[Show abstract][Hide abstract] ABSTRACT: A central issue in biology concerns the presence, timing and nature of phylotypic periods of development, but whether, when and why species exhibit conserved morphologies remains unresolved. Here, we construct a developmental morphospace to show that amniote faces share a period of reduced shape variance and convergent growth trajectories from prominence formation through fusion, after which phenotypic diversity sharply increases. We predict in silico the phenotypic outcomes of unoccupied morphospaces and experimentally validate in vivo that observed convergence is not due to developmental limits on variation but instead from selection against novel trajectories that result in maladaptive facial clefts. These results illustrate how epigenetic factors such as organismal geometry and shape impact facial morphogenesis and alter the locus of adaptive selection to variation in later developmental events.
Development 03/2014; 141(5):1059-63. · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FGF signaling mutations are a frequent contributor to craniofacial malformations including midfacial anomalies and craniosynostosis. FGF signaling has been shown to control cellular mechanisms that contribute to facial morphogenesis and growth such as proliferation, survival, migration and differentiation. We hypothesized that FGF signaling controls not only the magnitude of growth during facial morphogenesis but also regulates the direction of growth via cell polarity. To test this idea, we infected migrating neural crest cells of chicken embryos with RCAS virus expressing both FgfR2(C278F), a receptor mutation found in Crouzon syndrome, and the ligand Fgf8. Treated embryos exhibited craniofacial malformations resembling facial dysmorphologies in craniosynostosis syndrome. Consistent with our hypothesis, ectopic activation of FGF signaling resulted in decreased cell proliferation, increased expression of the Sprouty class of FGF signaling inhibitors, and repressed phosphorylation of ERK/MAPK. Furthermore, quantification of cell polarity in facial mesenchymal cells showed that while orientation of the Golgi body matches the direction of facial prominence outgrowth in normal cells, in FGF-treated embryos this direction is randomized, consistent with aberrant growth that we observed. Together these data demonstrate that FGF signaling regulates cell proliferation and cell polarity, and that together these cell processes contribute to facial morphogenesis.
Human Molecular Genetics 08/2013; · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Craniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus) embryo model. Eggs were incubated in either hypoxic (7%, 9%, 11%, 13%, 15%, 17%, or 19% O2) or normoxic (21% O2) conditions. Embryos were photographed for morphological analysis at days three through six. For analysis of skeletal development, 13-day embryos were cleared and stained in alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early stage embryos (day two) were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostained for cleaved caspase 3, while later stage embryos (days four and six) were sectioned in paraffin for analysis of cell proliferation (BrdU), apoptosis (TUNEL), and metabolic stress (phospho-AMPK). Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation, and in early stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development may induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are critical to normal craniofacial morphogenesis.
Disease Models and Mechanisms 04/2013; · 4.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Craniofacial development requires an exquisitely timed and positioned cross-talk between the embryonic cephalic epithelia and mesenchyme. This cross-talk underlies the precise translation of patterning processes and information into distinct, appropriate skeletal morphologies. The molecular and cellular dialogue includes communication via secreted signaling molecules, including Fgf8, and effectors of their interpretation. Herein, we use genetic attenuation of Fgf8 in mice and perform gain-of-function mouse-chick chimeric experiments to demonstrate that significant character states of the frontonasal and optic skeletons are dependent on Fgf8. Notably, we show that the normal orientation and polarity of the nasal capsules and their developing primordia are dependent on Fgf8. We further demonstrate that Fgf8 is required for midfacial integration, and provide evidence for a role for Fgf8 in optic capsular development. Taken together, our data highlight Fgf8 signaling in craniofacial development as a plausible target for evolutionary selective pressures.
[Show abstract][Hide abstract] ABSTRACT: Like other tissue injuries, bone fracture triggers an inflammatory response, which plays an important role in skeletal repair. Inflammation is believed to have both positive and negative effects on bone repair, but the underlying cellular mechanisms are not well understood. To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. These results suggested that the inflammatory response could influence skeletal cell differentiation after fracture. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, and which we previously showed is a potential regulator of cell fate decisions during fracture repair. Mmp9(-/-) mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9(-/-) compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9(-/-) mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9(-/-) stroma and periosteum. Following transplantation, Mmp9(-/-) mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Moreover, Mmp9(-/-) periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9(-/-) hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9(-/-) mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. Taken together, these results show that MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation.
[Show abstract][Hide abstract] ABSTRACT: Multiple fibroblast growth factor (Fgf) ligands are expressed in the forebrain and facial ectoderm, and vascular endothelial growth factor (VEGF) is expressed in the facial ectoderm. Both pathways activate the MAP kinase cascade and can be suppressed by SU5402. We placed a bead soaked in SU5402 into the brain after emigration of neural crest cells was complete.
Within 24 hr we observed reduced pMEK and pERK staining that persisted for at least 48 hr. This was accompanied by significant apoptosis in the face. By day 15, the upper beaks were truncated. Molecular changes in the FNP were also apparent. Normally, Shh is expressed in the frontonasal ectodermal zone and controls patterned growth of the upper jaw. In treated embryos, Shh expression was reduced. Both the structural and molecular deficits were mitigated after transplantation of FNP-derived mesenchymal cells.
Thus, mesenchymal cells actively participate in signaling interactions of the face, and the absence of neural crest cells in neurocristopathies may not be merely structural.
[Show abstract][Hide abstract] ABSTRACT: The Frontonasal Ectodermal Zone (FEZ) is a signaling center in the face that expresses Sonic hedgehog (Shh) and regulates patterned growth of the upper jaw. Blocking SHH in the forebrain blocks Shh expression in the FEZ and creates malformations resembling holoprosencephaly (HPE), while inhibition of BMP signaling in the mesenchyme blocks FEZ formation and causes similar dysmorphology. Thus, the brain could regulate FEZ formation by SHH or BMP signaling, and if so, activating one of these pathways in the face might alleviate the effects of repression of SHH in the brain.
We blocked SHH signaling in the brain while adding SHH or BMP between the neural and facial ectoderm of the frontonasal process. When applied early, SHH restored Shh expression in the FEZ and significantly improved shape outcomes, which contrasts with our previous experiments that showed later SHH treatments have no effect. BMP-soaked beads introduced early and late caused apoptosis that exacerbated malformations. Finally, removal of Smoothened from neural crest cells did not inhibit Shh expression in the FEZ.
Collectively, this work suggests that a direct, time-sensitive SHH signal from the brain is required for the later induction of Shh in the FEZ. We propose a testable model of FEZ activation and discuss signaling mediators that may regulate these interactions.
[Show abstract][Hide abstract] ABSTRACT: Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice, after application of bioactive molecules, after altering physiological parameters of healing, after modifying the amount or time of stabilization, after distraction osteogenesis, after creation of a non-union, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays an important role during skeletal development and fracture healing. Previous experimental studies have shown that VEGF applied immediately after injury can stimulate bone repair in animal fracture nonunion models. However, the effectiveness of VEGF on an established fracture non-union has not been determined. the goal of this work was to test the ability of VEGF applied at a later stage on the healing of fracture nonunions.
In this study, a murine non-union model was induced by rapid distraction of a tibia osteotomy. this model exhibits radiological and histological evidence of impaired fracture healing at 7 days after the completion of distraction. VEGF (10 µg in 20 µl Pbs/day, n=10) or control (20 µl Pbs/day, n=10) was injected directly into the distraction gap through the posterior musculature on three consecutive days (7, 8, and 9 days after completing distraction). A third group of animals (n=10) with rapid distraction, but no injections, served as non-treated controls. Fracture healing was analyzed by x-ray, histology, and histomorphometry at 27 days after the last round of distraction.
radiographs showed that half of the VEGF treated animals (5/10) achieved bony healing whereas the majority of Pbs treated (7/10) and non-treated controls (8/10) did not exhibit bone bridging. Histological and histomorphometric analyses demonstrated that VEGF increased, but not significantly, the amount of bone formed in the distraction gap (1.35 ± 0.35 mm(3)), compared to the saline treated (0.77 ± 0.25 mm(3), p=0.19) and non-treated animals (0.79 ± 0.23mm(3), p=0.12).
