Pleiotropic patterning response to activation of Shh signaling in the limb apical ectodermal ridge.

Sonic hedgehog (Shh) signaling in the limb plays a central role in coordination of limb patterning and outgrowth. Shh expression in the limb is limited to the cells of the zone of polarizing activity (ZPA), located in posterior limb bud mesoderm. Shh is not expressed by limb ectoderm or apical ectodermal ridge (AER), but recent studies suggest a role for AER-Shh signaling in limb patterning.

Expression and function of Dlx genes in the osteoblast lineage.

Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5(-/-)/Dlx6(-/-) mice have more severe craniofacial and limb defects than Dlx5(-/-), some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5(-/-) mice.

Transcriptional regulation of Msx2 in the AERs of developing limbs is dependent on multiple closely spaced regulatory elements.

In developing limb buds, Msx2 transcripts are expressed in the apical ectodermal ridge (AER) and in various regions of the limb mesenchyme. To identify DNA sequences responsible for Msx2 expression in the AER, we characterized the expression of LacZ reporter constructs driven by chicken Msx2 regulatory sequences in transgenic mice. We have identified a 55-bp enhancer that can direct AER-specific reporter gene expression.

Function of BMPs in the apical ectoderm of the developing mouse limb.

Several bone morphogenetic proteins (BMPs) are expressed in the apical ectodermal ridge (AER), a critical signaling center that directs the outgrowth and patterning of limb mesoderm, but little is known about their function. To study the functions of apical ectodermal BMPs, an AER-specific promoter element from the Msx2 gene was used to target expression of the potent BMP antagonist noggin to the apical ectoderm of the limbs of transgenic mice. Msx2-noggin mutant mice have severely malformed limbs characterized by syndactyly, postaxial polydactyly, and dorsal transformations of ventral structures indicated by absence of ventral footpads and presence of supernumerary ventral nails.

Region- and stage-specific effects of FGFs and BMPs in chick mandibular morphogenesis.

The mandibular processes are specified as at least two independent functional regions: two large lateral regions where morphogenesis is dependent on fibroblast growth factor (FGF)-8 signaling, and a small medial region where morphogenesis is independent of FGF-8 signaling. To gain insight into signaling pathways that may be involved in morphogenesis of the medial region, we have examined the roles of pathways regulated by FGFs and bone morphogenetic proteins (BMPs) in morphogenesis of the medial and lateral regions of the developing chick mandible. Our results show that, unlike in the lateral region, the proliferation and growth of the mesenchyme in the medial region is dependent on signals derived from the overlying epithelium.

Studies on the role of Cux1 in regulation of the onset of joint formation in the developing limb.

Joint formation, the onset of which is characterized by the segmentation of continuous skeletal rudiments into two or more separate elements, is a fundamental aspect of limb pattern formation, playing a critical role in determining the size, shape, and number of individual skeletal elements. Joint formation is initiated by conversion of differentiated chondrocytes at sites of presumptive joints into densely packed nonchondrogenic cells of the joint interzone. This conversion is accompanied by loss of Alcian blue-staining cartilage matrix and downregulation of cartilage-specific gene expression.

MSX2 expression in the apical ectoderm ridge is regulated by an MSX2 and Dlx5 binding site.

The apical ectodermal ridge (AER) is a specialized ectodermal region essential for limb outgrowth. Msx2 expression patterns in limb development strongly suggest an important role for Msx2 in the AER. Our previous studies identified a 348-bp fragment of the chicken Msx2 gene with AER enhancer activity.

Effects of BMP-7 on mouse tooth mesenchyme and chick mandibular mesenchyme.

BMP-7 is a member of the BMP family of signaling molecules that are thought to play key roles in mediating inductive events during embryogenesis. In the present study the possible roles of BMP-7 in mediating inductive events during the initiation phase of odontogenesis and mandibular morphogenesis were investigated. To do so, we have examined the effects of agarose beads soaked in recombinant BMP-7 on E11 mouse molar-forming mesenchyme and stage 23 chick mandibular mesenchyme, and analyzed the patterns of expression of Bmp-7 in developing mouse and chick first branchial arches.

Complete primary structure of the chicken alpha1(V) collagen chain.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

Odontogenic epithelium induces similar molecular responses in chick and mouse mandibular mesenchyme.

Previous observations have shown that, during the initiation phase of odontogenesis, signals from mouse odontogenic epithelium can elicit teeth in non-odontogenic but neural crest-derived mesenchyme isolated from ectopic sites including chick mandibular mesenchyme. In the present study the formation of ectopic tooth buds and dental mesenchyme in chick mandibular mesenchyme was examined using heterospecific recombinations between E11 mouse odontogenic epithelium and stage 23 chick lateral mandibular mesenchyme. Both morphological criteria and chick-specific probes for Msx-1, Msx-2, and Bmp-4 mRNAs were used as markers for early dental mesenchyme.

DNase I hypersensitive sites in the chromatin of the chicken Msx2 gene differ in anterior and posterior limb mesenchyme, calvarial osteoblasts and embryonic fibroblasts.

To identify candidate cis-acting regulatory regions involved in regulation of the Msx2 gene in anterior limb mesenchyme and calvarial osteoblasts, DNase I hypersensitive sites (DHSs) from -6.1 kb to +8.4 kb relative to the translation start site of the chicken Msx2 gene were identified in anterior and posterior limb mesenchyme, calvarial osteoblasts, and embryonic fibroblasts.

Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits.

EGF does not induce Msx-1 and Msx-2 in dental mesenchyme.

Previous heterospecific tissue recombinations indicate that mandibular epithelium exerts the first known inductive signal for odontogenesis in mouse embryos. BMP-4 and EGF are two growth factors implicated as signaling molecules mediating the initial inductive epithelial-mesenchymal interactions during odontogenesis. The purpose of the present study was to examine and compare the effects of these growth factors and mouse mandibular epithelium on expression of Msx-1 and Msx-2 genes in molar-forming mesenchyme.

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins.

Differential regulation of COL2A1 expression in developing and mature chondrocytes.

To investigate the regulation of type II collagen gene expression in cells undergoing chondrogenic differentiation, we have employed a 5-kbp genomic fragment of the human type II collagen gene which contains 1.8kbp of upstream sequences, the transcription start site, the first exon and 3 kbp of intronic sequences, fused to either lac Z or chloramphenicol acetyl transferase-reporter gene. Transient expression studies revealed a parallel increase in transgene activity and endogenous type II collagen mRNA levels during the onset of the cartilage differentiation of limb mesenchymal cells in high-density micromass cultures.

The expression pattern of the Distal-less homeobox-containing gene Dlx-5 in the developing chick limb bud suggests its involvement in apical ectodermal ridge activity, pattern formation, and cartilage differentiation.

Here we report the isolation from a chick limb bud cDNA library of a cDNA that contains the full coding sequence of chicken Dlx-5, a member of the Distal-less (Dlx) family of homeobox-containing genes that encode homeodomains highly similar to that of the Drosophila Distal-less gene, a gene that is required for limb development in the Drosophila embryo. The expression pattern of Dlx-5 in the developing chick limb bud suggests that it may be involved in several aspects of limb morphogenesis. Dlx-5 is expressed in the apical ectodermal ridge (AER) which directs the outgrowth and patterning of underlying limb mesoderm.

Identification of a spatially specific enhancer element in the chicken Msx-2 gene that regulates its expression in the apical ectodermal ridge of the developing limb buds of transgenic mice.

Msx-2 is a member of the Msx family of homeobox-containing genes expressed in a variety of embryonic tissues involved in epithelial-mesenchymal interactions and pattern formation. In the developing chick limb bud, Msx-2 is expressed in the apical ectodermal ridge, which plays a crucial role in directing the growth and patterning of limb mesoderm. In addition, Msx-2 is expressed in the anterior nonskeletal-forming mesoderm of the limb bud, in the posterior necrotic zone, and in the interdigital mesenchyme.

Characterization of chicken syndecan-3 as a heparan sulfate proteoglycan and its expression during embryogenesis.

Syndecan-3 is one of four identified members of a family of transmembrane proteoglycans (the syndecans) that possess highly similar cytoplasmic and transmembrane domains and may function as extracellular matrix receptors and/or low affinity receptors for signaling molecules such as FGF. We previously reported the cloning of a partial cDNA for chicken syndecan-3. Here we report the isolation of a syndecan-3 cDNA containing additional 5' sequence which includes a potential methionine start codon and putative signal sequence.

The chicken homeobox gene Hoxd-11 encodes two alternatively spliced RNA species.

Two forms of cDNA clones corresponding to Hoxd-11 were obtained by screening a chicken limb bud cDNA library. Comparison of the two cDNA sequences with the mouse cDNA and genomic sequence, shows that the shorter cDNA is missing 3 nucleotides, which encode an alanine residue, at the junction between exons 1 and 2. Analysis of the chicken genomic sequence shows two tandem CAG repeats at the intron 1-exon 2 junction, both of which could serve as splice acceptor sequences.

Experimental analysis of Msx-1 and Msx-2 gene expression during chick mandibular morphogenesis.

Homeobox-containing genes are thought to be involved in regulating pattern formation in a variety of tissues during embryogenesis. We have examined the expression of the homeobox-related genes Msx-1 and Msx-2 during the development of the chick mandibular arch. Northern blot hybridization indicates that transcripts for both Msx-1 (1.

Enhancement of avian mandibular chondrogenesis in vitro in the absence of epithelium.

The roles of mandibular epithelium in chondrogenesis and growth of mandibular mesenchyme were examined in organ cultures. Epithelium and mesenchyme were separated from the mandibular arches of chick embryos at stages before and after the onset of chondrogenesis in vivo (stages 18-28). Isochronic and heterochronic tissue recombinations were prepared.

Molecular cloning and analysis of the protein modules of aggrecans.

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan.

Molecular cloning and analysis of the protein modules of aggrecans.

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan.

Ectoderm from various regions of the developing chick limb bud differentially regulates the expression of the chicken homeobox-containing genes GHox-7 and GHox-8 by limb mesenchymal cells.

The apical ectodermal ridge expresses high amounts of the homeobox gene GHox-8 when placed upon dissociated limb mesenchymal cells in culture and induces high expression of GHox-7, but only low expression of GHox-8, in the underlying mesenchymal cells. Ectoderm from the proximal anterior border of the limb induces high expression of both GHox-7 and GHox-8, while ectoderm from the proximal posterior border does not induce expression of either gene. Thus, ectoderm in various regions of the limb bud has distinct regulatory activities and may be involved in controlling the regionally specific expression of GHox-7 and GHox-8 in the mesoderm.