Comparative analysis of the expression patterns of <Emphasis Type="Italic">Wnts</Emphasis> during chick limb development

Wnts control a number of processes during limb development—from initiating outgrowth and controlling patterning, to regulating cell differentiation in a number of tissues. We analyzed the expression pattern of various Wnts (4, 5a, 5b, 6, 11, and 14) in whole mount in situ hybridization during chick wing development. From HH stage 26, expression of Wnt 4 is observed in the central elbow region and wrist-forming regions, and during later stages, expression is seen in the joint-forming regions of the whole limb. Wnt 5a is expressed throughout the limb mesenchyme during early limb developmental stages, and later, at HH stage 23, it becomes predominantly confined to the distal tip, leaving low expression levels proximally. At HH stage 29, expression at the distal tip is restricted to the interdigital regions, and at day 8, expression is seen in the region surrounding the phalanges. Wnt 5b expression is first observed in the AER at HH stage 20 and later in the dorsal and ventral mesenchyme surrounding the cartilage elements of the limb. Expression of Wnt 6 is observed from HH stage 17 until day 8 in the dorsal and ventral ectoderm and also in the dorsoventral limb boundaries. Expression of Wnt 11 is observed in the proximal dorsal mesenchyme of the limb from HH stage 23 onward and later in the dorsal and ventral subectodermal mesenchyme and in the regions adjacent to the digits at day 8. Weak expression of Wnt 14 is observed at the proximal mesenchyme of the limb at HH stage 23; later, it extends as a transverse strip surrounding the cartilage elements as well as in the interdigital mesenchyme.This paper is dedicated to Professor Dr. W. Zenker on the occasion of his 80th birthday.     

The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb.

In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.     

Nonuniform distribution of fibronectin during avian limb development

Using indirect immunofluorescence we have examined the distribution of the cell surface and extracellular matrix glycoprotein fibronectin at the epithelial-mesenchymal interface and in the mesenchyme of developing chick and duck wing buds. At all stages examined, in both species, staining for fibronectin is greatly enhanced in the basement membrane subjacent to the apical ectodermal ridge (AER), a site of inductive tissue interaction, relative to the epithelial basement membranes in the noninductive dorsal and ventral limb epithelial-mesenchymal interfaces. In stage 23, 25, and 28 chick limb buds, staining for fibronectin is uniform in the least mature distal mesenchyme, retained between more proximal cells undergoing precartilage condensation and lost in those regions undergoing myogenesis, and persistent in all but the most mature cartilage present at the latest stage examined. These results are consistent with a role for fibronectin in AER-induced limb outgrowth, and with a transient role for the glycoprotein in the formation of the skeletal pattern of the limb.     

Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro

Cartilage formation in the chick limb follows rapid proliferation, condensation and differentiation of limb mesenchyme. The control of these early events is poorly understood. Platelet-derived growth factor receptor alpha (PDGFR-alpha) is present throughout the mesenchyme of early chick limb buds, while its ligand, PDGF-A, is expressed in the surrounding epithelium. PDGFR-alpha is down-regulated in areas that will not give rise to cartilage and is then lost from cartilage forming areas after they begin to differentiate. PDGF-A increases chondrogenesis in micromass cultures of stage-20-24 limb buds, but not stage 25, where it inhibits chondrogenesis. Ectopic PDGF-A in the chick wing can lead to either a localized increase in cartilage formation, or an inhibition. Inhibition of PDGF signalling in the chick limb results in the loss of cartilage. These data demonstrate that PDGF-A functions to promote chondrogenesis at early stages of limb development and suggest that it inhibits chondrogenesis at later stages.     

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 to 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.     

The dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs.

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.