Ectodermal FGFs Induce Perinodular Inhibition of Limb Chondrogenesis in Vitro and in Vivo via FGF Receptor 2

Abstract

The formation of cartilage elements in the developing vertebrate limb, where they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm formed continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produced one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but not a mesodermal FGF (FGF7), substituted for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, rendered FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides blocked appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis and, when introduced into the limb buds of developing embryos, led to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements.

Comprehensive analysis of fibroblast growth factor receptor expression patterns during chick forelimb development

“…Our observation of FgfR2 expression surrounding cartilage elements during limb development is in agreement with the suggestion that FgfR2 activation elicits a lateral inhibition of chondrogenesis that limits the expansion of developing skeletal elements (Moftah et al 2002). Finally, although mouse homozygous for targeted FgfR4 mutation was normal, double homozygous disruption for both FgfR3 and 4 showed defects in long bone growth indicating that FgfR4 could also be a positive regulator of long bone growth (Weinstein et al 1998;Lazarus et al 2007).…”

“…Activating FgfR3 in mice can mimic human dwarfism ) and lack of FgfR3 causes skeletal overgrowth Deng et al 1996), indicating that FgfR3 acts as a negative regulator of bone development. The downstream pathways responsible for the negative regulation are the MAP kinase pathway that inhibits chondrocyte differentiation and the Stat1 pathway that inhibits chondrocyte proliferation (Murakami et al 2004).Our observation of FgfR2 expression surrounding cartilage elements during limb development is in agreement with the suggestion that FgfR2 activation elicits a lateral inhibition of chondrogenesis that limits the expansion of developing skeletal elements (Moftah et al 2002). Finally, although mouse homozygous for targeted FgfR4 mutation was normal, double homozygous disruption for both FgfR3 and 4 showed defects in long bone growth indicating that FgfR4 could also be a positive regulator of long bone growth (Weinstein et al 1998;Lazarus et al 2007).…”

Mechanisms of digit formation: Human malformation syndromes tell the story

“…Signaling by both these factors inhibit differentiation of e.g. cartilage (Rudnicki and Brown, 1997;Moftah et al, 2002) and account for the maintenance of naïve mesenchymal progenitors, however in regulating cellular differentiation both factors have distinct functions (reviewed in Yang, 2009). A failure in AER signaling leading to a collapse in the progenitor cell pool causes severe limb malformations such as the group of ectrodactyly syndromes also called split-hand/foot malformations (reviewed e.g., in Duijf et al, 2003).…”

Before programs: The physical origination of multicellular forms

“…Since then evidence has accumulated for a role for this class of mechanism in vertebrate axis formation (Meinhardt, 2001), formation of pigment stripes on the skin of fish (Kondo and Asai, 1995), formation of feather patterns in avian skin (Jiang et al, 1999), breaking of left-right symmetry in the vertebrate embryo (Solnica-Krezel, 2003) and generation of the skeletal pattern in the vertebrate limb (Hentschel et al, 2004, Miura and Shiota, 2000a, Miura and Shiota, 2000b, Moftah et al, 2002. The seven stripes of Even-skipped in the Drosophila embryo have the appearance of a reaction-diffusion pattern but are actually generated in a more complex fashion (Akam, 1989, Clyde et al, 2003, Small et al, 1991.…”