Leg bud mesoderm retains morphogenetic potential to express limb‐like characteristics (“limbness”) in collagen gel culture
Authors: Isokawa Keitaro , Krug Edward L. , Fallon John F. , Markwald Roger R. 
Published: February 2005

Recent in situ hybridization studies have correlated expression of potential regulatory genes with pattern formation in limb bud mesoderm (Tabin: Cell 66:199–217, 1991); however, the mechanism(s) controlling their expression in mesoderm and their relevance to the establishment of a limb morphogenetic pattern remain unknown. One likely candidate for regulating patterning events in limb mesoderm is the apical ectodermal ridge, as its removal in ovo results in a graded truncation of limb skeletal elements in the proximal‐distal axis dependent upon the time of excision (Rowe and Fallon: J Embryol Exp Morph 68:1–7, 1982). In the present study, we investigate whether the hypothetical imprint of ridge ectoderm is retained in cultured mesoderm. Specifically, we sought to determine if a subpopulation of limb mesoderm that forms in collagen gel culture (Markwald et al: Anat Rec 226:91–107, 1990), retains any expression of “limbness” in the absence of limb ectoderm as characterized by the formation of a predictable number and distribution of limb‐like chondrogenic elements in comparison to the temporal and spatial relationships of the in situ proximal, hindlimb skeletal structures. Accordingly, explants of undissociated mesoderm from stage 18–22 chicken leg buds were cultured without ectoderm on collagen gel lattices and the central subpopulation of mesoderm was examined morphologically. We show that this central subset of mesoderm will form chondrogenic cells which were not expressed uniformly throughout the subset, but rather distinct nodules or elements of cartilage were elaborated. Moreover, the number of elements expressed by the central subset increased with the age of the mesoderm at the time of explantation; spatially and temporally, the sequence of elements that formed always proceeded from the proximal, anterior margin of the subset to its distal, posterior border. The shapes of the initial elements (designated I and II) resembled the forms of in situ proximal skeletal structures (girdle and femur‐like), whereas more distal elements (III‐V) were often fused and without structural similarity to in situ skeletal structures. When cultures were established from the posterior mesoderm of stage 19/20 or 21 mesoblasts, the frequency of element I formation was reduced approximately one‐half, whereas formation of more distal elements was unaffected. Conversely, element formation from the central subset established from isolated anterior mesoderm was virtually identical to intact mesoblasts, indicating a capacity to regulate for the loss of mesoderm as occurs in situ (Hampé: Archs Anat Microsc Morph Exp 48:345–378, 1959).

We interpret these findings to mean that limb mesoderm which forms the central subset is not merely competent to express in the absence of continuous ectodermal co‐culture, a chondrogenic phenotype, but also retains intrinsic, limblike morphogenetic potential to form, regulate, and, to a variable extent, shape a reproducibly specific number of cartilage elements. Because the number of chondrogenic elements that formed in culture appeared stage‐specific, a role for the apical ridge in specifying chondrogenic elements prior to its removal is supported. Indeed, our temporal findings correlate closely with the predictable truncation of leg skeletal structures following removal of the apical ridge in ovo, indicating that the endogenous ectodermal influence upon limb mesodermal morphogenesis can be assayed in vitro without the requirement of their continuous co‐culture. © 1992 Wiley‐Liss, Inc.