+/CD105+ chondro-progenitors, in contrast, mature vessels were found stabilized by ¿-SMA+ smooth muscle cells.ConclusionVascularization of ossification centres of the growth plate was mediated by sprouting of capillaries coming from the bone collar or by intussusception rather than by de-novo vessel formation involving endothelial progenitor cells. Vascular invasion of the joint anlage was temporally delayed compared to the surrounding joint tissue.

PMID 25164565

Histogenesis of Japanese quail bone and cartilage tissues at the final stages of embryonic development in microgravity

Aviakosm Ekolog Med. 2013 Nov-Dec;47(6):24-8.

[Article in Russian] Komissarova DV, Dadasheva OA, Gurieva TS, Sychev VN. Abstract The article reports the results of comparative histological studies of skeleton development in 14- and 16-day Japanese quail embryos grown in space flight and in the 1-g gravity. The investigation revealed retardation of cartilage replacement by bone in the femur and tibia in space embryos as compared with their controls. Perichondral ossification metaphysis was reached by day 14 both in the space and control embryos. Destruction of cartilaginous diaphysis advanced onto the proximal and distal parts including the periphery. Tibia and femur cartilaginous cores in space embryos were destructed worse than in the controls in consequence of insufficient minerals supply. Perichondral ossification in the 16-day space and control embryos was alike close to completion spreading as far as the epiphysis. Long bones metaphysis was abundant in monomorphic cells as a growth bank existing during and 7 days post hatching. However, absence, in contrast to the controls, of osteogensis sites in long bones epiphysis, suggested retardation of chondrocytes calcification in these areas, as well as of ossification in space embryos. PMID 24660239



A unified anatomy ontology of the vertebrate skeletal system

PLoS One. 2012;7(12):e51070. doi: 10.1371/journal.pone.0051070. Epub 2012 Dec 10.

Dahdul WM, Balhoff JP, Blackburn DC, Diehl AD, Haendel MA, Hall BK, Lapp H, Lundberg JG, Mungall CJ, Ringwald M, Segerdell E, Van Slyke CE, Vickaryous MK, Westerfield M, Mabee PM. Source Department of Biology, University of South Dakota, Vermillion, South Dakota, United States of America ; National Evolutionary Synthesis Center, Durham, North Carolina, United States of America.


The skeleton is of fundamental importance in research in comparative vertebrate morphology, paleontology, biomechanics, developmental biology, and systematics. Motivated by research questions that require computational access to and comparative reasoning across the diverse skeletal phenotypes of vertebrates, we developed a module of anatomical concepts for the skeletal system, the Vertebrate Skeletal Anatomy Ontology (VSAO), to accommodate and unify the existing skeletal terminologies for the species-specific (mouse, the frog Xenopus, zebrafish) and multispecies (teleost, amphibian) vertebrate anatomy ontologies. Previous differences between these terminologies prevented even simple queries across databases pertaining to vertebrate morphology. This module of upper-level and specific skeletal terms currently includes 223 defined terms and 179 synonyms that integrate skeletal cells, tissues, biological processes, organs (skeletal elements such as bones and cartilages), and subdivisions of the skeletal system. The VSAO is designed to integrate with other ontologies, including the Common Anatomy Reference Ontology (CARO), Gene Ontology (GO), Uberon, and Cell Ontology (CL), and it is freely available to the community to be updated with additional terms required for research. Its structure accommodates anatomical variation among vertebrate species in development, structure, and composition. Annotation of diverse vertebrate phenotypes with this ontology will enable novel inquiries across the full spectrum of phenotypic diversity.

PMID 23251424

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification

Bone. 2012 Dec 26. pii: S8756-3282(12)01434-2. doi: 10.1016/j.bone.2012.12.016. [Epub ahead of print]

Lu C, Wan Y, Cao J, Zhu X, Yu J, Zhou R, Yao Y, Zhang L, Zhao H, Li H, Zhao J, He L, Ma G, Yang X, Yao Z, Guo X. Source Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, 200240, China.


The role of Wnt signaling is extensively studied in skeletal development and postnatal bone remodeling, mostly based on the genetic approaches of β-catenin manipulation. However, given their independent function, a requirement for β-catenin is not the same as that for Wnt. Here, we investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Taken together, our studies reveal that Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate. Copyright © 2012. Published by Elsevier Inc.

