Regulation of Stem Cell Proliferation and Cell Fate Specification by Wingless/Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries

Intestinal stem cell (ISC) self-renewal and proliferation are directed by Wnt/β-catenin signaling in mammals, whereas aberrant Wnt pathway activation in ISCs triggers the development of human colorectal carcinoma. Herein, we have utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. We provide evidence that the Wg signaling pathway, activation of which peaks at each of the major compartment boundaries of the adult intestine, has essential functions. Wg pathway activation in the intestinal epithelium is required not only to specify cell fate near compartment boundaries during development, but also to control ISC proliferation within compartments during homeostasis. Further, in contrast with the previous focus on Wg pathway activation within ISCs, we demonstrate that the primary mechanism by which Wg signaling regulates ISC proliferation during homeostasis is non-autonomous. Activation of the Wg pathway in absorptive enterocytes is required to suppress JAK-STAT signaling in neighboring ISCs, and thereby their proliferation. We conclude that Wg signaling gradients have essential roles during homeostasis and development of the adult intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries

Erect Wing Facilitates Context-Dependent Wnt/Wingless Signaling by Recruiting the Cell-Specific Armadillo-TCF Adaptor Earthbound to Chromatin

During metazoan development, the Wnt/Wingless signal transduction pathway is activated repetitively to direct cell proliferation, fate specification, differentiation and apoptosis. Distinct outcomes are elicited by Wnt stimulation in different cellular contexts; however, mechanisms that confer context specificity to Wnt signaling responses remain largely unknown. Starting with an unbiased forward genetic screen in Drosophila, we recently uncovered a novel mechanism by which the cell-specific co-factor Earthbound 1 (Ebd1), and its human homolog jerky, promote interaction between the Wnt pathway transcriptional co-activators B-catenin/Armadillo and TCF to facilitate context-dependent Wnt signaling responses. Here, through the same genetic screen, we find an unanticipated requirement for Erect Wing (Ewg), the fly homolog of the human sequence-specific DNA-binding transcriptional activator nuclear respiratory factor 1 (NRF1), in promoting contextual regulation of Wingless signaling. Ewg and Ebd1 functionally interact with the Armadillo-TCF complex and mediate the same context-dependent Wingless signaling responses. In addition, Ewg and Ebd1 have similar cell-specific expression profiles, bind to each other directly and also associate with chromatin at shared genomic sites. Furthermore, recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. Our findings provide in vivo evidence that recruitment of a cell-specific co-factor complex to specific chromatin sites, coupled with its ability to facilitate Armadillo-TCF interaction and transcriptional activity, promotes contextual regulation of Wnt/Wingless signaling responses

Adenomatous Polyposis Coli is Present Near the Minimal Level Required for Accurate Graded Responses to the Wingless Morphogen

The mechanisms by which the Wingless (Wg) morphogen modulates the activity of the transcriptional activator Armadillo (Arm) to elicit precise, concentration-dependent cellular responses remain uncertain. Arm is targeted for proteolysis by the Axin/Adenomatous polyposis coli (Apc1 and Apc2)/Zeste-white 3 destruction complex, and Wg-dependent inactivation of destruction complex activity is crucial to trigger Arm signaling. In the prevailing model for Wg transduction, only Axin levels limit destruction complex activity, whereas Apc is present in vast excess. To test this model, we reduced Apc activity to different degrees, and analyzed the effects on three concentration-dependent responses to Arm signaling that specify distinct retinal photoreceptor fates. We find that both Apc1 and Apc2 negatively regulate Arm activity in photoreceptors, but that the relative contribution of Apc1 is much greater than that of Apc2. Unexpectedly, a less than twofold reduction in total Apc activity, achieved by loss of Apc2, decreases the effective threshold at which Wg elicits a cellular response, thereby resulting in ectopic responses that are spatially restricted to regions with low Wg concentration. We conclude that Apc activity is not present in vast excess, but instead is near the minimal level required for accurate graded responses to the Wg morphogen