Results from this study demonstrate that VEGF potentially promotes bone repair, warranting further research in this direction.
[Show abstract][Hide abstract] ABSTRACT: Numerous factors can affect skeletal regeneration, including the extent of bone injury, mechanical loading, inflammation and exogenous molecules. Bisphosphonates are anticatabolic agents that have been widely used to treat a variety of metabolic bone diseases. Zoledronate (ZA), a nitrogen-containing bisphosphonate (N-BP), is the most potent bisphosphonate among the clinically approved bisphosphonates. Cases of bisphosphonate-induced osteonecrosis of the jaw have been reported in patients receiving long term N-BP treatment. Yet, osteonecrosis does not occur in long bones. The aim of this study was to compare the effects of zoledronate on long bone and cranial bone regeneration using a previously established model of non-stabilized tibial fractures and a new model of mandibular fracture repair. Contrary to tibial fractures, which heal mainly through endochondral ossification, mandibular fractures healed via endochondral and intramembranous ossification with a lesser degree of endochondral ossification compared to tibial fractures. In the tibia, ZA reduced callus and cartilage formation during the early stages of repair. In parallel, we found a delay in cartilage hypertrophy and a decrease in angiogenesis during the soft callus phase of repair. During later stages of repair, ZA delayed callus, cartilage and bone remodeling. In the mandible, ZA delayed callus, cartilage and bone remodeling in correlation with a decrease in osteoclast number during the soft and hard callus phases of repair. These results reveal a more profound impact of ZA on cartilage and bone remodeling in the mandible compared to the tibia. This may predispose mandible bone to adverse effects of ZA in disease conditions. These results also imply that therapeutic effects of ZA may need to be optimized using time and dose-specific treatments in cranial versus long bones.
PLoS ONE 01/2012; 7(2):e31771. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The goal of this study was to determine to what extent mechanical stability affects vascular repair during fracture healing.
Stabilized and nonstabilized tibia fractures were created in adult mice. Fracture tissues were collected at multiple time points during early fracture healing. Vasculature in fractured limbs was visualized by immunohistochemistry with an anti-PECAM-1 antibody on tissue sections and then quantified with stereology. Oxygen tension, vascular endothelial growth factor expression, and lactate accumulation at the fracture site were measured. Gene expression was compared between stabilized and nonstabilized fractures by microarray analysis.
We found that new blood vessel formation was robust by 3 days after fracture. Quantitative analysis showed that nonstabilized fractures had higher length density and surface density than stabilized fractures at 3 days after injury, suggesting that nonstabilized fractures were more vascularized. Oximetry analysis did not detect a significant difference in oxygen tension at the fracture site between stabilized and nonstabilized fractures during the first 3 days after injury. Further microarray analysis was performed to determine the effects of mechanical stability on the expression of angiogenic factors. No significant difference in the expression of vascular endothelial growth factors and other angiogenic factors was detected between stabilized and nonstabilized fractures.
Mechanical instability promotes angiogenesis during early fracture healing and further research is required to determine the underlying mechanisms.
Journal of orthopaedic trauma 08/2011; 25(8):494-9. · 1.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of the brain on the morphology of the face has long been recognized in both evolutionary biology and clinical medicine. In this work, we describe factors that are active between the development of the brain and face and how these might impact craniofacial variation. First, there is the physical influence of the brain, which contributes to overall growth and morphology of the face through direct structural interactions. Second, there is the molecular influence of the brain, which signals to facial tissues to establish signaling centers that regulate patterned growth. Importantly, subtle alterations to these physical or molecular interactions may contribute to both normal and abnormal variation. These interactions are therefore critical to our understanding of how a diversity of facial morphologies can be generated both within species and across evolutionary time.