PMID 23274346


Bone vs. fat: embryonic origin of progenitors determines response to androgen in adipocytes and osteoblasts

Bone. 2011 Oct;49(4):662-72. doi: 10.1016/j.bone.2011.06.010. Epub 2011 Jun 17.

Wiren KM1, Hashimoto JG, Semirale AA, Zhang XW.


Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male and not in female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal and in not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSC profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest. Published by Elsevier Inc. PMID 21704206

Role of GSK-3β in the Osteogenic Differentiation of Palatal Mesenchyme

PLoS One. 2011;6(10):e25847. Epub 2011 Oct 14.

Nelson ER, Levi B, Sorkin M, James AW, Liu KJ, Quarto N, Longaker MT. Source Hagey Laboratory for Pediatric Regenerative Medicine, Plastic and Reconstructive Surgery Division, Department of Surgery, Stanford University School of Medicine, Stanford, California, United States of America.


INTRODUCTION: The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification.

METHODS: Palates were harvested from GSK-3β, embryonic days 15.0-18.5 (e15.0-e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and -/- e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists.

RESULTS: GSK-3β null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β -/- palate cultures were "rescued" with the Wnt inhibitor, Dkk-1.

CONCLUSIONS: Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation.

PMID 22022457 [PubMed - in process] PMCID: PMC3194817


Developing bones are differentially affected by compromised skeletal muscle formation

Bone. 2010 May;46(5):1275-85. Epub 2009 Nov 27.

Nowlan NC, Bourdon C, Dumas G, Tajbakhsh S, Prendergast PJ, Murphy P.

Department of Zoology, School of Natural Sciences, Trinity College Dublin, Ireland. Abstract Mechanical forces are essential for normal adult bone function and repair, but the impact of prenatal muscle contractions on bone development remains to be explored in depth in mammalian model systems. In this study, we analyze skeletogenesis in two 'muscleless' mouse mutant models in which the formation of skeletal muscle development is disrupted; Myf5(nlacZ/nlacZ):MyoD(-/-) and Pax3(Sp/Sp) (Splotch). Ossification centers were found to be differentially affected in the muscleless limbs, with significant decreases in bone formation in the scapula, humerus, ulna and femur, but not in the tibia. In the scapula and humerus, the morphologies of ossification centers were abnormal in muscleless limbs. Histology of the humerus revealed a decreased extent of the hypertrophic zone in mutant limbs but no change in the shape of this region. The elbow joint was also found to be clearly affected with a dramatic reduction in the joint line, while no abnormalities were evident in the knee. The humeral deltoid tuberosity was significantly reduced in size in the Myf5(nlacZ/nlacZ):MyoD(-/-) mutants while a change in shape but not in size was found in the humeral tuberosities of the Pax3(Sp/Sp) mutants. We also examined skeletal development in a 'reduced muscle' model, the Myf5(nlacZ/+):MyoD(-/-) mutant, in which skeletal muscle forms but with reduced muscle mass. The reduced muscle phenotype appeared to have an intermediate effect on skeletal development, with reduced bone formation in the scapula and humerus compared to controls, but not in other rudiments. In summary, we have demonstrated that skeletal development is differentially affected by the lack of skeletal muscle, with certain rudiments and joints being more severely affected than others. These findings indicate that the response of skeletal progenitor cells to biophysical stimuli may depend upon their location in the embryonic limb, implying a complex interaction between mechanical forces and location-specific regulatory factors affecting bone and joint development.

Copyright (c) 2009 Elsevier Inc. All rights reserved.

PMID: 19948261


J Osteoporos. 2010 Sep 29;2010:941419.

Silverman SL.

Cedars-Sinai, UCLA, OMC Clinical Research Center, 8641 Wilshire Boulevard, Suite 301, Beverly Hills, CA 90211, USA. Abstract The striking clinical benefits of intermittent parathyroid hormone in osteoporosis have begun a new era of skeletal anabolic agents. One potential new agent is monoclonal antibody to sclerostin, a potent inhibitor of osteoblastogenesis.

The Wnt signaling pathway demonstrates a complex network of proteins well known for their roles in embryogenesis but also involving normal physiologic processes of bone formation in response to loading and unloading [1]. The Wnt pathway involves a large network of proteins that can regulate the production of Wnt signaling molecules [2]. Several proteins that inhibit Wnt signaling [2] have been described. One such protein is sclerostin which binds low-density lipoprotein receptor-related protein (LRP) and inhibits Wnt signaling.