The ADP-Ribose Polymerase Tankyrase Regulates Adult Intestinal Stem Cell Proliferation During Homeostasis in Drosophila

Wnt/β-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the β-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease

GATA- and Smad1-Dependent Enhancers in the Smad7 Gene Differentially Interpret Bone Morphogenetic Protein Concentrations

Smad7, an inhibitor of transforming growth factor beta superfamily signaling, is induced by bone morphogenetic protein (BMP) in an inhibitory feedback loop. Here, we identify multiple BMP response elements (BREs) in the Smad7 gene and demonstrate that they function differentially to interpret BMP signals in a cell type-specific manner. Two BREs (BRE-1 and -2) reside in the promoter region. One of these contains several conserved Smad1 and Smad4 binding sites that cooperate to mediate BMP-dependent induction, most likely in the absence of DNA binding partners. The third BRE (I-BRE) resides in the first intron and contains GATA factor binding sites. GATA-1, -5, or -6 is required for strong activation of I-BRE, and we show that they assemble with Smad1 on the I-BRE in living cells. Activation of the I-BRE is mediated by a specific region in GATA-5 and -6 but does not require direct physical interaction with Smad1. Comparison of I-BRE to BRE-1 showed that I-BRE is more responsive to low BMP concentrations. Moreover, analysis by chromatin immunoprecipitation experiments demonstrates that the endogenous I-BRE is occupied more robustly by endogenous Smad1 than is BRE-1. This correlates with regulation of the Smad7 gene, which is induced at lower BMP concentrations in GATA-expressing cell lines compared to non-GATA-expressing lines. These data thus define how cooperative and noncooperative Smad-dependent transcriptional regulation can function to interpret different BMP concentrations

Wingless/Wnt Signaling in Intestinal Development, Homeostasis, Regeneration and Tumorigenesis: A Drosophila Perspective

In mammals, the Wnt/β-catenin signal transduction pathway regulates intestinal stem cell maintenance and proliferation, whereas Wnt pathway hyperactivation, resulting primarily from the inactivation of the tumor suppressor Adenomatous polyposis coli (APC), triggers the development of the vast majority of colorectal cancers. The Drosophila adult gut has recently emerged as a powerful model to elucidate the mechanisms by which Wingless/Wnt signaling regulates intestinal development, homeostasis, regeneration, and tumorigenesis. Herein, we review recent insights on the roles of Wnt signaling in Drosophila intestinal physiology and pathology

Essential long-range action of Wingless/Wnt in adult intestinal compartmentalization.

Signal transduction activated by Wingless/Wnt ligands directs cell proliferation and fate specification in metazoans, and its overactivation underlies the development of the vast majority of colorectal cancers. In the conventional model, the secretion and movement of Wingless to cells distant from its source of synthesis are essential for long-range signaling in tissue patterning. However, this model was upended recently by an unanticipated finding: replacement of wild-type Drosophila Wingless with a membrane-tethered form produced viable adults with largely normal external morphology, which suggested that Wingless secretion and movement are dispensable for tissue patterning. Herein, we tested this foundational principle in the adult intestine, where Wingless signaling gradients coincide with all major boundaries between compartments. We find that the critical roles of Wingless during adult intestinal development, which include regulation of target gene activation, boundary formation, stem cell proliferation, epithelial cell fate specification, muscle differentiation, gut folding, and signaling crosstalk with the Decapentaplegic pathway, are all disrupted by Wingless tethering. These findings provide new evidence that supports the requirement for the direct, long-range action of Wingless in tissue patterning, with relevance for animal development, tissue homeostasis and Wnt-driven disease

Wg pathway is activated primarily in ECs, but not ISCs, during adult homeostasis.

