Embryonic Skeletal Development Research Articles

A large duplication involving the IHH locus mimics acrocallosal syndrome

Indian hedgehog (Ihh) signaling is a major determinant of various processes during embryonic development and has a pivotal role in embryonic skeletal development. A specific spatial and temporal expression of Ihh within the developing limb buds is essential for accurate digit outgrowth and correct digit number. Although missense mutations in IHH cause brachydactyly type A1, small tandem duplications involving the IHH locus have recently been described in patients with mild syndactyly and craniosynostosis. In contrast, a ∼600-kb deletion 5' of IHH in the doublefoot mouse mutant (Dbf) leads to severe polydactyly without craniosynostosis, but with craniofacial dysmorphism. We now present a patient resembling acrocallosal syndrome (ACS) with extensive polysyndactyly of the hands and feet, craniofacial abnormalities including macrocephaly, agenesis of the corpus callosum, dysplastic and low-set ears, severe hypertelorism and profound psychomotor delay. Single-nucleotide polymorphism (SNP) array copy number analysis identified a ∼900-kb duplication of the IHH locus, which was confirmed by an independent quantitative method. A fetus from a second pregnancy of the mother by a different spouse showed similar craniofacial and limb malformations and the same duplication of the IHH-locus. We defined the exact breakpoints and showed that the duplications are identical tandem duplications in both sibs. No copy number changes were observed in the healthy mother. To our knowledge, this is the first report of a human phenotype similar to the Dbf mutant and strikingly overlapping with ACS that is caused by a copy number variation involving the IHH locus on chromosome 2q35.

Fibrodysplasia ossificans progressiva: a human genetic disorder of extraskeletal bone formation, or—how does one tissue become another?

Fibrodysplasia ossificans progressiva (FOP) is a rare human genetic disease in which de novo osteogenesis—a developmental process occurring during embryonic skeletal formation—is induced aberrantly and progressively beginning during early childhood in soft connective tissues. Episodic initiation of spontaneous bone-forming lesions occurs over time, affecting a generally predictable sequence of body locations following a pattern similar to that of the developing embryonic skeleton. The heterotopic (extraskeletal) bone formation in FOP can also be induced by connective tissue injury. At the tissue level, an initial tissue degradation phase is followed by a tissue formation phase during which soft connective tissues are replaced by bone tissue through endochondral osteogenesis. This extraskeletal bone is physiologically normal and develops through the same series of tissue differentiation events that occur during normal embryonic skeletal development. The underlying genetic mutation in FOP alters the signals that regulate induction of cell differentiation leading to bone formation. In addition to postnatal heterotopic ossification, FOP patients show specific malformations of skeletal elements indicating effects on bone formation during embryonic development as well. Nearly all cases of FOP are caused by the identical mutation in the ACVR1 gene that causes a single amino acid substitution, R206H, in the bone morphogenetic protein (BMP) type I receptor ACVR1 (formerly known as ALK2). This mutation causes mild constitutive activation of the BMP signaling pathway and identifies ACVR1 as a key regulator of cell fate decisions and bone formation, providing opportunities to investigate previously unrecognized functions for this receptor during tissue development and homeostasis.

Lrp5 and Lrp6 redundantly control skeletal development in the mouse embryo

The role of Wnt signaling in osteoblastogenesis in the embryo remains to be fully established. Although β-catenin, a multifunctional protein also mediating canonical Wnt signaling, is indispensable for embryonic osteoblast differentiation, the roles of the key Wnt co-receptors Lrp5 and Lrp6 are unclear. Indeed, global deletion of either Lrp5 or Lrp6 did not overtly affect osteoblast differentiation in the mouse embryo. Here, we generated mice lacking both receptors specifically in the embryonic mesenchyme and observed an absence of osteoblasts in the embryo. In addition, the double-deficient embryos developed supernumerary cartilage elements in the zeugopod, revealing an important role for mesenchymal Lrp5/6 signaling in limb patterning. Importantly, the phenotypes of the Lrp5/6 mutant closely resembled those of the β-catenin-deficient embryos. These phenotypes are likely independent of any effect on the adherens junction, as deletion of α-catenin, another component of the complex, did not cause similar defects. Thus, Lrp5 and 6 redundantly control embryonic skeletal development, likely through β-catenin signaling.

