Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination.

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. We previously identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat's regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. We here characterize a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, we find that Dlish binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. We further show that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex.

Big roles for Fat cadherins.

To create an intricately patterned and reproducibly sized and shaped organ, many cellular processes must be tightly regulated. Cell elongation, migration, metabolism, proliferation rates, cell-cell adhesion, planar polarization and junctional contractions all must be coordinated in time and space. Remarkably, a pair of extremely large cell adhesion molecules called Fat (Ft) and Dachsous (Ds), acting largely as a ligand-receptor system, regulate, and likely coordinate, these many diverse processes. Here we describe recent exciting progress on how the Ds-Ft pathway controls these diverse processes, and highlight a few of the many questions remaining as to how these enormous cell adhesion molecules regulate development.

The palmitoyltransferase Approximated promotes growth via the Hippo pathway by palmitoylation of Fat.

The large protocadherin Fat functions to promote Hippo pathway activity in restricting tissue growth. Loss of Fat leads to accumulation of the atypical myosin Dachs at the apical junctional region, which in turn promotes growth by inhibiting Warts. We previously identified Approximated (App), a DHHC domain palmitoyltransferase, as a negative regulator of Fat signaling in growth control. We show here that App promotes growth by palmitoylating the intracellular domain of Fat, and that palmitoylation negatively regulates Fat function. Independently, App also recruits Dachs to the apical junctional region through protein-protein association, thereby stimulating Dachs's activity in promoting growth. Further, we show that palmitoylation by App functions antagonistically to phosphorylation by Discs-overgrown, which activates Fat. Together, these findings suggest a model in which App promotes Dachs activity by simultaneously repressing Fat via posttranslational modification and recruiting Dachs to the apical junctional region, thereby promoting tissue growth.

The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs.

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, we have performed a structure-function analysis of Dachs, and used this to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein we have named Dlish. We found that Dlish is regulated by Fat and Dachs, that Dlish also binds Fat and the Dachs regulator Approximated, and that Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. Our evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs.

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

Planar cell polarity: the importance of getting it backwards.

The core and Fat-Dachsous signaling systems locally align planar cell polarities in Drosophila epithelia. Three recent papers address how coupling between these systems can be altered and reversed by the products of the gene prickle.

Closing the Social Class Achievement Gap for First-Generation Students in Undergraduate Biology.

Many students start college intending to pursue a career in the biosciences, but too many abandon this goal because they struggle in introductory biology. Interventions have been developed to close achievement gaps for underrepresented minority students and women, but no prior research has attempted to close the gap for first-generation students, a population that accounts for nearly a fifth of college students. We report a values affirmation intervention conducted with 798 U.S. students (154 first-generation) in an introductory biology course for majors. For first-generation students, values affirmation significantly improved final course grades and retention in the second course in the biology sequence, as well as overall GPA for the semester. This brief intervention narrowed the achievement gap between first-generation and continuing generation students for course grades by 50% and increased retention in a critical gateway course by 20%. Our results suggest that educators can expand the pipeline for first-generation students to continue studying in the biosciences with psychological interventions.

The Drosophila WIF1 homolog Shifted maintains glypican-independent Hedgehog signaling and interacts with the Hedgehog co-receptors Ihog and Boi.

Hedgehog (Hh) family proteins are secreted signaling ligands whose short- and long-range activities transform cellular fates in multiple contexts in organisms ranging from metazoans to humans. In the developing Drosophila wing, extracellular Hh binds to cell-bound glypican heparan sulfate proteoglycans (HSPGs) and the secreted protein Shifted (Shf), a member of Wnt inhibitory factor 1 (WIF1) family. The glypicans and Shf are required for long-range Hh movement and signaling; it has been proposed that Shf promotes long-range Hh signaling by reinforcing binding between Hh and the glypicans, and that much or all of glypican function in Hh signaling requires Shf. However, we will show here that Shf maintains short-range Hh signaling in the wing via a mechanism that does not require the presence of or binding to the Drosophila glypicans Dally and Dally-like protein. Conversely, we demonstrate interactions between Hh and the glypicans that are maintained, and even strengthened, in the absence of Shf. We present evidence that Shf binds to the CDO/BOC family Hh co-receptors Interference hedgehog (Ihog) and Brother of Ihog, suggesting that Shf regulates short-range Hh signaling through interactions with the receptor complex. In support of a functional interaction between Ihog and members of the Shf/WIF1 family, we show that Ihog can increase the Wnt-inhibitory activity of vertebrate WIF1; this result raises the possibility of interactions between WIF1 and vertebrate CDO/BOC family members.

