A regulatory network of two galectins mediates the earliest steps of avian limb skeletal morphogenesis

Abstract

BackgroundThe skeletal elements of vertebrate embryonic limbs are prefigured by rod- and spot-like condensations of precartilage mesenchymal cells. The formation of these condensations depends on cell-matrix and cell-cell interactions, but how they are initiated and patterned is as yet unresolved.ResultsHere we provide evidence that galectins, β-galactoside-binding lectins with β-sandwich folding, play fundamental roles in these processes. We show that among the five chicken galectin (CG) genes, two, CG-1A, and CG-8, are markedly elevated in expression at prospective sites of condensation in vitro and in vivo, with their protein products appearing earlier in development than any previously described marker. The two molecules enhance one another's gene expression but have opposite effects on condensation formation and cartilage development in vivo and in vitro: CG-1A, a non-covalent homodimer, promotes this process, while the tandem-repeat-type CG-8 antagonizes it. Correspondingly, knockdown of CG-1A inhibits the formation of skeletal elements while knockdown of CG-8 enhances it. The apparent paradox of mutual activation at the gene expression level coupled with antagonistic roles in skeletogenesis is resolved by analysis of the direct effect of the proteins on precartilage cells. Specifically, CG-1A causes their aggregation, whereas CG-8, which has no adhesive function of its own, blocks this effect. The developmental appearance and regulation of the unknown cell surface moieties ("ligands") to which CG-1A and CG-8 bind were indicative of specific cognate- and cross-regulatory interactions.ConclusionOur findings indicate that CG-1A and CG-8 constitute a multiscale network that is a major mediator, earlier-acting than any previously described, of the formation and patterning of precartilage mesenchymal condensations in the developing limb. This network functions autonomously of limb bud signaling centers or other limb bud positional cues.

‘Biogeneric’ developmental processes: drivers of major transitions in animal evolution

“…In the mouse, the spatio-temporal expression of the cartilage master transcription factor Sox9 in vitro and in vivo depends on its dynamical interaction with the two toolkit morphogens Bmp2 and Wnt (the 'BSW network') [82]. In the chicken, galectin-1a and galectin-8, two members of the galectin family of carbohydrate-binding proteins that may have also mediated some forms of triploblasty (see §3), form a multiscale skeletal pattern formation network [83,84]. The extent to which the various tetrapod classes share these two reaction -diffusion patterning mechanisms is as yet unknown.…”

“…In the sarcopterygians, moreover, the galectin-8 gene acquired new cis-regulatory motifs with binding sites for what were later limb-associated transcription factors [86]. Although these data do not permit strong inferences regarding the fin-to-limb transition, the following scenario is consistent with the available information: (i) a galectin-1 -galectin-8 regulatory network in cartilaginous fish and their ancestors are/were capable of generating the repeated parallel cartilage elements and plates in the fins of those species; (ii) the common ancestor of those fish with the ray-finned fish and sarcopterygians diverged by protein-and gene-regulatory element evolution to produce a galectin-1-galectin-8 network in the former (ray-finned) group that generates fin cartilaginous elements less regularly arrayed and less consistent from species to species than in the cartilaginous fish, and in the latter (sarcopterygian) group, the propensity to produce small numbers of parallel elements in a highly controlled fashion [83,84,86].…”

From Skeletal Development to Tissue Engineering: Lessons from the Micromass Assay

“…3) and quantifying the numbers and spacing of condensations in the culture as a measure of patterning. 79,80 For example, a developing tissue is subjected to geometric boundary conditions by neighboring tissues.…”

Making waves: the rise and fall and rise of quantitative developmental biology

“…He explained how the condensation of mesenchymal cells, which is one of the earliest steps in the formation of cartilage and bone in the developing limb, can be recapitulated in vitro as a uniform layer of embryonic cells selforganises into patterned condensations that are first visible as patterned differences in the concentration of extracellular matrix components and galectins, without the influence of external signals (Bhat et al, 2011). This suggests a radical revision of the prevailing model, such that the conserved morphogens and cell state regulators, which have been the focus of so much attention in the limb field in the past 30 years, function to refine and sharpen underlying patterns generated by self-organising processes similar to those envisioned by Turing.…”