Morphogenesis of precursor subpopulations of chicken limb mesenchyme in three dimensional collagen gel culture
Authors: Markwald Roger R. , Bolender David L. , Krug Edward L. , Lepera Ross 
Published: February 2005

Although homogeneous in appearance, several lines of evidence suggest early (stage 17—19) limb mesenchymal cells are committed to particular cell lineages, e.g., myogenic or chondrogenic. However, subsequent expression of cell or tissue phenotype in the developing limb does not occur in a randomized process but rather in a spatially specific pattern. The potential regulatory mechanisms controlling the “patterned” expression of tissue phenotype in the limb have not been resolved. The purpose of this study was to determine if, prior to the formation of an apical ectodermal ridge, nondissociated limb mesenchyme has inherent morphogenetic potential to form nonrandomized patterns of tissue organization. The hypotheses to be tested were that, if provided a spatially permissive culture environment, (1) mesenchymal cells committed to a particular lineage would segregate into precursor (sub)populations prior to overt expression of phenotype and (2) the ultimate expression of a tissue phenotype may be regulated, in part, by histogenic interactions between the precursor cell groups. For these studies, mesoblasts (intact mesenchyme minus ectoderm) from stage 17—19 hindlimb buds were explanted intact to the surface of a 1—3 mm thick hydrated lattice of repolymerized type I collagen and incubated for 2—11 days. Examination of cultures at variable intervals revealed three distinct temporal sequences (periods) which were arbitrarily termed early morphogenesis (0—3 days), cytodifferentiation (3—5.5 days), and primitive tissue formation (5.5—11 days) based on similarities to in situ limb development. By the end of the first period, the mesenchymal cells had sorted into three distinct precursor populations: (1) an epithelial‐like outgrowth of premyogenic and prefibrogenic cells at the surface of the gel lattice (termed the “surface subset”) which circumscribed, (2) a centrally positioned prechondrogenic condensate (“central subset”), and overlaid (3) a dispersed, population of free cells that invaded the collagen lattice (“seeded subset”). Subsequent cytodifferentiation led to the appearance of multinucleated myotubes within the surface subset and chondrification of the central subset. Cells of the seeded subset remained dispersed within the collagen lattice. Primitive histogenic events were initiated during the final period of development including (1) at sites where surface cells established boundaries with the central subset, collectives or “bundles” of variable sized myotubes were formed which became partially ensheathed by the attenuated processes of fibroblastlike cells; and (2) a secondary site of chondrogenic activity was initiated within the gel lattice at the boundary between the central and seeded cell populations. Transformation of seeded fibroblasts into chondroblasts accompanied expansion of the secondary chondrogenic element within the gel lattice. Remaining cells of the seeded population which did not become incorporated (transformed) into the secondary site of chondrogenesis persisted as a dense stratified network of fibrous connective tissue. Removing the central subset after 2 days of culture inhibited the transformation of seeded fibroblasts into chondrogenic cells.

These findings indicate that intact limb mesenchyme prior to the formation of a mature apical ridge and in the absence of dorsal/ventral ectoderm has the endogenous potential to segregate, cytodifferentiate, and interact to intiate tissue formation similar to that seen in the intact limb.