Cell polarity: overdosing on PCPs.

Several spatial cues combine to influence cell polarity within the plane of the Drosophila wing epithelium, orienting two separable mechanisms of short-range intercellular communication, one utilizing the 'core' polarity proteins, and another utilizing the protocadherins Dachsous and Fat, and the atypical myosin Dachs.

Crossveinless d is a vitellogenin-like lipoprotein that binds BMPs and HSPGs, and is required for normal BMP signaling in the Drosophila wing.

The sensitivity of the posterior crossvein in the pupal wing of Drosophila to reductions in the levels and range of BMP signaling has been used to isolate and characterize novel regulators of this pathway. We show here that crossveinless d (cv-d) mutations, which disrupt BMP signaling during the development of the posterior crossvein, mutate a lipoprotein that is similar to the vitellogenins that comprise the major constituents of yolk in animal embryos. Cv-d is made in the liver-like fat body and other tissues, and can diffuse into the pupal wing via the hemolymph. Cv-d binds to the BMPs Dpp and Gbb through its Vg domain, and to heparan sulfate proteoglycans, which are well-known for their role in BMP movement and accumulation in the wing. Cv-d acts over a long range in vivo, and does not have BMP co-receptor-like activity in vitro. We suggest that, instead, it affects the range of BMP movement in the pupal wing, probably as part of a lipid-BMP-lipoprotein complex, similar to the role proposed for the apolipophorin lipid transport proteins in Hedgehog and Wnt movement.

Separating planar cell polarity and Hippo pathway activities of the protocadherins Fat and Dachsous.

The giant Drosophila protocadherin Fat (Ft) affects planar cell polarity (PCP). Ft also inhibits the overgrowth of imaginal discs via the Hippo pathway, repressing the activity of the transcription co-factor Yorkie (Yki). Much of Ft activity is likely to be mediated by its intracellular domain (Ft ICD). However, the links between the Ft ICD and either PCP or Hippo activity are poorly understood, and the role of the Hippo pathway in PCP is ambiguous. We have performed a structure-function analysis of the Ft ICD. We found that the effects of the Ft ICD on PCP and the Hippo pathway are largely separable. Surprisingly, the domains required for PCP and Hippo activities do not map to any of the previously identified protein interaction domains, nor, with one exception, to the regions that are highly conserved in mammalian Fat4. We also found that the extracellular domain of Ft can act independently of the Ft ICD in PCP and can trigger dominant-negative and boundary effects on Hippo activity, probably via binding to the protocadherin Dachsous.

The role of glypicans in Wnt inhibitory factor-1 activity and the structural basis of Wif1's effects on Wnt and Hedgehog signaling.

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding "EGF-like" domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the "WIF" domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling.

The extracellular regulation of bone morphogenetic protein signaling.

In many cases, the level, positioning and timing of signaling through the bone morphogenetic protein (BMP) pathway are regulated by molecules that bind BMP ligands in the extracellular space. Whereas many BMP-binding proteins inhibit signaling by sequestering BMPs from their receptors, other BMP-binding proteins cause remarkably context-specific gains or losses in signaling. Here, we review recent findings and hypotheses on the complex mechanisms that lead to these effects, with data from developing systems, biochemical analyses and mathematical modeling.

Phosphorylation of the tumor suppressor fat is regulated by its ligand Dachsous and the kinase discs overgrown.