[Show abstract][Hide abstract] ABSTRACT: The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., (1, 2, 3, 4)). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick (5) or mouse-chick (6) chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues (7). Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras (8). The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.
[Show abstract][Hide abstract] ABSTRACT: Variation is an intrinsic feature of biological systems, yet developmental biology does not frequently address population-level phenomena. Sonic hedgehog (SHH) signaling activity in the vertebrate forebrain and face is thought to contribute to continuous variation in the morphology of the upper jaw, but despite its potential explanatory power, this idea has never been quantitatively assessed. Here, we test this hypothesis with an experimental design that is explicitly focused on the generation and measurement of variation in multivariate shape, tissue growth, cellular behavior and gene expression. We show that the majority of upper jaw shape variation can be explained by progressive changes in the spatial organization and mitotic activity of midfacial growth zones controlled by SHH signaling. In addition, nonlinearity between our treatment doses and phenotypic outcomes suggests that threshold effects in SHH signaling may play a role in variability in midfacial malformations such as holoprosencephaly (HPE). Together, these results provide novel insight into the generation of facial morphology, and demonstrate the value of quantifying variation for our understanding of development and disease.
Development 10/2010; 137(20):3405-9. · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Age significantly reduces the regenerative capacity of the skeleton, but the underlying causes are unknown. Here, we tested whether the functional status of inflammatory cells contributes to delayed healing in aged animals. We created chimeric mice by bone marrow transplantation after lethal irradiation. In this model, chondrocytes and osteoblasts in the regenerate are derived exclusively from host cells while inflammatory cells are derived from the donor. Using this model, the inflammatory system of middle-aged mice (12 month old) was replaced by transplanted bone marrow from juvenile mice (4 weeks old), or age-matched controls. We found that the middle-aged mice receiving juvenile bone marrow had larger calluses and more bone formation during early stages and faster callus remodeling at late stages of fracture healing, indicating that inflammatory cells derived from the juvenile bone marrow accelerated bone repair in the middle-aged animals. In contrast, transplanting bone marrow from middle-aged mice to juvenile mice did not alter the process of fracture healing in juvenile mice. Thus, the roles of inflammatory cells in fracture healing may be age-related, suggesting the possibility of enhancing fracture healing in aged animals by manipulating the inflammatory system.
Journal of Orthopaedic Research 08/2010; 28(8):1000-6. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After bone injury, developmental processes such as endochondral and intramembranous ossification are recapitulated as the skeleton regenerates. In contrast to development, skeletal healing involves inflammation. During the early stages of healing a variety of inflammatory cells infiltrate the injured site, debride the wound, and stimulate the repair process. Little is known about the inflammatory process during bone repair. In this work, we examined the effect of a pro-inflammatory cytokine, Interleukin-1 beta (IL-1beta), on osteoblast and stem cell differentiation and on intramembranous and endochondral ossification, because IL-1beta exerts effects on skeletal homeostasis and is upregulated in response to fracture. We determined that IL-1beta stimulated proliferation of osteoblasts and production of mineralized bone matrix, but suppressed proliferation and inhibited differentiation of bone marrow derived MSCs. We next performed loss- and gain-of-function experiments to determine if altering IL-1beta signaling affects fracture healing. We did not detect any differences in callus, cartilage, and bone matrix production during healing of nonstabilized or stabilized fractures in mice that lacked the IL-1beta receptor compared to wild-type animals. We observed subtle alterations in the healing process after administering IL-1beta during the early phases of repair. At day 10 after injury, the ratio of cartilage to callus was increased, and by day 14, the proportion of cartilage to total callus and to bone was reduced. These changes could reflect a slight acceleration of endochondral ossification, or direct effects on cartilage and bone formation.
