Bmps and BmpRs are expressed in the limb mesenchyme prior to aggregation of prechondrogenic condensations, and then, in or around condensations. BMPs

Control st 25-26

Control EtO

Injected E

Injected E7

FIG. 4. Noggin misexpression results in disappearance of the cartilage after its formation. (A) Control limbs stained with alcian blue to indicate the extent of cartilage formation prior to Noggin misexpression. (B,C) E10 wholemount, and (E,F) E7 sections of limbs stained with alcian blue. (B,E) Non-injected control limbs. (C,F) RCAS-Noggi┬╗ virus injection at stage 24 into the hindlimb zeugopod results in loss of the tibia (C) and loss of the fibula and truncation of the tibia (F). The length of the infected zeugopod is approximately one-third that of control (E, F; depicted by brackets). The overall extent of cartilage formation is represented schematically (not to scale) for control (D) and infected (G) limbs. (D-G) Anterior at left, distal at top. (E,F) Composites of 100xbright field photographs. F, femur; fi, fibula; M, metatarsals; P, phalanx; T, tibia; ta, tarsals.

are thus good candidates to direct early stages of cartilage formation. Here, we have examined their role(s) during limb chondrogenesis by misexpressing Noggin. This prevents the appearance of prechondrogenic condensations and also blocks their differentiation into chondrocytes. Moreover, once chondrocytes are present, Noggin misexpression leads to their disappearance.

Specificity of the Noggin-mediated antagonism

Activin and transforming growth factor (TGF)^2 have been shown to be potential regulators of early steps of chondrogenesis in the chick autopod, based on their expression pattern and ability to stimulate de novo chondrogenesis. Their effects are suggested to be mediated through induction of BmpRIB expression, only then allowing BMPs to perform later functions (Merino et al 1998,1999b). GDF5 may also participate in the initial stages of cartilage formation and is expressed in both zeugopod and autopod (Francis-West et al 1999). Noggin binds BMP2, BMP4 and BMP7, preventing them from interacting with their receptors, but does not bind activin or TGF^ (Zimmerman et al 1996). However, Noggin also binds GDF5, blocking its activity (Merino et al 1999a). Hence, BMP signalling aside, GDF5 signalling could have been antagonized by Noggin misexpression. Could the loss of GDF5 function account for the described phenotypes?

In the autopod, Gdf5 is expressed in presumptive joint regions and around the well-developed condensations, but not in or around early condensations (Merino et al 1999a, S. Pizette & L. Niswander, unpublished observations). Accordingly, digit condensations in the Gdf5 loss-of-function mouse mutant, Brachypodism, are formed but are thinner than normal (Gruneberg & Lee 1973, Storm et al 1994). These data suggest a restricted role for GDF5 in recruitment of cells to preexisting cartilaginous condensations of the autopod. We thus conclude that the total absence of autopodial prechondrogenic condensations in the Noggin-induced phenotype arises from loss of BMP signalling.

In the zeugopod, Gdf5 is expressed in the joint-forming area of the precartilaginous condensations (Fig. 3B). GDF5 has been implicated in joint formation as well as in proliferation of the adjacent immature chondrocytes, and chondrocyte differentiation is delayed in the autopod of the Brachypodism mutant (Storm & Kingsley 1996, 1999, Merino et al 1999a, Francis-West et al 1999). Therefore, the later effects of Noggin misexpression on chondrogenesis may occur through loss of GDF and/or BMP signalling and the specific contribution can not be inferred from other studies. Indeed, GDF5 and BMPs share the same receptors and may form heterodimers (Erlacher et al 1998, Chang & Hemmati-Brivanlou 1999), indicating that alterations of the activity of one of these factors could alter the activity of the others.

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