The Drosophila tumor suppressor gene fat encodes a large cadherin that regulates growth and a form of tissue organization known as planar cell polarity (PCP). Fat regulates growth via the Hippo kinase pathway, which controls expression of genes promoting cell proliferation and inhibiting apoptosis (reviewed in). The Hippo pathway is highly conserved and is implicated in the regulation of mammalian growth and cancer development. Genetic studies suggest that Fat activity is regulated by binding to another large cadherin, Dachsous (Ds). The tumor suppressor discs overgrown (dco)/Casein Kinase I delta/epsilon also regulates Hippo activity and PCP. The biochemical nature of how Fat, Ds, and Dco interact to regulate these pathways is poorly understood. Here we demonstrate that Fat is cleaved to generate 450 kDa and 110 kDa fragments (Fat(450) and Fat(110)). Fat(110) contains the cytoplasmic and transmembrane domain. The cytoplasmic domain of Fat binds Dco and is phosphorylated by Dco at multiple sites. Importantly, we show Fat forms cis-dimers and that Fat phosphorylation is regulated by Dachsous and Dco in vivo. We propose that Ds regulates Dco-dependent phosphorylation of Fat and Fat-associated proteins to control Fat signaling in growth and PCP.

The DHHC palmitoyltransferase approximated regulates Fat signaling and Dachs localization and activity.

Signaling via the large protocadherin Fat (Ft), regulated in part by its binding partner Dachsous (Ds) and the Golgi-resident kinase Four-jointed (Fj), is required for a variety of developmental functions in Drosophila. Ft and, to a lesser extent, Ds suppress overgrowth of the imaginal discs from which appendages develop and regulate the Hippo pathway [1-5] (reviewed in [6]). Ft, Ds, and Fj are also required for normal planar cell polarity (PCP) in the wing, abdomen, and eye and for the normal patterning of appendages, including the spacing of crossveins in the wing and the segmentation of the leg tarsus (reviewed in [7-9]). Ft signaling was recently shown to be negatively regulated by the atypical myosin Dachs [10, 11]. We identify here an additional negative regulator of Ft signaling in growth control, PCP, and appendage patterning, the Approximated (App) protein. We show that App encodes a member of the DHHC family, responsible for the palmitoylation of selected cytoplasmic proteins, and provide evidence that App acts by controlling the normal subcellular localization and activity of Dachs.

The BMP-binding protein Crossveinless 2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila.

In Drosophila, the secreted BMP-binding protein Short gastrulation (Sog) inhibits signaling by sequestering BMPs from receptors, but enhances signaling by transporting BMPs through tissues. We show that Crossveinless 2 (Cv-2) is also a secreted BMP-binding protein that enhances or inhibits BMP signaling. Unlike Sog, however, Cv-2 does not promote signaling by transporting BMPs. Rather, Cv-2 binds cell surfaces and heparan sulfate proteoglygans and acts over a short range. Cv-2 binds the type I BMP receptor Thickveins (Tkv), and we demonstrate how the exchange of BMPs between Cv-2 and receptor can produce the observed biphasic response to Cv-2 concentration, where low levels promote and high levels inhibit signaling. Importantly, we show also how the concentration or type of BMP present can determine whether Cv-2 promotes or inhibits signaling. We also find that Cv-2 expression is controlled by BMP signaling, and these combined properties enable Cv-2 to exquisitely tune BMP signaling.

Wing vein patterning in Drosophila and the analysis of intercellular signaling.

The positioning and elaboration of ectodermal veins in the wing of Drosophila melanogaster rely on widely utilized developmental signals, including those mediated by EGF, BMP, Hedgehog, Notch, and Wnt. Analysis of vein patterning mutants, using the molecular and genetic mosaic techniques available in Drosophila, has provided important insights into how a combination of short-range and long-range signaling can pattern a simple epidermal tissue. Moreover, venation has become a powerful system for isolating and analyzing novel components in these signaling pathways. I here review the basic events of vein patterning and give examples of how changes in venation have been used to identify important features of cell signaling pathways.