Wnt signaling has been reported to block apoptosis and regulate differentiation of mesenchymal progenitors through inhibition of glycogen synthase kinase 3 and stabilization of ␤-catenin. The effects of Wnt in preadipocytes may be mediated through Frizzled (Fz) 1 and/or Fz2 as these Wnt receptors are expressed in preadipocytes and their expression declines upon induction of differentiation. We ectopically expressed constitutively active chimeras between Wnt8 and Fz1 or Fz2 in preadipocytes and mesenchymal precursor cells. Our results indicated that activated Fz1 increases stability of ␤-catenin, inhibits apoptosis, induces osteoblastogenesis, and inhibits adipogenesis. Although activated Fz2 does not influence apoptosis or osteoblastogenesis, it inhibits adipogenesis through a mechanism independent of ␤-catenin. An important mediator of the ␤-catenin-independent pathway appears to be calcineurin because inhibitors of this serine/threonine phosphatase partially rescue the block to adipogenesis caused by Wnt3a or activated Fz2. These data supported a model in which Wnt signaling inhibits adipogenesis through both ␤-catenin-dependent and ␤-catenin-independent mechanisms.

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.adipogenesis ͉ TCF ͉ Wntless

Ectopic expression of Wnt-1 in 3T3-L1 preadipocytes stabilizes ␤-catenin, activates TCF-dependent gene transcription, and blocks adipogenesis. Here we report that upon serum withdrawal, Wnt-1 causes 3T3-L1 cells to resist apoptosis through a mechanism that is partially dependent on phosphatidylinositol 3-kinase. Although activation of Wnt signaling by inhibition of GSK-3 activity or ectopic expression of dominant stable ␤-catenin blocks apoptosis, inhibition of Wnt signaling through expression of dominant negative TCF-4 increases apoptosis. Wnt-1 stimulates 3T3-L1 preadipocytes to secrete factors that increase PKB/Akt phosphorylation at levels comparable with treatment with 10% serum. With DNA microarrays, we identified several secreted antiapoptotic genes that are induced by Wnt-1, notably insulinlike growth factor I (IGF-I) and IGF-II. Consistent with IGFs mediating the antiapoptotic effects of Wnt-1 in preadipocytes, conditioned medium from Wnt-1 expressing 3T3-L1 cells was unable to promote protein kinase B phosphorylation after the addition of recombinant IG-FBP-4. Thus, we demonstrated that Wnt-1 induces expression of antiapoptotic genes in 3T3-L1 preadipocytes such as IGF-I and IGF-II, which allows these cells to resist apoptosis in response to serum deprivation.

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We have cloned T-cell factor 4N (TCF-4N

Prop1 is one of several transcription factors important for the development of the pituitary gland. Downstream targets of PROP1 and other critical pituitary transcription factors remain largely unknown. We have generated a partial expression profile of the developing pituitary gland containing over 350 transcripts, using cDNA subtractive hybridization between Prop1(df/df) and wild-type embryonic pituitary gland primordia. Numerous classes of genes including transcription factors, membrane associated molecules, and cell cycle regulators were identified in this study. Of the transcripts, 34% do not have sequence similarity to known genes, but are similar to ESTs, and 4% represent novel sequences. Pituitary gland expression of a number of clones was verified using in situ hybridization. Several members of the Wnt signaling pathway were identified in the developing pituitary gland. The frizzled2 receptor, Apc, beta-catenin, groucho, and a novel isoform of TCF4 (officially named Tcf7l2) were identified in developing pituitary libraries. Three N-terminal alternatively spliced Tcf7l2 isoforms are reported here, each of which lacks a DNA-binding domain. Functional studies indicate that these isoforms can act as endogenous inhibitors of Wnt signaling in some contexts. This is the first report of Tcf7l2 and Fzd2 expression in the developing pituitary. These molecules may be important in mediating Wnt signaling during pituitary ontogeny. We expect other transcripts from these libraries to be involved in pituitary gland development.

MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally silence gene expression by binding to target mRNAs. Previous studies have identified the miRNA miR-8 as a pleiotropic regulator of Drosophila development, controlling body size and neuronal survival by targeting multiple mRNAs. Here we demonstrate that miR-8 is required for proper spatial patterning of pigment on the adult abdominal cuticle. Female adult flies lacking miR-8 exhibit decreased pigmentation of the dorsal abdomen, with a pattern of pigmentation similar to wild type flies grown at higher temperatures. This pigmentation defect in miR-8 mutants is independent of the previously reported body size defect, and miR-8 acts directly in the developing cuticle to regulate pigmentation patterning. Loss of miR-8 increases thermal sensitivity of Drosophila for both pigmentation patterning and the ability to eclose. Together, these data suggest miR-8 acts as a buffer to stabilize gene expression patterns in the midst of environmental variation.

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