Mechanisms of GDF-5 action during skeletal development.
(1/511)
Mutations in GDF-5, a member of the TGF-beta superfamily, result in the autosomal recessive syndromes brachypod (bp) in mice and Hunter-Thompson and Grebe-type chondrodysplasias in humans. These syndromes are all characterised by the shortening of the appendicular skeleton and loss or abnormal development of some joints. To investigate how GDF-5 controls skeletogenesis, we overexpressed GDF-5 during chick limb development using the retrovirus, RCASBP. This resulted in up to a 37.5% increase in length of the skeletal elements, which was predominantly due to an increase in the number of chondrocytes. By injecting virus at different stages of development, we show that GDF-5 can increase both the size of the early cartilage condensation and the later developing skeletal element. Using in vitro micromass cultures as a model system to study the early steps of chondrogenesis, we show that GDF-5 increases chondrogenesis in a dose-dependent manner. We did not detect changes in proliferation. However, cell suspension cultures showed that GDF-5 might act at these stages by increasing cell adhesion, a critical determinant of early chondrogenesis. In contrast, pulse labelling experiments of GDF-5-infected limbs showed that at later stages of skeletal development GDF-5 can increase proliferation of chondrocytes. Thus, here we show two mechanisms of how GDF-5 may control different stages of skeletogenesis. Finally, our data show that levels of GDF-5 expression/activity are important in controlling the size of skeletal elements and provides a possible explanation for the variation in the severity of skeletal defects resulting from mutations in GDF-5. (+info)
Expression of the paired-box genes Pax-1 and Pax-9 in limb skeleton development.
(2/511)
Vertebrate Pax genes encode a family of transcription factors that play important roles in embryonic patterning and morphogenesis. Two closely related Pax genes, Pax-1 and Pax-9, are associated with early axial and limb skeleton development. To investigate the role of these genes in cartilage formation we have examined the expression profiles of Pax-1 and Pax-9 in developing chick limb mesenchyme in vivo and in vitro. Both transcripts are detected by reverse transcription polymerase chain reaction and Northern blotting throughout chick limb development, from the early bud stages (Hamburger-Hamilton 20-23) to fully patterned appendages (stage 30). Whole-mount in situ hybridization reveals complex, nonoverlapping expression domains of these two genes. Pax-1 transcripts first appear at the anterior proximal margin of the limb buds, while Pax-9 is expressed more distally at what will be the junction of the autopod and the zeugopod. In situ hybridization to serial sections of the girdles reveals that in the pectoral region Pax-1 is expressed proximally in condensed mesenchyme surrounding the junction of the developing scapula, humerus, and coracoid. In the pelvis, Pax-1 is expressed between the femur and the developing acetabulum and along the ventral edge of the ischium; this transcript was also found in the distal hindlimb along the posterior edge of the fibula. Pax-9 transcripts were not detected in the pectoral girdle at any stage, and only weakly in the pelvis along the ventral ischial margin. In the distal parts of both wings and legs, however, Pax-9 is strongly expressed between the anterior embryonic cartilages (e.g., distal radius or tibia) and the anterior ectodermal ridge. The expression of both genes was strongest in undifferentiated cells of precartilage condensations or at the margins of differentiated cartilages, and was absent from cartilage itself. In micromass cultures of chondrifying limb bud mesenchyme expression of Pax-1 and Pax-9 is maintained for up to 3 days in vitro, most strongly at the end of the culture period during chondrogenic differentiation. As seen in vivo, transcripts are found in loose mesenchyme cells at the outer margins of developing cartilage nodules, and are absent from differentiated chondrocytes at the nodule center. Taken together, these investigations extend previous studies of Pax-1 and Pax-9 expression in embryonic limb development while validating limb bud mesenchyme culture as an accessible experimental system for the study of Pax gene function and regulation. Our in vivo and in vitro observations are discussed with reference to 1) the relationship between somitic and limb expression of these two Pax genes, 2) what regulates this expression in different regions of the embryo, and 3) the putative cellular functions of Pax-1 and Pax-9 in embryonic skeletogenesis. (+info)
Multiple cis-acting regulatory regions are required for restricted spatio-temporal Hoxa5 gene expression.