Loss of Gremlin Delays Primordial Follicle Assembly but Does Not Affect Female Fertility in Mice1

The transforming growth factor beta (TGFB) protein family is renowned for its diverse roles in developmental biology including reproduction. Gremlin is a member of the differential screening-selected gene aberrative in neuroblastoma (DAN)/cerberus family of bone morphogenetic protein (BMP) antagonists. Recent studies on gremlin focus on its involvement in embryonic skeletal, lung, and kidney development. To define the role of gremlin (Grem1) in female reproduction, we analyzed postnatal folliculogenesis using global and conditional knockout (cKO) mice for gremlin. Grem1(-/-) mice die within 48 h after birth, and ovaries collected from neonatal Grem1(-/-) mice demonstrated reduced oocyte numbers and delayed primordial follicle development. Transplanting Grem1(-/-) neonatal ovaries showed that folliculogenesis proceeded to large antral follicle stage, but Grem1(-/-) ovaries contained corpora lutea-like structures not found in control-transplanted ovaries. However, Grem1 cKO mice had comparable fertility to control mice. These data suggest that gremlin plays a previously uncharacterized role in the regulation of oocyte numbers and the timing of primordial follicle development, but either it is not required for later folliculogenesis or its loss is possibly compensated by other BMP antagonists.

Generation and characterization of Col10a1‐mcherry reporter mice

We report here on the generation of a new fluorescent protein reporter transgenic mouse line, Col10a1-mCherry, which can be used as a tool to study chondrocyte biology and pathology. Collagen, Type X, alpha 1 (Col10a1) is highly expressed in hypertrophic chondrocytes and commonly used as a gene marker for this cell population. The Col10a1-mCherry reporter line was generated using a bacterial recombination strategy with the mouse BAC clone RP23-192A7. To aid in the characterization of this animal model, we intercrossed Col10a1-mCherry mice with Collagen, Type II, alpha 1 (Col2a1) enhanced cyan fluorescent protein (ECFP) reporter mice and characterized the expression of both chondrocyte reporters during embryonic skeletal development from days E10.5 to E17.5. Additionally, at postnatal day 0, Col10a1-mCherry reporter expression was compared to endogenous Col10a1 mRNA expression in long bones and revealed that mCherry fluorescence extended past the Col10a1 expression domain. However, in situ hybridization for mCherry was consistent with the zone of Col10a1 mRNA expression, indicating that the persistent detection of mCherry fluorescence was a result of the long protein half life of mCherry in conjunction with a very rapid phase of skeletal growth and not due to aberrant transcriptional regulation. Taking advantage of the continued fluorescence of hypertrophic chondrocytes at the chondro-osseus junction, we intercrossed Col10a1-mCherry mice with two different Collagen, Type 1, alpha 1, (Col1a1) osteoblast reporter mice, pOBCol3.6-Topaz and pOBCol2.3-Emerald to investigate the possibility that hypertrophic chondrocytes transdifferentiate into osteoblasts. Evaluation of long bones at birth suggests that residual hypertrophic chondrocytes and osteoblasts in the trabecular zone exist as two completely distinct cell populations. genesis 49:410-418, 2011.

Mechanobiology of embryonic skeletal development: Insights from animal models.

A range of clinical conditions in which fetal movement is reduced or prevented can have a severe effect on skeletal development. Animal models have been instrumental to our understanding of the interplay between mechanical forces and skeletal development, particularly the mouse and the chick model systems. In the chick, the most commonly used means of altering the mechanical environment is by pharmaceutical agents which induce paralysis, whereas genetically modified mice with nonfunctional or absent skeletal muscle offer a valuable tool for examining the interplay between muscle forces and skeletogenesis in mammals. This article reviews the body of research on animal models of bone or joint formation in vivo in the presence of an altered or abnormal mechanical environment. In both immobilized chicks and "muscleless limb" mice, a range of effects are seen, such as shorter rudiments with less bone formation, changes in rudiment and joint shape, and abnormal joint cavitation. However, although all bones and synovial joints are affected in immobilized chicks, some rudiments and joints are unaffected in muscleless mice. We propose that extrinsic mechanical forces from movements of the mother or littermates impact on skeletogenesis in mammals, whereas the chick embryo is reliant on intrinsic movement for mechanical stimulation. The insights gained from animal models into the mechanobiology of embryonic skeletal development could provide valuable cues to prospective tissue engineers of cartilage and bone and contribute to new or improved treatments to minimize the impact on skeletal development of reduced movement in utero.

Functional analysis of Klf2 during embryonic skeletal development

Functional analysis of Klf2 during embryonic skeletal development

Fgf-9 is required for angiogenesis and osteogenesis in long bone repair.