PMID: 20981340

FGFs in endochondral skeletal development

J Cell Biochem. 2010 Aug 1;110(5):1046-57.

Degnin CR, Laederich MB, Horton WA. Shriners Hospital and Molecular & Medical Genetics and Cell & Developmental Biology, Oregon Health & Sciences University, Portland, Oregon 97239, USA.

Abstract The mammalian skeleton developments and grows through two complementary pathways: membranous ossification, which gives rise to the calvarial bones and distal clavicle, and endochondral ossification, which is responsible for the bones of the limbs, girdles, vertebrae, face and base of the skull and the medial clavicle. Fibroblast growth factors (FGFs) and their cognate FGF receptors (FGFRs) play important roles in regulating both pathways. However, the details of how FGF signals are initiated, propagated and modulated within the developing skeleton are only slowly emerging. This prospect will focus on the current understanding of these events during endochondral skeletal development with special attention given to concepts that have emerged in the past few years. Published 2010 Wiley-Liss, Inc.

PMID: 20564212

Control of Dkk-1 ameliorates chondrocyte apoptosis, cartilage destruction, and subchondral bone deterioration in osteoarthritic knees

Arthritis Rheum. 2010 May;62(5):1393-402.

Weng LH, Wang CJ, Ko JY, Sun YC, Wang FS.

Chang Gung Memorial Hospital-Kaohsiung Medical Center and Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Kaohsiung, Taiwan. Abstract OBJECTIVE: Perturbation of Wnt signaling components reportedly regulates chondrocyte fate and joint disorders. The Wnt inhibitor Dkk-1 mediates remodeling of various tissue types. We undertook this study to examine whether control of Dkk-1 expression prevents joint deterioration in osteoarthritic (OA) knees.

METHODS: Anterior cruciate ligament transection-and collagenase-induced OA in rat knees was treated with end-capped phosphorothioate Dkk-1 antisense oligonucleotide (Dkk-1-AS). Articular cartilage destruction, cartilage degradation markers, bone mineral density (BMD), and subchondral trabecular bone volume of injured knee joints were measured using Mankin scoring, enzyme-linked immunosorbent assay, dual x-ray absorptiometry, and histomorphometry. Dkk-1-responsive molecule expression and apoptotic cells in knee tissue were detected by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and TUNEL staining.

RESULTS: Up-regulated Dkk-1 expression was associated with increased Mankin score and with increased serum levels of cartilage oligomeric matrix protein and C-telopeptide of type II collagen (CTX-II) during OA development. Dkk-1-AS treatment alleviated OA-associated increases in Dkk-1 expression, Mankin score, cartilage fibrillation, and serum cartilage degradation markers. Dkk-1-AS also alleviated epiphyseal BMD loss and subchondral bone exposure associated with altered serum levels of osteocalcin and CTX-I. The treatment abrogated chondrocyte/osteoblast apoptosis and subchondral trabecular bone remodeling in OA. Dkk-1 knockdown increased levels of nuclear beta-catenin and phosphorylated Ser(473)-Akt but attenuated expression of inflammatory factors (Toll-like receptor 4 [TLR-4], TLR-9, interleukin-1beta, and tumor necrosis factor alpha), the apoptosis regulator Bax, matrix metalloproteinase 3, and RANKL in OA knee joints.

CONCLUSION: Interference with the cartilage- and bone-deleterious actions of Dkk-1 provides therapeutic potential for alleviating cartilage destruction and subchondral bone damage in OA knee joints.

PMID: 20131282


Osteogenesis imperfecta: questions and answers

Curr Opin Pediatr. 2009 Dec;21(6):709-16.

Shapiro JR, Sponsellor PD.

Department Physical Medicine and Rehabilitation, Johns Hopkins University, Kennedy Krieger Institute, Baltimore, Maryland 21205, USA. Abstract PURPOSE OF REVIEW: Considerable attention has recently been focused on the pathogenesis, diagnosis and treatment of osteogenesis imperfecta. Two new genes have been defined in patients with recessive severe or lethal osteogenesis imperfecta types. Diagnostic concerns involve testing procedures, either skin biopsies or DNA analysis. Bisphosphonates have been accepted as 'standard of care' for children with osteogenesis imperfecta. However, questions remain as to the selection of patients for treatment, effectiveness in fracture prevention, which bisphosphonates should be used and the duration of treatment. Orthopedic intervention occurs on several levels: including the immediate treatment of fractures, the treatment of scoliosis and the use of intramedullary rods.

