Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: Their relationships in the signalling cascade involved in limb patterning
TL;DR: The dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a–p patterning of limb buds in the chick mutant ta3 mutant.
Abstract: The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds.
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TL;DR: It is shown here that transgenic mice overexpressing SHH in the skin develop many features of basal cell nevus syndrome, demonstrating that SHH is sufficient to induce basal cell carcinomas in mice, suggesting thatSHH may have a role in human tumorigenesis.
Abstract: Mutations in the tumor suppressor gene PATCHED (PTC) are found in human patients with the basal cell nevus syndrome, a disease causing developmental defects and tumors, including basal cell carcinomas. Gene regulatory relationships defined in the fruit fly Drosophila suggest that overproduction of Sonic hedgehog (SHH), the ligand for PTC, will mimic loss of ptc function. It is shown here that transgenic mice overexpressing SHH in the skin develop many features of basal cell nevus syndrome, demonstrating that SHH is sufficient to induce basal cell carcinomas in mice. These data suggest that SHH may have a role in human tumorigenesis.
727 citations
TL;DR: Results indicate BMP signaling actively mediates cell death in the embryonic limb and Expression of dnBMPR in chicken embryonic hind limbs greatly reduced interdigital apoptosis and resulted in webbed feet.
Abstract: Interdigital cell death leads to regression of soft tissue between embryonic digits in many vertebrates. Although the signals that regulate interdigital apoptosis are not known, BMPs—signaling molecules of the transforming growth factor-β superfamily—are expressed interdigitally. A dominant negative type I BMP receptor (dnBMPR-IB) was used here to block BMP signaling. Expression of dnBMPR in chicken embryonic hind limbs greatly reduced interdigital apoptosis and resulted in webbed feet. In addition, scales were transformed into feathers. The similarity of the webbing to webbed duck feet led to studies that indicate that BMPs are not expressed in the duck interdigit. These results indicate BMP signaling actively mediates cell death in the embryonic limb.
564 citations
TL;DR: It is demonstrated that the two type I BMPRs, BM PR-IA and BMPR-IB, regulate distinct processes during chick limb development and regulates apoptosis.
Abstract: The bone morphogenetic proteins (BMPs), TGFβ superfamily members, play diverse roles in embryogenesis, but how the BMPs exert their action is unclear and how different BMP receptors (BMPRs) contribute to this process is not known. Here we demonstrate that the two type I BMPRs, BMPR-IA and BMPR-IB, regulate distinct processes during chick limb development. BmpR-IB expression in the embryonic limb prefigures the future cartilage primordium, and its activity is necessary for the initial steps of chondrogenesis. During later chondrogenesis, BmpR-IA is specifically expressed in prehypertrophic chondrocytes. BMPR-IA regulates chondrocyte differentiation, serving as a downstream mediator of Indian Hedgehog (IHH) function in both a local signaling loop and a longer-range relay system to PTHrP. BMPR-IB also regulates apoptosis: Expression of activated BMPR-IB results in increased cell death, and we showed previously that dominant-negative BMPR-IB inhibits apoptosis. Our studies indicate that in TGFβ signaling systems, different type I receptor isoforms are dedicated to specific functions during embryogenesis.
517 citations
Cites background from "Expression of genes encoding bone m..."
...…BMPR-IA and BMPR-IB have similar but not identical binding affinities in vitro for the BMP ligands that are expressed in the developing limb (Francis et al. 1994; Koenig et al. 1994; ten Dijke et al. 1994b; Francis-West et al. 1995; Liu et al. 1995; Nohno et al. 1995; Rosenzweig et al. 1995)....
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...Prior to the formation of cartilage elements, Bmp2, Bmp4, and Bmp7 are expressed in spatially and temporally dynamic patterns (Francis et al. 1994; Francis-West et al. 1995)....
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TL;DR: The analysis of specific gene functions has revealed the existence of complex interactions between signaling pathways operated by secreted factors of the HH, TGF-beta/BMP, WNT, and FGF superfamilies, which interact with many other genetic networks to control limb positioning, outgrowth, and patterning.
Abstract: ■ Abstract Vertebrate limb buds are embryonic structures for which much molecular and cellular data are known regarding the mechanisms that control pattern formation during development. Specialized regions of the developing limb bud, such as the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the non-ridge ectoderm, direct and coordinate the development of the limb bud along the anteriorposterior (AP), dorsal-ventral (DV), and proximal-distal (PD) axes, giving rise to a stereotyped pattern of elements well conserved among tetrapods. In recent years, specific gene functions have been shown to mediate the organizing and patterning activities of the ZPA, the AER, and the non-ridge ectoderm. The analysis of these gene functions has revealed the existence of complex interactions between signaling pathways operated by secreted factors of the HH, TGF-fl/BMP, WNT, and FGF superfamilies, which interact with many other genetic networks to control limb positioning, outgrowth, and patterning. The study of limb development has helped to establish paradigms for the analysis of pattern formation in many other embryonic structures and organs.