Bone healing requires a complex interaction of growth factors that establishes an environment for efficient bone regeneration. Among these, FGFs have been considered important for intrinsic bone-healing capacity. In this study, we analyzed the role of Fgf-9 in long bone repair. One-millimeter unicortical defects were created in tibias of Fgf-9(+/-) and wild-type mice. Histomorphometry revealed that half-dose gene of Fgf-9 markedly reduced bone regeneration as compared with wild-type. Both immunohistochemistry and RT-PCR analysis revealed markedly decreased levels of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (Runx2), osteocalcin, Vega-a, and platelet endothelial cell adhesion molecule 1 (PECAM-1) in Fgf-9(+/-) defects. muCT angiography indicated dramatic impairment of neovascularization in Fgf-9(+/-) mice as compared with controls. Treatment with FGF-9 protein promoted angiogenesis and successfully rescued the healing capacity of Fgf-9(+/-) mice. Importantly, although other pro-osteogenic factors [Fgf-2, Fgf-18, and bone morphogenic protein 2 (Bmp-2)] still were present in Fgf-9(+/-) mice, they could not compensate for the haploinsufficiency of the Fgf-9 gene. Therefore, endogenous Fgf-9 seems to play an important role in long bone repair. Taken together our data suggest a unique role for Fgf-9 in bone healing, presumably by initiating angiogenesis through Vegf-a. Moreover, this study further supports the embryonic phenotype previously observed in the developing limb, thus promoting the concept that healing processes in adult organisms may recapitulate embryonic skeletal development.

The Orphan Nuclear Receptor Estrogen Receptor-related Receptor γ Negatively Regulates BMP2-induced Osteoblast Differentiation and Bone Formation

Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.

Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment.

Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

Classic and atypical fibrodysplasia ossificans progressiva (FOP) phenotypes are caused by mutations in the bone morphogenetic protein (BMP) type I receptor ACVR1

Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant human disorder of bone formation that causes developmental skeletal defects and extensive debilitating bone formation within soft connective tissues (heterotopic ossification) during childhood. All patients with classic clinical features of FOP (great toe malformations and progressive heterotopic ossification) have previously been found to carry the same heterozygous mutation (c.617G>A; p.R206H) in the glycine and serine residue (GS) activation domain of activin A type I receptor/activin-like kinase 2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor. Among patients with FOP-like heterotopic ossification and/or toe malformations, we identified patients with clinical features unusual for FOP. These atypical FOP patients form two classes: FOP-plus (classic defining features of FOP plus one or more atypical features) and FOP variants (major variations in one or both of the two classic defining features of FOP). All patients examined have heterozygous ACVR1 missense mutations in conserved amino acids. While the recurrent c.617G>A; p.R206H mutation was found in all cases of classic FOP and most cases of FOP-plus, novel ACVR1 mutations occur in the FOP variants and two cases of FOP-plus. Protein structure homology modeling predicts that each of the amino acid substitutions activates the ACVR1 protein to enhance receptor signaling. We observed genotype-phenotype correlation between some ACVR1 mutations and the age of onset of heterotopic ossification or on embryonic skeletal development.

PAX3/7 expression coincides with myod during chronic skeletal muscle overload

Paired box (Pax) proteins 3 and 7 are key determinants for embryonic skeletal muscle development by initiating myogenic regulatory factor (MRF) gene expression. We show that Pax3 and 7 participate in adult skeletal muscle plasticity during the initial responses to chronic overload (< or =7 days) and appear to coordinate MyoD expression, a member of the MRF family of genes. Pax3 and 7 mRNA were higher than control within 12 h after initiation of overload, preceded the increase in MyoD mRNA on day 1, and peaked on day 2. On days 3 and 7, Pax7 mRNA remained higher than control, suggesting that satellite cell self-renewal was occurring. Pax3 and 7 and MyoD protein levels were higher than control on days 2 and 3. These data indicate that Pax3 and 7 coordinate the recapitulation of developmental-like regulatory mechanisms in response to growth-inducing stimuli in adult skeletal muscle, presumably through activation of satellite cells.

Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers (e.g., type X collagen) by MSCs undergoing chondrogenesis raises concern for a tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biologic basis of MSC hypertrophy, we examined the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy-associated markers. Human MSC pellet cultures were predifferentiated for 2 weeks in a chondrogenic medium, and hypertrophy was induced by withdrawing transforming growth factor beta (TGFbeta), reducing the concentration of dexamethasone, and adding thyroid hormone (T3). Cultures were characterized by histologic, immunohistochemical, and biochemical methods, and gene expression was assessed using quantitative reverse transcription-polymerase chain reaction. The combination of TGFbeta withdrawal, a reduction in the level of dexamethasone, and the addition of T3 was essential for hypertrophy induction. Cytomorphologic changes were accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various markers of hypertrophy, including type X collagen, fibroblast growth factor receptors 1-3, parathyroid hormone-related protein receptor, retinoic acid receptor gamma, matrix metalloproteinase 13, Indian hedgehog, osteocalcin, and the proapoptotic gene p53. However, hypertrophy was not induced uniformly throughout the pellet culture, and distinct regions of dedifferentiation were observed. Chondrogenically differentiating MSCs behave in a manner functionally similar to that of growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC-derived chondrocytes underwent a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophy-inhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering.

