Distinct spatiotemporal roles of hedgehog signalling during chick and mouse cranial base and axial skeleton development.

The cranial base exerts a supportive role for the brain and includes the occipital, sphenoid and ethmoid bones that arise from cartilaginous precursors in the early embryo. As the occipital bone and the posterior part of the sphenoid are mesoderm derivatives that arise in close proximity to the notochord and floor plate, it has been assumed that their development, like the axial skeleton, is dependent on Sonic hedgehog (Shh) and modulation of bone morphogenetic protein (Bmp) signalling. Here we examined the development of the cranial base in chick and mouse embryos to compare the molecular signals that are required for chondrogenic induction in the trunk and head. We found that Shh signalling is required but the molecular network controlling cranial base development is distinct from that in the trunk. In the absence of Shh, the presumptive cranial base did not undergo chondrogenic commitment as determined by the loss of Sox9 expression and there was a decrease in cell survival. In contrast, induction of the otic capsule occurred normally demonstrating that induction of the cranial base is uncoupled from formation of the sensory capsules. Lastly, we found that the early cranial mesoderm is refractory to Shh signalling, likely accounting for why development of the cranial base occurs after the axial skeleton. Our data reveal that cranial and axial skeletal induction is controlled by conserved, yet spatiotemporally distinct mechanisms that co-ordinate development of the cranial base with that of the cranial musculature and the pharyngeal arches.

Identification of synergistic signals initiating inner ear development.

Tissue manipulation experiments in amphibians more than 50 years ago showed that induction of the inner ear requires two signals: a mesodermal signal followed by a neural signal. However, the molecules mediating this process have remained elusive. We present evidence for mesodermal initiation of otic development in higher vertebrates and show that the mesoderm can direct terminal differentiation of the inner ear in rostral ectoderm. Furthermore, we demonstrate the synergistic interactions of the extracellular polypeptide ligands FGF-19 and Wnt-8c as mediators of mesodermal and neural signals, respectively, initiating inner ear development.

Active GSK3ß and an intact ß-catenin TCF complex are essential for the differentiation of human myogenic progenitor cells.

Wnt-ß-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle remains unknown. Here we have used human primary CD56Pos satellite cell-derived myogenic progenitors obtained from healthy individuals to study the role of Wnt-ß-catenin signalling in myogenic differentiation. We show that dephosphorylated ß-catenin (active-ß-catenin), the central effector of the canonical Wnt cascade, is strongly upregulated at the onset of differentiation and undergoes nuclear translocation as differentiation progresses. To establish the role of Wnt signalling in regulating the differentiation process we manipulated key nodes of this pathway through a series of ß-catenin gain-of-function (GSK3 inhibition and ß-catenin overexpression) or loss-of-function experiments (dominant negative TCF4). Our data showed that manipulation of these critical pathway components led to varying degrees of disruption to the normal differentiation phenotype indicating the importance of Wnt signalling in regulating this process. We reveal an independent necessity for active-ß-catenin in the fusion and differentiation of human myogenic progenitors and that dominant negative inhibition of TCF4 prevents differentiation completely. Together these data add new mechanistic insights into both Wnt signalling and adult human myogenic progenitor differentiation.

The ectodermal control in chick limb development: Wnt-7a, Shh, Bmp-2 and Bmp-4 expression and the effect of FGF-4 on gene expression.

We have manipulated the chick limb bud by dorsoventrally inverting the ectoderm, by grafting the AER to the dorsal or ventral ectoderm and by insertion of an FGF-4 soaked heparin bead into the mesoderm. After dorso-ventral reversal of the ectoderm, Wnt-7a expression is autonomous from an early stage of limb development in the original dorsal ectoderm. Exogenous FGF-4 causes ectopic Wnt-7a expression and induces ectopic Shh. In addition, exogenous FGF-4 increases the thickness of cartilages and also shortens them, and both Bmp-2 and Bmp-4 may mediate this effect. The ectoderm outside the AER can regulate not only the dorso-ventral polarity of the underlying mesenchyme cells but also the cartilage formation, and both Bmp-2 and Bmp-4 may mediate this control.

Signalling interactions during facial development.

The development of the vertebrate face is a dynamic multi-step process which starts with the formation of neural crest cells in the developing brain and their subsequent migration to form, together with mesodermal cells, the facial primordia. Signalling interactions co-ordinate the outgrowth of the facial primordia from buds of undifferentiated mesenchyme into the intricate series of bones and cartilage structures that, together with muscle and other tissues, form the adult face. Some of the molecules that are thought to be involved have been identified through the use of mouse mutants, data from human craniofacial syndromes and by expression studies of signalling molecules during facial development. However, the way that these molecules control the epithelial-mesenchymal interactions which mediate facial outgrowth and morphogenesis is unclear. The role of neural crest cells in these processes has also not yet been well defined. In this review we discuss the complex interaction of all these processes during face development and describe the candidate signalling molecules and their possible target genes.

Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb.

Bone morphogenetic proteins are members of the transforming growth factor beta (TGF beta) superfamily which are involved in a range of developmental processes including modelling of the skeleton. We show here that Bmp-2 is expressed in mesenchyme surrounding early cartilage condensations in the developing chick limb, and that Bmp-4 is expressed in the perichondrium of developing cartilage elements. To investigate their roles during cartilage development, BMP-2 and BMP-4 were expressed ectopically in developing chick limbs using retroviral vectors. Over-expression of BMP-2 or BMP-4 led to a dramatic increase in the volume of cartilage elements, altered their shapes and led to joint fusions. This increase in volume appeared to result from an increase in the amount of matrix and in the number of chondrocytes. The latter did not appear to be due to increased proliferation of chondrocytes, suggesting that it may result from increased recruitment of precursors. BMP-2 and BMP-4 also delayed hypertrophy of chondrocytes and formation of the osteogenic periosteum. These data provide insights into how BMP-2 and BMP-4 may model and control the growth of skeletal elements during normal embryonic development, suggesting roles for both molecules in recruiting non-chondrogenic precursors to chondrogenic fate.

Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud.

In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.

Bone morphogenetic proteins in the development and healing of synovial joints.

OBJECTIVES: To review current knowledge of the role of bone morphogenetic proteins (BMPs) in joint formation and how this may be relevant to healing in adult joints. METHOD: Review of published literature using a search of the PubMed database (1966 to 2000) made available by the National Library of Medicine. Additional articles of historical interest were identified from the bibliographies of published literature. RESULTS: BMPs and a related family, the growth and differentiation factors (GDFs), are stimulators of bone and cartilage formation in the developing skeleton. They, together with their antagonists, play key roles in the specification of the joint site and cavitation of synovial joints during embryonic development. Disruption of the GDF-5 gene in mice and humans is associated with abnormal joint formation. In situ hybridization studies have shown that BMPs are expressed during formation of synovial joints in the embryo. However, excessive BMP activity leads to obliteration of joints because of cartilage overgrowth. BMPs are being considered as therapeutic agents to stimulate healing of articular cartilage after damage. Evidence suggests that BMPs are present in adult joints and have roles in healing and maintenance. However, inflammatory cytokines and growth factors present in damaged joints modulate the actions of BMPs. CONCLUSIONS: BMPs, and in particular GDF-5, are involved in synovial joint formation. They may also have effects on the maintenance and healing of adult joints, but factors present after damage may alter their effectiveness. RELEVANCE: Articular cartilage heals poorly after damage. BMPs may be useful therapeutically to stimulate healing of damaged articular cartilage. Increased knowledge of their role in joint formation will improve understanding of how to use them. Semin Arthritis Rheum 31:33-42.

Craniofacial development: the tissue and molecular interactions that control development of the head.

The molecular cascades that control craniofacial development have until recently been little understood. The paucity of data that exists has in part been due to the complexity of the head, which is the most intricate regions of the body. However, the generation of mouse mutants and the identification of gene mutations that cause human craniofacial syndromes, together with classical embryological approaches in other species, have given significant insight into how the head develops. These studies have emphasized how unique the head actually is, with each individual part governed by a distinct set of signalling interactions, again demonstrating the complexity of this region of the body. This review discussed the tissue and molecular interactions that control each region of the head. The processes that control neural tube closure together with correct development of the skull, midline patterning, neural crest generation and migration, outgrowth, patterning, and differentiation of the facial primordia and the branchial arches are thus discussed. Defects in these processes result in a number of human syndromes such as exencephaly, holoprosencephaly, musculoskeletal dysplasias, first arch syndromes such as Riegers and Treacher-Collins syndrome, and neural crest dysplasias such as DiGeorge syndrome. Our current knowledge of the genes responsible for these human syndromes together with how the head develops, is rapidly advancing so that we will soon understand the complex set of molecular and tissue interactions that build a head.

Growth/differentiation factor-5 (GDF-5) and skeletal development.

