Abstract: Four and a half LIM domain 2 (FHL2) is a multifunctional scaffolding protein of well?known function
regulating cell signalling cascades and gene transcription in cancer tissues. However, its function
in embryonic systems is poorly characterized. Here, we show that Fhl2 is involved in the
differentiation of connective tissues of developing limb autopod. We show that Fhl2 exhibits spatially
restricted and temporally dynamic expression around the tendons of developing digits,
interphalangeal joint capsules, and fibrous peridigital tissue. Immunolabelling analysis of the skeletal
progenitors identified a predominant, but not exclusive, cytoplasmic distribution of FHL2
being associated with focal adhesions and actin cytoskeleton. In the course of chondrogenic differentiation
of cultures of limb skeletal progenitors, the expression of Fhl2 is down?regulated.
Furthermore, cultures of skeletal progenitors overexpressing Fhl2 take on a predominant
fibrogenic appearance. Both gain?of?function and loss?of?function experiments in the micromass
culture assays revealed a positive transcriptional influence of Fhl2 in the expression of fibrogenic
markers including Scleraxis, Tenomodulin, Tenascin C, ?ig?h3, and Tgif1. We further show that the
expression of Fhl2 is positively regulated by profibrogenic signals including Tgf?2, all?transretinoic
acid, and canonical Wnt signalling molecules and negatively regulated by
prochondrogenic factors of the bone morphogenetic protein family. Expression of Fhl2 is also regulated
negatively in immobilized limbs, but this influence appears to be mediated by other connective
tissue markers, such as Tgf?s and Scleraxis.