Novel point mutations in GDF5 associated with two distinct limb malformations in Chinese: brachydactyly type C and proximal symphalangism

Growth/differentiation factor 5 (GDF5) is a secreted growth factor that plays a key regulatory role in embryonic skeletal and joint development. Mutations in the GDF5 gene can cause different types of skeletal dysplasia, including brachydactyly type C (BDC) and proximal symphalangism (SYM1). We report two novel mutations in the GDF5 gene in Chinese families with distinct limb malformations. In one family affected with BDC, we identified a novel nonsense mutation, c.1461T > G (p.Y487X), which is predicted to truncate the GDF5 precursor protein by deleting 15 amino acids at its C-terminus. In one family with SYM1, we found a novel missense mutation, c.1118T > G (p.L373R), which changes a highly conserved amino acid in the prodomain of GDF5. We transfected COS-7 cells with retroviral constructs to express human wild-type or mutant GDF5 cDNAs. The mature GDF5 protein was detected, as in the wild-type, in supernatant derived from the p.L373R mutant GDF5 transfected cells, but not in the supernatant from the p.Y487X mutant transfected cells, indicating that the two mutations led to different fates of the mutant GDF5 proteins, thereby producing distinct limb phenotypes.

Redundant function of the heparan sulfate 6‐O‐endosulfatases Sulf1 and Sulf2 during skeletal development

Modification of the sulfation pattern of heparan sulfate (HS) during organ development is thought to regulate binding and signal transduction of several growth factors. The secreted sulfatases, Sulf1 and Sulf2, desulfate HS on 6-O-positions extracellularly. We show that both sulfatases are expressed in overlapping patterns during embryonic skeletal development. Analysis of compound mutants of Sulf1 and Sulf2 derived from gene trap insertions and targeted null alleles revealed subtle but distinct skeletal malformations including reduced bone length, premature vertebrae ossification and fusions of sternebrae and tail vertebrae. Molecular analysis of endochondral ossification points to a function of Sulf1 and Sulf2 in delaying the differentiation of endochondral bones. Penetrance and severity of the phenotype increased with reduced numbers of functional alleles indicating redundant functions of both sulfatases. The mild skeletal phenotype of double mutants suggests a role for extracellular modification of 6-O-sulfation in fine-tuning rather than regulating the development of skeletal structures.

Forkhead Proteins Are Critical for Bone Morphogenetic Protein-2 Regulation and Anti-tumor Activity of Resveratrol

Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer.

Mechanobiology of Embryonic Limb Development

Considerable evidence exists to support the hypothesis that mechanical forces have an essential role in healthy embryonic skeletal development. Clinical observations and experimental data indicate the importance of muscle contractions for limb development. However, the influence of these forces is seldom referred to in biological descriptions of bone development, and perhaps this is due to the fact that the hypothesis that mechanical forces are essential for normal embryonic skeletal development is difficult to test and elaborate experimentally in vivo, particularly in humans. Computational modeling has the potential to address this issue by simulating embryonic growth under a range of loading conditions but the potential of such models has yet to be fully exploited. In this article, we review the literature on mechanobiology of limb development in three main sections: (a) experimental alteration of the mechanical environment, (b) mechanical properties of embryonic tissues, and (c) the use of computational models. Then we analyze the main issues, and suggest how experimental and computational fields could work closer together to enhance our understanding of mechanobiology of the embryonic skeleton.