(3/511)
Genetic analyses have revealed the essential role of the murine Hoxa5 gene for the correct specification of the cervical and upper thoracic region of the skeleton, and for the normal organogenesis and function of the respiratory tract, both structures expressing Hoxa5 during embryogenesis. To understand how the expression domains of the Hoxa5 gene are established during development, we have analyzed the cis-acting control regions mediating Hoxa5 gene expression using a transgenic approach. Four transcripts are derived from the Hoxa5 locus. The shortest and most abundant one displays a specific spatio-temporal profile of expression at earlier stages and in more anterior structures along the embryonic axis than the larger forms. We established that an 11.1 kilobase pair (kb) genomic fragment, extending from position -3.8 kb to +7.3 kb relative to Hoxa5 transcription initiation site, was sufficient to reproduce the temporal expression and substantially reconstitute the spatial pattern of the major Hoxa5 transcript. By deletion analyses, we identified a 2.1 kb fragment located downstream of the Hoxa5 gene that possesses mesodermal enhancer activity. Overall, the findings demonstrate that cis-acting regulatory elements essential for the correct expression of the major Hoxa5 transcript are located both upstream and downstream of the Hoxa5 coding sequences. (+info)
Selective expression of purinoceptor cP2Y1 suggests a role for nucleotide signalling in development of the chick embryo.
(4/511)
Responses to extracellular nucleotides (e.g., ATP, ADP, etc.) have been demonstrated in a number of embryonic cell types suggesting they may be important signalling molecules during embryonic development. Here the authors describe for the first time the expression of a G-protein-coupled receptor for extracellular ATP, chick P2Y1 (cP2Y1), during embryonic development of the chick. During the first 10 days of embryonic development, cP2Y1 is expressed in a developmentally regulated manner in the limb buds, mesonephros, brain, somites, and facial primordia, suggesting that this receptor may have a role in the development of each of these systems. (+info)
Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity.
(5/511)
In spite of recent breakthroughs in understanding limb patterning, the genetic factors determining the differences between the forelimb and the hindlimb have not been understood. The genes Pitx1 and Tbx4 encode transcription factors that are expressed throughout the developing hindlimb but not forelimb buds. Misexpression of Pitx1 in the chick wing bud induced distal expression of Tbx4, as well as HoxC10 and HoxC11, which are normally restricted to hindlimb expression domains. Wing buds in which Pitx1 was misexpressed developed into limbs with some morphological characteristics of hindlimbs: the flexure was altered to that normally observed in legs, the digits were more toe-like in their relative size and shape, and the muscle pattern was transformed to that of a leg. (+info)
Discrete roles for secreted and transmembrane semaphorins in neuronal growth cone guidance in vivo.
(6/511)
From the initial stages of axon outgrowth to the formation of a functioning synapse, neuronal growth cones continuously integrate and respond to multiple guidance cues. To investigate the role of semaphorins in the establishment of appropriate axon trajectories, we have characterized a novel secreted semaphorin in grasshopper, gSema 2a. Sema 2a is expressed in a gradient in the developing limb bud epithelium during Ti pioneer axon outgrowth. We demonstrate that Sema 2a acts as chemorepulsive guidance molecule critical for axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Ti axon projection. Interestingly, simultaneous perturbation of both secreted Sema 2a and transmembrane Sema I results in a broader range and increased incidence of abnormal Ti pioneer axon phenotypes, indicating that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo. (+info)
Limb and skin abnormalities in mice lacking IKKalpha.
(7/511)
The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB. (+info)
Morphological clues from multilegged frogs: are retinoids to blame?
(8/511)
Morphological analysis was performed on multilegged deformed frogs representing five species from 12 different localities in California, Oregon, Arizona, and New York. The pattern of duplicated limbs was consistent with mechanical perturbation by trematode infestation but not with the effects of retinoids. (+info)