400 citations
Cites background from "Expression of genes encoding bone m..."
...The molecular characterization of the genes mutated in chicktalpid mutants (Izpis´ua Belmonte et al. 1992, Francis-West et al. 1995, Rodr´ıguez et al. 1996, Caruccio et al. 1999, Lewis et al. 1999) has the potential to reveal new elements of the SHH pathway, because in these mutantsShhexpression is…...
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TL;DR: The 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 G DF-5.
Abstract: 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.
365 citations
Cites methods from "Expression of genes encoding bone m..."
...Embryos were fixed in 4% (w/v) paraformaldehyde, processed to methanol for whole-mount in situ hybridisation or embedded in wax for in situ hybridisation of tissue sections as previously described (Francis-West et al., 1995)....
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References
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TL;DR: The preparation of a series of normal stages of the chick embryo does not need justification at a time when chick ernbryos are not only widely used in descriptive and experimental embryology but are proving to be increasingly valuable in medical research, as in work on viruses and cancer.
Abstract: FORTY-FIVE FIGURES The preparation of a series of normal stages of the chick embryo does not need justification at a time when chick ernbryos are not only widely used in descriptive and experimental embryology but are proving to be increasingly valuable in medical research, as in work on viruses and cancer. The present series was planned in connection with the preparation of a new edition of Lillie’s DeueZopmerzt of the Chick by the junior author. It is being published separately to make it accessible immediately to a large group of workers. Ever since Aristotle “discovered” the chick embryo as the ideal, object for embryological studies, the embryos have been described in terms of the length of time of incubation, and this arbitrary method is still in general use, except for the first three days of incubation during which more detailed characteristics such as the numbers of somites are applied. The shortcomings of a classification based on chronological age are obvious to every worker in this field, for enormous variations may occur in embryos even though all eggs in a setting are plmaced in the incubator at the same time. Many factors are responsible for the lack of correlation between chronological and structural age. Among these are : genetic differences in the rate of development of different breccls (eg., the embryo of the White Leghorn breed develops more 49
12,079 citations
TL;DR: A vertebrate gene related to the Drosophila segment polarity gene hedgehog, which is expressed specifically in the ZPA and in other regions of the embryo, that is capable of polarizing limbs in grafting experiments is isolated.
2,368 citations
TL;DR: It is reported that chick limb buds contain endogenous RA and it is shown that RA, but not its biosynthetic precursor retinol, forms a concentration gradient across the limb anlage with a high-point in the posterior domain of the limb bud, the part that also contains the ZPA.
Abstract: All-trans-retinoic acid (RA) induces striking digit pattern duplications when locally applied to the developing chick limb bud1–4. Instead of the normal digit pattern (234) a mirror-symmetrical 432234 pattern can be specified. Hence, RA closely mimics posterior limb bud tissue (the zone of polarizing activity, ZPA) that causes very similar duplications when grafted to an anterior site of a host limb bud5. This resemblance raises an intriguing possibility: that RA is related to the as yet unidentified inducer substance thought to be released by the ZPA1,6. Here we report that chick limb buds contain endogenous RA and we show that RA, but not its biosynthetic precursor retinol, forms a concentration gradient across the limb anlage with a high-point in the posterior domain of the limb bud, the part that also contains the ZPA.
929 citations
TL;DR: Drosophila limbs are subdivided into anterior and posterior compartments which derive from adjacent cell populations founded early in development, and evidence is provided that posterior cells organize growth and cell patterning in both compartments by secreting hedgehog protein.
Abstract: Drosophila limbs are subdivided into anterior and posterior compartments which derive from adjacent cell populations founded early in development. Evidence is now provided that posterior cells organize growth and cell patterning in both compartments by secreting hedgehog protein and that hedgehog protein acts indirectly by inducing neighbouring anterior cells to secrete decapentaplegic or wingless protein.
863 citations
TL;DR: The authors' results indicate that Sonic hedgehog initiates expression of secondary signaling molecules, including Bmp-2 in the mesoderm and Fgf-4 in the ectoderm, which provide a basis for understanding the integration of growth and patterning in the developing limb.
838 citations