Journal of Orthopaedic Research 06/2010; 28(6):778-84. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory cells to sites of injury occurs in response to specific signaling pathways. The CC chemokine receptor type 2 (CCR2) is crucial for recruiting macrophages, as well as regulating osteoclast function. In this study, we examined fracture healing in Ccr2-/- mice. We first demonstrated that the expression of Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2-/- mice compared with wild-type controls at 3 days after injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after injury in mutant mice. At day 14, Ccr2-/- mice had less bone in their calluses. At day 21, Ccr2-/- mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2-/- osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2-/- mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.
Disease Models and Mechanisms 03/2010; 3(7-8):451-8. · 4.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Integrins are heterodimeric transmembrane proteins that mediate cell-matrix interactions and modulate cell behavior. Beta3 subunit is a component of alphaIIbeta3 and alphaVbeta3 integrins. In this study, we first determined that beta3 transcripts are expressed by cells within fracture calluses at 7 and 10 days after injury in a mouse model. We then analyzed fracture healing in mice deficient of beta3 integrin with molecular, histomorphometric, and biomechanical techniques. We found that lack of beta3 integrin results in an extended bleeding time and leads to more bone formation and accelerated cartilage maturation at 7 days after injury. However, beta3 deficiency does not appear to affect later fracture healing. At days 14 and 21, histological appearance or biomechanical properties of fracture calluses are similar between wild type and mutant mice. We also found that altered fracture healing in beta3-null mice is not associated with accelerated angiogenesis, because no significant difference of length density and surface density of blood vessels in fracture limbs was detected at 3 days after injury between wild type and beta3-null mice. In conclusion, our findings demonstrate that beta3 integrin plays an important role during early fracture healing. Further research is required to determine the underlying mechanisms.
Journal of Orthopaedic Research 08/2009; 28(1):32-7. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interactions among the forebrain, neural crest and facial ectoderm regulate development of the upper jaw. To examine these interactions, we activated the Sonic hedgehog (SHH) pathway in the brain. Beginning 72 hours after activation of the SHH pathway, growth within the avian frontonasal process (FNP) was exaggerated in lateral regions and impaired in medial regions. This growth pattern is similar to that in mice and superimposed a mammalian-like morphology on the upper jaw. Jaw growth is controlled by signals from the frontonasal ectodermal zone (FEZ), and the divergent morphologies that characterize birds and mammals are accompanied by changes in the FEZ. In chicks there is a single FEZ spanning the FNP, but in mice both median nasal processes have a FEZ. In treated chicks, the FEZ was split into right and left domains that resembled the pattern present in mice. Additionally, we observed that, in the brain, fibroblast growth factor 8 (Fgf8) was downregulated, and signals in or near the nasal pit were altered. Raldh2 expression was expanded, whereas Fgf8, Wnt4, Wnt6 and Zfhx1b were downregulated. However, Wnt9b, and activation of the canonical WNT pathway, were unaltered in treated embryos. At later time points the upper beak was shortened owing to hypoplasia of the skeleton, and this phenotype was reproduced when we blocked the FGF pathway. Thus, the brain establishes multiple signaling centers within the developing upper jaw. Changes in organization of the brain that occur during evolution or as a result of disease can alter these centers and thereby generate morphological variation.
Development 12/2008; 136(1):107-16. · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic proteins (BMPs) regulate many aspects of development including skeletogenesis. Here, we examined the response of neural crest-derived cells to ectopic BMP signaling by infecting avian embryos with retroviruses encoding Bmp-2 or Bmp-4 at various times of development. Infection at stages 10 and 15 transformed large areas of the skull into cartilage by day 13. At this time cartilage condensations were still forming, which revealed the presence of uncommitted mesenchymal cells. By day 19, hypertrophic chondrocytes were present in the cartilage possibly due to changes in the perichondrium that relieved repression on hypertrophy. While these cells expressed Sox9, Collagen-2, Runx2, Ihh, Noggin, and Collagen-10, cartilage was not replaced by bone. Whether this is an intrinsic property of the skull cartilage, or results from sustained Bmp signaling is not known.