Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod, and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of Retinoic Acid (RA) and Fibroblast Growth Factor (FGF) signaling define the expression domains of putative segment markers, Meis1, Hoxa11, and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces novel interzone-like structures and its loss-of-function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and consequently, formation of repositioned joints. Our data suggest that during early limb bud development, Meis1 and Hoxa11 expression domains are overlapping while Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and Barx1 expression domain becomes congruent with the border of these two putative segment markers.