High-dose estrogen-induced osteogenesis is decreased in aged RUNX2+/− mice

Runx2 is a transcription factor that is not only critical in embryonic skeletal development but also important in regulating osteoblast function in the adult. Heterozygosity of RUNX2 ( RUNX2 +/−) leads to haploinsufficiency and manifests as a condition with distinctive skeletal features in humans and mice. Aged but not young RUNX2 +/− adult mice may also display reduced intramembranous bone formation. To clarify the role of Runx2 in intramembranous bone formation in adult mice a histomorphometric study was performed to compare the osteogenic response to high-dose estrogen in RUNX2 +/− and wild-type mice. Young (10 weeks) and aged (26 weeks) RUNX2 +/− and wild-type littermate mice were treated with vehicle or high-dose estrogen (0.5 mg/animal/week) by subcutaneous injection for 4 weeks. Mice were divided into 8 groups according to age, genotype and treatment with 6 animals per group. Following sacrifice, longitudinal tibial sections were prepared and examined by static and dynamic histomorphometry. Estrogen treatment induced formation of new cancellous bone in both wild-type and RUNX2 +/− mice. This occurred to the same extent in young mice of both genotypes. However, in the aged RUNX2 +/− mice this response as assessed by bone volume (BV/TV%) was decreased by over 70% ( p < 0.001) when compared to aged wild-type mice. Furthermore, significant reductions in cancellous double-labelled surfaces (dls/TV, 1.7 ± 0.2 vs 1.0 ± 0.4 mm 2/mm 3, p < 0.05) and mineral apposition rate (1.8 ± 0.1 vs 1.4 ± 0.1 μm/day, p < 0.01) were observed in aged RUNX2 +/− mice compared to wild-types. Aged RUNX2 +/− mice display an abrogated osteogenic response to high-dose estrogen. This may have occurred through combined reductions in recruitment of osteoprogenitor cells, osteoblast activity and mineralization. Since the characteristic histological changes in the marrow cavity which precede the formation of cancellous bone following estrogen treatment was seen in the aged RUNX2 +/− mice we suggest that they may eventually be capable of a full osteogenic response but haploinsufficiency leads to delayed bone formation.

Functional Study of Gene using Inducible Cre System

Gene manipulation by disrupting important genes using homologous recombination in mammals has provided important insights into their function and development with regard to disease. However, many questions related to the genetic pathways that regulate cellular differentiation and function remain to be clarified. In particular, analysis of genetic pathways that control embryonic skeletal development is often hindered by the disruption of critical genes that function in early embryogenesis, thereby leading to embryonic or perinatal death and thus preventing study of the role of these genes in skeletal development and physiology postnatally. To overcome this problem gene-targeting methods, using site- and time-specific recombination based methods with the Cre/loxP system, have been used to delete particular genes in specific tissues and stages during development. Thus, the generation and characterization of transgenic mice expressing Cre recombinase, under the control of a tissue-specific and stage-specific promoter, has become prerequisite for study of the physiology and homeostasis of specific tissues during a specific time frame. In this report, we introduce the principles and methods of site-specific and time-specific recombination using the Cre/loxP and inducible Cre system, and discuss the potential applications for applying this system to the study of the development and physiology of the skeletal system. (J Kor Endocrinol Soc 21:364~369, 2006)

Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler.

Parathyroid hormone (PTH)-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR) play an essential role in controlling growth plate development. The aim of the present study was to use the deer antler as a model to determine whether PTHrP and PPR may also have a function in regulating cartilage and bone regeneration in an adult mammal. Antlers are the only mammalian appendages that are able to undergo repeated cycles of regeneration, and their growth from a blastema involves a modified endochondral process. Immunohistochemistry was used to establish sites of localization of PTHrP and PPR in antlers at different stages of development. The pattern of Indian Hedgehog (IHH) and transforming growth factor-beta1 (TGF beta1) distribution was also investigated, because PTHrP expression in the developing limb is regulated by IHH and during embryonic growth plate formation TGF beta1 acts upstream of PTHrP to regulate the rate of chondrocyte differentiation. In the antler blastema (<10 days of development), PTHrP, PPR, and TGF beta1 were localized in epidermis, dermis, regenerating epithelium, and in mesenchymal cells but IHH expression was not detected. In the rapidly growing antler (weeks 4-8 of development), PTHrP, PPR, and TGF beta1 were localized in skin, perichondrium, undifferentiated mesenchyme, recently differentiated chondrocytes, and in perivascular cells in cartilage but not in fully differentiated hyperytrophic chondrocytes. IHH was restricted to recently differentiated chondrocytes and to perivascular cells in cartilage. In mineralized cartilage and bone, PTHrP, PPR, IHH, and TGF beta1 were immunolocalized in perivascular cells and differentiated osteoblasts. PTHrP and PPR were also present in the periosteum. TGF beta1 in vitro stimulated PTHrP synthesis by cells from blastema, perichondrium, and cartilage. The findings of this study suggest that molecules which regulate embryonic skeletal development and postnatal epiphyseal growth may also control blastema formation, chondrogenesis, and bone formation in the regenerating deer antler. This finding is further evidence that developmental signaling pathways are recapitulated during adult mammalian bone regeneration.