BACKGROUND: Growth/differentiation factor-5 (GDF-5) has been shown to be essential for normal appendicular skeletal and joint development in humans and mice. In brachypod, a Gdf-5 gene mouse mutant, the defect is first apparent during early chondrogenesis, with the cartilage blastema already reduced in size by E12.5. This defect is associated with changes in the expression of cell surface molecules. METHODS: To understand further how GDF-5 controls cartilage formation, we first mapped the expression of the Gdf-5 gene during skeletal development (please note that the abbreviation for the gene is given in italics and the abbreviation for the protein expressed by the gene is given in capital letters). Subsequently, we over-expressed GDF-5 in the developing chick embryo using a replication competent retrovirus, RCAS(BP). We determined its effects on skeletal development by histological examination and its effects on early growth by autoradiography of proliferating cells. In addition, we examined the effect of GDF-5 on chondrogenic differentiation using micromass and single cell suspension cultures of limb mesenchymal cells, RESULTS: These studies show that the Gdf-5 gene is expressed in the early cartilage condensation, the perichondrium, and the joint interzone. Over-expresSion of GDF-5 in chick limb buds, during the condensation stage or later when the skeletal elements have formed, increased the size of the affected elements. In both cases, the increase in size was associated with an increase in cell number and, at later stages, this was correlated with an increase in S-phase cells. In vitro studies showed that GDF-5 could increase cell adhesiveness, and this may be a mechanism through which GDF-5 initiates condensation formation. CONCLUSION: These studies show that GDF-5 acts at two stages of skeletal development and by two distinct mechanisms. First, GDF-5 promotes the initial stages of chondrogenesis by promoting cell adhesion, which is consistent with the expression of Gdf-5 in the cartilage condensation. Second, GDF-5 can increase the size of the skeletal elements by increasing proliferation within the epiphyseal cartilage adjacent to its expression within the joint interzone.

Differential regulation of GDF-5 and FGF-2/4 by immobilisation in ovo exposes distinct roles in joint formation.

Members of the fibroblast growth factor (FGF) family and growth and differentiation factor 5 (GDF-5) have been implicated in joint specification, but their roles in subsequent cavity formation are not defined. Cavity formation (cavitation) depends upon limb movement in embryonic chicks and factors involved in joint formation are often identified by their expression at the joint-line. We have sought support for the roles of FGF-2, FGF-4, and GDF-5 in cavitation by defining expression patterns, immunohistochemically, during joint formation and establishing whether these are modified by in ovo immobilisation. We found that FGF-2 exhibited low level nuclear expression in chondrocytes and fibrocartilage cells close to presumptive joints, but showed significantly higher expression levels in cells at, and directly bordering, the forming joint cavity. This high-level joint line FGF-2 expression was selectively diminished in immobilised limbs. In contrast, we show that FGF-4 does not exhibit differential joint-line expression and was unaffected by immobilisation. GDF-5 protein also failed to show joint-line selective labelling, and although immobilisation induced a cartilaginous fusion across presumptive joints, it did not affect cellular GDF-5 expression patterns. Examining changes in GDF-5 expression in response to a direct mechanical strain stimulus in primary embryonic chick articular surface (AS) cells in vitro discloses only small mechanically-induced reductions in GDF-5 expression, suggesting that GDF-5 does not exert a direct positive contribution to the mechano-dependent joint cavitation process. This notion was supported by retroviral overexpression of UDPGD, a characteristic factor involved in hyaluronan (HA) accumulation at presumptive joint lines, which was also found to produce small decreases in AS cell GDF-5 expression. These findings support a direct mechano-dependent role for FGF-2, but not FGF-4, in the cavitation process and indicate that GDF-5 is likely to influence chondrogenesis positively without contributing directly to joint cavity formation.

Expression patterns of the bone morphogenetic protein genes Bmp-4 and Bmp-2 in the developing chick face suggest a role in outgrowth of the primordia.

Bone morphogenetic proteins BMP-4 and BMP-2 are closely-related members of the transforming growth factor-beta superfamily that have been implicated in signalling in a number of developmental systems. To determine whether they could be involved in the epithelial-mesenchymal interactions that control face development, we mapped the distribution of Bmp-4 and Bmp-2 gene transcripts in the developing chick facial primordia. At stages when primordia were becoming established, Bmp-4 transcripts were present in specific regions of epithelium in all facial primordia, but were undetectable in the mesenchyme. Bmp-4 transcripts appeared subsequently in specific regions of mesenchyme at the distal tips of the primordia. This mesenchymal expression first appeared in the frontonasal mass and then, in turn, in the lateral nasal processes, the maxillary primordia and the mandibular primordia. There was a complex relationship between domains of epithelial and mesenchymal Bmp-4 expression, and at many sites there was an inverse correlation between epithelial and mesenchymal Bmp-4 expression. Bmp-2 transcripts were found in the epithelium and mesenchyme of the maxillary and mandibular primordia at early stages in facial development. Bmp-2 transcripts appeared in the frontonasal mass and lateral nasal processes at later stages, with epithelial expression preceding mesenchymal expression. In general, mesenchymal Bmp-2 expression was associated with overlying epithelial Bmp-2 expression. The domains of Bmp-4 expression overlapped with those of Bmp-2, but detailed examination showed that there was no precise correlation between the expression patterns of the two genes. Indeed, in some places the Bmp-4 and Bmp-2 expression domains were complementary. The expression of the Bmp-4 and Bmp-2 genes in the epithelium and distal mesenchyme of the facial primordia suggests that BMP-4 and BMP-2 may be involved in the epithelial-mesenchymal interactions that control outgrowth of these primordia.

