Jennifer A. Kennell

Wnt Signaling Inhibits Adipogenesis through β-Catenin-dependent and -independent Mechanisms

Wnt signaling has been reported to block apoptosis and regulate differentiation of mesenchymal progenitors through inhibition of glycogen synthase kinase 3 and stabilization of β-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 β-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 β-catenin. An important mediator of the β-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 β-catenin-dependent and β-catenin-independent mechanisms.

The microRNA miR-8 is a conserved negative regulator of Wnt signaling

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.

Identification of members of the Wnt signaling pathway in the embryonic pituitary gland.

Abstract.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 Prop1df/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,β-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.

The microRNA miR-8 is a positive regulator of pigmentation and eclosion in Drosophila.

Background: 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. In this study we demonstrate that miR‐8 is also required for proper spatial patterning of pigment on the adult abdominal cuticle in females but not males. Results: 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. The decrease in pigmentation in miR‐8 mutants was more pronounced in flies grown at higher temperatures. We also found that loss of miR‐8 dramatically affected the ability to eclose at higher temperatures. Conclusion: Loss of miR‐8 increased the sensitivity of Drosophila to higher temperatures for both pigmentation patterning and the ability to eclose. Together, these data suggest that miR‐8 acts as a buffer to stabilize gene expression patterns in the midst of environmental variation. Developmental Dynamics 241:161–168, 2012.

APC and β-Catenin Degradation

The adenomatous polyposis coli (APC) gene encodes a tumor suppressor that is mutated in familial adenomatous polyposis (FAP) colon cancer as well as the large majority of sporadic colorectal cancers (reviewed in ref 1, Chapter by Sansom, Kwong and Dove). Most of the mutations in APC associated with increased incidence of cancer generate a truncated form of APC.2, 3 While many studies have implicated the APC protein in the regulation of the cytoskeleton (reviewed in ref 4, Chapter by Morrison, Caldwell and Kaplan et al. ), there is also compelling evidence that APC is a key component of Wnt/β-catenin signaling (reviewed in refs. 3, 5). Consistent with this, loss of APC1 and APC2 genes in Drosophila result in elevated Wnt/β-catenin signaling.6–8 Mutations of APC in mice or zebrafish also increase the susceptibility of these animals to developing colorectal cancer with elevation of Wnt/β-catenin signaling in the tumors.9,10 In this chapter, we will focus on the role of APC as a negative regulator of the Wnt/β-catenin pathway.

Regulation of cuticle pigmentation in drosophila by the nutrient sensing insulin and TOR signaling pathways

Background: Insect pigmentation is a phenotypically plastic trait that plays a role in thermoregulation, desiccation tolerance, mimicry, and sexual selection. The extent and pattern of pigmentation of the abdomen and thorax in Drosophila melanogaster is affected by environmental factors such a growth temperature and access to the substrates necessary for melanin biosynthesis. This study aimed to determine the effect of nutritional status during development on adult pigmentation and test whether nutrient sensing through the Insulin/IGF and target of rapamycin (TOR) pathways regulates the melanization of adult cuticle in Drosophila. Results: Flies reared on low quality food exhibit decreased pigmentation, which can be phenocopied by inhibiting expression of the Insulin receptor (InR) throughout the entire fly during mid to late pupation. The loss of Insulin signaling through PI3K/Akt and FOXO in the epidermis underlying the developing adult cuticle causes a similar decrease in adult pigmentation, suggesting that Insulin signaling acts in a cell autonomous manner to regulate cuticle melanization. In addition, TOR signaling increases pigmentation in a cell autonomous manner, most likely through increased S6K activity. Conclusion: These results suggest that nutrient sensing through the Insulin/IGF and TOR pathways couples cuticle pigmentation of both male and female Drosophila with their nutritional status during metamorphosis. Developmental Dynamics 243:393–401, 2014.

MiR-8 modulates cytoskeletal regulators to influence cell survival and epithelial organization in Drosophila wings

The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling - pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate.