Fibroblast Growth Factor 4 Directs Gap Junction Expression in the Mesenchyme of the Vertebrate Limb

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell–cell communication and polarizing signaling are intimately connected. Address all correspondence to Anne Warner, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT UK. Tel. : 171-380-7279. Fax: 171-419-3014. E-mail: a.warner@ucl.ac. uk 1. Abbreviations used in this paper : AER, apical ectodermal ridge; Cx, connexin; FGF, fibroblast growth factor; HH, Hamilton Hamburger. P atterning of the skeleton in the vertebrate limb involves a number of cellular interactions. Antero– posterior patterning is controlled by the polarizing region (Saunders and Gasseling, 1968), a group of posterior mesenchyme cells near the tip of the limb bud that induce pattern duplication when transplanted to the anterior mesenchyme of a host limb bud. The signaling process is highly conserved because polarizing region cells can reprogram anterior mesenchyme across vertebrate species (e.g., Tickle et al., 1976). The tip of the limb bud mesenchyme is rimmed by the apical ectodermal ridge (AER). 1 When the ridge is removed, outgrowth and pattern formation within the limb bud ceases (Saunders, 1948; Summerbell, 1974). Growth factor signaling plays a central role in limb bud patterning. Posterior apical ridge cells express fibroblast growth factor 4 (FGF4) transcripts, and FGF4 can maintain polarizing activity in the absence of the ridge (Niswander et al., 1993; Vogel and Tickle, 1993; Fallon et al., 1994). Cells in the polarizing region express sonic hedgehog gene transcripts, which are associated with polarizing activity (Riddle et al., 1993; Laufer et al., 1994). FGF4 drives Shh expression in the polarizing region (Laufer et al., 1994; Niswander et al., 1994); when the apical ridge is removed, Shh expression is reduced. Growth factor signals in the developing limb may be linked to cell–cell interactions through gap junctions, which have been implicated in mediating cell patterning in the limb bud. Allen, Tickle, and Warner (1990) used antibodies to gap junction protein to interfere with communication through gap junctions; they showed that when communication between polarizing cells and anterior mesenchyme cells was prevented, duplication of the digits was substantially reduced. Green et al. (1994) noted a reduction in gap junction labeling at the tip of the limb bud when the AER was removed. We need to understand the cellular mechanisms that contribute to limb bud signaling. This requires unraveling of both the hierarchy of the many signals now recognized to operate in the developing limb bud and the way in which they interact with each other. In this paper, we begin such a mechanistic analysis by testing the hypothesis that FGF4 controls the expression of gap junctions and functional communication in the mesenchyme. on July 7, 2017 jcb.rress. org D ow nladed fom