The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest.

The pattern of skeletal structures and muscles in the branchial region of the head is profoundly influenced by the neural crest, whose cells arise at discrete segmental levels of the chick hindbrain: specifically, rhombomeres (r)1+2, r4 and r6, whereas r3 and r5 are crest-depleted. We have demonstrated that an interaction between even-numbered rhombomeres and r3/r5 effects this depletion of neural crest, resulting in the sculpting of discrete migratory streams of neural crest. This mechanism acts through increased expression of msx2 and the induction of apoptosis in dorsal cells of r3 and r5 (ref. 3) (Fig. 1A). Here we demonstrate that the signalling molecule Bmp4 is expressed in r3 and r5 and is dependent on the neighbouring rhombomeres. Addition of recombinant BMP4 protein to explant cultures of r3 or r5, which produce neural crest when isolated from their neighbouring rhombomeres, upregulates msx2 and reinstates apoptosis in the neural crest population.

Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia.

The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The signals that control facial outgrowth and patterning are largely unknown. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. We show that expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2 and that ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. We correlate this activation of gene expression with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel's cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of shh and bifurcation of the palatine bone. We also show that application of BMP-2 can increase cell proliferation of the mandibular primordia. Our data suggest that BMP-2 and BMP-4 are part of a signalling cascade that controls outgrowth and patterning of the facial primordia.

The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain.

In the avian hindbrain, the loss of premigratory neural crest cells from rhombomeres 3 and 5 (r3, r5) through programmed cell death contributes to the patterning of emigrant crest cells into three discrete streams. Programmed cell death is induced by the upregulation of Bmp4 and Msx2 in r3 and r5. We show that cSFRP2, a WNT antagonist, is expressed in the even-numbered rhombomeres and that over-expression of cSfrp2 inhibits Bmp4 expression in r3 and r5, preventing programmed cell death. By contrast, depleting cSFRP2 function in r4 results in elevated levels of Msx2 expression and ectopic programmed cell death, as does overexpression of Wnt1. We propose that programmed cell death in the rhombencephalic neural crest is modulated by pre-patterned cSfrp2 expression and a WNT-BMP signalling loop.

Activation of Fgf-4 and HoxD gene expression by BMP-2 expressing cells in the developing chick limb.

Bone morphogenetic protein-2 (BMP-2) has been implicated in the polarizing region signalling pathway, which specifies pattern across the antero-posterior of the developing vertebrate limb. Retinoic acid and Sonic Hedgehog (SHH) can act as polarizing signals; when applied anteriorly in the limb bud, they induce mirror-image digit duplications and ectopic Bmp-2 expression in anterior mesenchyme. In addition, the two signals can activate Fgf-4 expression in anterior ridge and HoxD expression in anterior mesenchyme. We tested the role of BMP-2 in this signalling cascade by ectopically expressing human BMP-2 (hBMP-2) at the anterior margin of the early wing bud using a replication defective retroviral vector, and found that ectopic expression of Fgf-4 was induced in the anterior part of the apical ectodermal ridge, followed later by ectopic expression of Hoxd-11 and Hoxd-13 in anterior mesenchyme. This suggests that BMP-2 is involved in regulating Fgf-4 and HoxD gene expression in the normal limb bud. Ectopically expressed hBMP-2 also induced duplication of digit 2 and bifurcation of digit 3, but could not produce the mirror-image digit duplications obtained with SHH-expressing cells. These results suggest that BMP-2 may be involved primarily in maintenance of the ridge, and in the link between patterning and outgrowth of the limb bud.

Expression of chick Barx-1 and its differential regulation by FGF-8 and BMP signaling in the maxillary primordia.

The vertebrate face develops from a series of primordia surrounding the primitive mouth and is thought to be patterned by the differential expression of homeobox-containing genes. Here we describe the isolation of the chick homologue of the homeobox-containing gene, Barx-1, and show its expression in the developing facial primordia, stomach, and appendicular skeleton. In the maxillary primordia, mesenchymal expression of Barx-1 is complementary to that of Msx-1, which correlate with overlying epithelial expression of Fgf-8 and Bmp-4, respectively. We show that epithelial signals are required to maintain Barx-1 expression and that FGF-8 can substitute for the epithelium. By contrast, BMPs reduce Barx-1 expression and can antagonize FGF-8 signaling. This suggests that in vivo, FGF-8/BMP signaling may regulate Barx-1 gene expression. This provides evidence that the differential expression of FGF-8 and BMPs may determine homeobox-containing gene expression and hence patterning of the facial primordia.