<p><b>(A-B”‘)</b><i>Nkd-lacZ</i> and <i>fz3-RFP</i> are expressed in partially distinct cell types. DAPI labels the nuclei and indicates their ploidy. Small Escargot^pos Prospero^neg diploid cells are progenitor cells (orange arrow), big polyploid cells are enterocytes (white arrow), small Escargot^neg Prospero^pos diploid cells are enteroendocrine cells (yellow arrow and arrowhead). <b>(A-A”‘)</b> <i>Nkd-lacZ</i> is primarily expressed inside enterocytes (white arrow) and a subpopulation of enteroendocrine cells (yellow arrow). Scale bar: 10μm. <b>(B-B”‘)</b> <i>Fz3-RFP</i> is expressed in both enterocytes (white arrow) and progenitors (orange arrow). Its expression is mostly absent in the enteroendocrine cells (yellow arrow). Scale bar: 10μm. <b>(C-G”‘)</b> Wg signaling is primarily transduced in enterocytes of the adult gut. MARCM clones of wild-type controls and Wg pathway mutants were induced during larval development and examined soon after eclosion. GFP marks the clones. <i>nkd-lacZ</i> or <i>fz3-RFP</i> represents Wg pathway activity. Orange arrows indicate progenitor cells while white arrows point to enterocytes. <b>(C-D”‘)</b> <i>Nkd-lacZ</i> expression is not affected in wild-type clones <b>(C-C”‘)</b> but specifically lost within enterocytes of <i>arr</i> clones at intestinal compartment boundaries <b>(D-D”‘)</b>. <b>(E-F”‘)</b> <i>Fz3-RFP</i> expression is not affected in wild-type clones <b>(E-E”‘)</b> but specifically lost within enterocytes of <i>dsh</i> clones at intestinal compartment boundaries <b>(F-F”‘)</b>. The mutant enterocytes retain normal cell morphology, nuclear morphology and cell-cell junctions, indicating that the loss of Wg pathway activation in this cell type is not due to cell death. Note that <i>Fz3-RFP</i> expression in progenitors is not dependent on Wg signaling. <b>(G-G”‘)</b> At the unique R5-HPZ boundary, Wg pathway is activated in both progenitors and enterocytes. Scale bar: 10μm.</p

All gut cell types are capable of responding to Wg exposure.

<p><b>(A-B”)</b> Compared with the wild-type anterior midgut, Wg signaling is ectopically induced in the <i>Apc1</i>^<i>Q8</i> mutant. <i>Fz3-RFP</i> serves as a reporter for Wg pathway activity. DAPI labels the gut cell nuclei. Anterior to the left. Scale bar: 100μm. <b>(C-C”‘)</b> GFP-marked <i>Apc2 Apc1</i> mutant MARCM clones exhibit aberrantly high <i>fz3-RFP</i> signals in all gut cell types at compartment boundaries, including progenitors (orange arrow), enterocytes (white arrow) and enteroendocrine cells (yellow arrow), as indicated by Arm and Prospero staining. Of note, despite the high-level Wg pathway activation that is already present at compartment boundaries, <i>Apc2 Apc1</i> double mutant cells display even higher levels of activation at all boundaries. Scale bar: 10μm. <b>(D and D’)</b> Wg originating from the visceral muscle is sufficient to activate signaling in the intestinal epithelium. <i>Wg</i> is expressed throughout the muscle using a temperature sensitive <i>dMef2-Gal4</i> driver <b>(D’)</b> alongside with wild-type control <b>(D)</b>. Flies were shifted to the permissive temperature for <i>wg</i> expression at eclosion and reared at this temperature for one week prior to analysis. Dramatic induction of <i>fz3-RFP</i> is detected in the epithelium, most pronouncedly in the anterior midgut. Scale bar: 100μm. <b>(E-E”‘)</b> Overexpression of <i>wg</i> in muscle during adulthood induces ectopic Wg pathway activation in all gut cell types, including progenitors (orange arrow), enterocytes (white arrow) and enteroendocrine cells (yellow arrow), as indicated by Arm and Prospero staining. Scale bar: 10μm.</p