WIF and Wnts Modulate Rod Photoreceptor Differentiation in vitro

Abstract

Abstract: : Purpose:Members of the Wnt protein family bind Frizzled-family transmembrane receptors and low-density lipoprotein-related protein (LRP) co-receptors to regulate embryogenesis, directing cell polarity and cell-fate specification. Additional molecules including Wnt-Inhibitory Factor-1 (WIF-1) are thought to bind soluble Wnts and render them biologically inactive. This study was undertaken to determine the expression and potential function of a Wnt4/LRP/WIF-1 complex in the retina during retinal morphogenesis. Methods:RT-PCR, in situ hybridization, protein transfer blot analysis, and immunolocalization experiments were used to investigate expression and localization of Wnt 4 and WIF-1 in developing retina. Immunoprecipitations were used to ascertain biochemical interactions between WIF and Wnt4. Dissociated postnatal retinal cells were co-cultured with either WIF-1 or Wnt 4-producing cells, or in the presence of purified WIF or anti-WIF-1 antibodies, to investigate modulatory effects on rod photoreceptor development. Results:Wnt4 expression becomes restricted to the outer retina as photoreceptor differentiation proceeds (P5-P8). In comparison, WIF-1 did not localize to the outer retina at this age; WIF-1 is present instead at the inner retina. Later, WIF-1 expression localizes to the outer retina. The co-culture of dissociated retinal cells with either WIF-1 protein or WIF-1-producing cells inhibits rod photoreceptor production; co-culture with anti-WIF antibodies increases rod production. In contrast, Wnt 4 protein increased rod photoreceptor expression. Co-precipitation of Wnt 4 and WIF-1 from adult retinae demonstrates a direct biochemical interaction between endogenous Wnt 4 and WIF-1. Consistent with the proposed role of LRP6 as a co-receptor for Wnt activation, LRP-knockout mice reveal fewer photoreceptor cells than wildtype mice. Conclusions: Together, these data suggest that the Wnt family of morphogens, modulatory effects of WIF-1, and the co-receptor LRP6, are important regulators of mammalian photoreceptor development.

Keywords: retinal development • extracellular matrix • Muller cells