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.
Highlights
The skeletal elements of vertebrate embryonic limbs are prefigured by rod- and spot-like condensations of precartilage mesenchymal cells
Our findings suggest that morphogenesis of individual condensations can be brought about by the action of chicken galectin (CG)-1A, but that condensation patterning depends on a multiscale local-autoactivation-lateral-inhibition network comprising CG-1A and CG-8
We first measured the relative levels of gene expression of all CGs in high density monolayer “micromass” cultures of freshly dissociated uncondensed leg precartilage mesenchymal cells by quantitative realtime PCR of reverse-transcribed mRNA
Summary
The 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. A key cellular event in the early developing limb is the formation of condensations - highdensity spot- or rod-shaped aggregates of precartilage mesenchymal cells [2,3,4]. These aggregates arise from and, once formed, are surrounded by, uncondensed mesenchymal cells embedded in a loose extracellular. Cell clusters that anticipate fully formed condensations (“protocondensations”) are seen in vitro as early as 17 h [13], at least 12 h before fibronectin is detectable
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