RECENT FINDINGS: The discovery of mutations involving CRTAP and LEPRE1 genes in severe/lethal and recessively inherited osteogenesis imperfecta has provided partial answers to questions about 'other' osteogenesis imperfecta genes in patients with an osteogenesis imperfecta phenotype but no COL1A1 and COL1A2 mutations. Current experience suggests that DNA analysis is a better test for diagnosis as compared with dermal biopsy. There are no standardized guidelines for initiating bisphosphonate treatment in children. Recent data suggest either intravenous or oral bisphosphonates are effective, but differences exist between different bisphosphonates. Two recent reports document the paucity of evidence-based data regarding the effectiveness of bisphosphonate treatment in fracture prevention.

SUMMARY: This report will update the medical and orthopedic approaches to care for children with osteogenesis imperfecta.

PMID: 19907330


Attenuation of WNT signaling by DKK-1 and -2 regulates BMP2-induced osteoblast differentiation and expression of OPG, RANKL and M-CSF

Mol Cancer. 2007 Oct 30;6:71.

Fujita K, Janz S.

Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. Abstract BACKGROUND: Enhanced osteoblast-dependent osteoclastogenesis due to inhibition of Wnt/beta-catenin signaling in bone morphogenic protein (BMP)-driven osteoprogenitors has been repeatedly implicated in the natural history of cancer-associated osteolytic lesions, but the mechanism of this bone loss is poorly understood.

METHODS: We examined the impact of secreted Wnt inhibitors from the Dickkopf (Dkk) family on pluripotent mesenchymal cells undergoing BMP2-induced osteoblastic differentiation.

RESULTS: We found that Dkk1 and -2 restored the Wnt3a-dependent reduction of alkaline phosphatase (ALP), Osterix and p53, indicating that mitigated Wnt/beta-catenin signaling promotes certain aspects of early osteoblastogenesis through the BMP-p53-Osterix-ALP axis. Dkk1 and -2 increased the expression of the osteoclast differentiation factors, receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), upon stimulation with Wnt3a/1,25-dihydroxyvitamine D3 and Wnt3a/BMP2, respectively. The decoy receptor of RANKL, osteoprotegerin (OPG), was down regulated under the latter conditions. These findings indicated that Dkk1 and -2 facilitate osteoclastogenesis by enhancing RANKL/RANK and M-CSF/c-Fms interactions. Dkk4 weakly shared activities of Dkk-1 and -2, whereas Dkk3 was ineffective.

CONCLUSION: Our results suggest that inhibited Wnt/beta-catenin signaling in BMP2-induced osteoprogenitors in vivo promotes, on balance, the heightened formation of osteoclasts. Focally increased Dkk1 production by tumor cells in the bone may thus lead to focal bone loss.

PMID: 17971207

Adult Bones Table

Vertebral column 26
Skull 22
Hyoid bone 1
Ribs and sternum 25

Upper extremities 64
Lower extremities 62

Auditory ossicles




Morphologic and radiological observations on the earliest bone marrow formation in human embryos and fetuses

Acta Pathol Jpn. 1983 May;33(3):439-46.

Enzan H, Hara H, Izumi T, Ohkita T.


Morphologic and radiologic studies were undertaken on 26 human embryos and fetuses to determine the stage and site of the earliest bone marrow formation. Up to the 10th week of gestation, primary bone marrow is not present anywhere although the intramembranous ossification occurs in the maxilla and mandible and also in the middle portion of the clavicle. At the 11th week of gestation, X-ray examination showed in two fetuses the bone formation in the clavicle, scapula, maxilla, mandible, and the diaphysis of the long bones. Serial sections of these fetuses revealed that the primary bone marrow occurs first in the middle portion of the clavicle. From a series of our embryological studies, the concept of the mononuclear phagocyte system which involves the bone-marrow-derived monocytic origin of tissue macrophages, is not accepted, at least, on the origin of Kupffer cells in human fetal livers.

PMID 6624441