The small GTPase Rap1 is a modulator of Hedgehog signaling.

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Dev Biol. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Dev Biol. 2016 January 1; 409(1): 84–94. doi:10.1016/j.ydbio.2015.10.020.

The Small GTPase Rap1 is a Modulator of Hedgehog Signaling Suresh Marada1, Ashley Truong1,2, and Stacey K. Ogden1,3 1Department

of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN

38105 2Rhodes

College Summer Plus Program, Rhodes College, Memphis TN 38112

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Abstract

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During development, the evolutionarily conserved Hedgehog (Hh) morphogen provides instructional cues that influence cell fate, cell affinity and tissue morphogenesis. To do so, the Hh signaling cascade must coordinate its activity with other morphogenetic signals. This can occur through engagement of or response to effectors that do not typically function as core Hh pathway components. Given the ability of small G proteins of the Ras family to impact cell survival, differentiation, growth and adhesion, we wanted to determine whether Hh and Ras signaling might intersect during development. We performed genetic modifier tests in Drosophila to examine the ability of select Ras family members to influence Hh signal output, and identified Rap1 as a positive modulator of Hh pathway activity. Our results suggest that Rap1 is activated to its GTPbound form in response to Hh ligand, and that the GTPase exchange factor C3G likely contributes to this activation. The Rap1 effector Canoe (Cno) also impacts Hh signal output, suggesting that a C3G-Rap1-Cno axis intersects the Hh pathway during tissue morphogenesis.

Keywords hedgehog; signal transduction; morphogenesis; GTPase; Rap1

Introduction

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The Hedgehog (Hh) signal transduction cascade governs tissue morphogenesis during development, contributes to post-developmental tissue homeostasis and, when corrupted, can lead to developmental disorders or cancer (Barakat et al., 2011; Ingham and McMahon, 2001; Jiang and Hui, 2008). To assure proper epithelial patterning and homeostasis, Hhmediated cell fate decisions must be temporally and spatially coordinated with other cellular processes and cell signaling events (De Celis, 2003; Dekanty and Milan, 2011). This can occur through Hh i) crosstalk with other signaling pathways, and ii) regulation of or

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Author for correspondence: Stacey K. Ogden, [email protected], St. Jude Children’s Research Hospital, Department of Cell and Molecular Biology, 262 Danny Thomas Place, MS #340, Memphis, TN 38105, Phone: 901-595-6281, Fax: 901-525-8025. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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response to effectors that are not typically considered to function in the classical Hh pathway (Ahn et al., 2010; Min et al., 2011; Ribes et al., 2009; Robbins et al., 2012).

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Small monomeric guanosine triphosphatases (GTPases, G proteins) are signaling effectors that function in a wide range of signaling cascades, thereby providing opportunity for crosspathway communication. Hundreds of distinct G proteins exist, and are divided into multiple superfamilies based upon their sequence homology and functionality (Bos and Zwartkruis, 1999; Wennerberg et al., 2005). The Ras superfamily is divided into distinct subbranches including Ras, Rho, Rab, Ran, Rheb and Arf (Bernards, 2003; Reuther and Der, 2000). Generally speaking, the Ras branch is dedicated to cell proliferation and differentiation, Rho to cytoskeletal regulation, Rab to membrane trafficking, Ran to nuclear transport, Rheb to mTOR signaling and Arf to vesicular transport (Wennerberg et al., 2005). To date, Rab, Arf and Rho subbranches have been clearly linked to the Hh pathway in either Drosophila or vertebrate systems. In flies, Rabs 4 and 5 facilitate vesicular transport and membrane recycling of pathway components including Hh, Dispatched, Costal2 (Cos2) and Smoothened (Smo) (Callejo et al., 2011; D’Angelo et al., 2015; Farzan et al., 2008). In vertebrates, Arl13B (Arf subfamily) and Rab23 impact pathway activity through influencing ciliary transport of Smo and activity of its Gli transcriptional effectors (Boehlke et al., 2010; Eggenschwiler et al., 2006; Evans et al., 2003; Larkins et al., 2011). In addition to being regulated by small G proteins, vertebrate Sonic Hh signaling can also activate them; Smo signals through the heterotrimeric G protein Gαi to activate RhoA and Rac1 to induce cell migration (Polizio et al., 2011a; Polizio et al., 2011b).

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Activation of all monomeric G proteins occurs through the stereotypical GTPase cycle, which is initiated by an upstream signal triggering a target G protein to exchange a bound GDP molecule for GTP. GTP binding switches the G protein to its “on” state, which facilitates its interaction with downstream signaling effectors. The cycle is completed by GTP hydrolysis to return the G protein to the GDP-bound “off” state (Wennerberg et al., 2005). The intrinsic nucleotide exchange and hydrolysis rates for a given monomeric G protein are typically quite slow (Wennerberg et al., 2005). However, these processes can be accelerated by GTP exchange factors (GEFs), which facilitate GDP release, and GTPase activating proteins (GAPs), which promote GTP hydrolysis (Bernards, 2003; Bos and Zwartkruis, 1999; Wennerberg et al., 2005). This allows for fine-tuning of G protein signaling, and assures that proper checks and balances are in place to prevent aberrant activity.

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Given the role of Hh in governing cell growth and differentiation during development, and the documented ability of GTPases of the Ras subbranch to affect such processes, we tested for genetic interactions between the Hh pathway and representative Ras subbranch members. A candidate genetic modifier test was performed in Drosophila against a smo sensitized genetic background generated by expression of a dominant negative Smo mutant (Collins and Cohen, 2005). Mutant alleles of Rala, Rheb, Ras85D, Ric and Rap1 were tested for the ability to enhance or suppress the wing phenotype induced by dominant negative Smo5A. Rap1, which stands for Ras proximate, scored as a positive interactor. This GTPase was originally identified through a screen for suppressors of the transforming capability of K-ras (Noda et al., 1989). In normal cell physiology, Rap1 is activated in response to a

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number of extracellular signals to control cell-cell junction formation, integrin-mediated cell adhesion, cell polarity and in some cases, epithelial to mesenchymal transition (EMT) (Boettner and Van Aelst, 2009; Frische and Zwartkruis, 2010; Kooistra et al., 2007; Pannekoek et al., 2009). Drosophila Rap1, originally referred to as Roughened (R), functions in tissue morphogenesis of the embryo, eye, ovary and wing (Asha et al., 1999; O’Keefe et al., 2009; O’Keefe et al., 2012).

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Herein, we demonstrate that Rap1 and its effector Canoe (Cno) function as modulators of Hh pathway activity during Drosophila wing development. Rap1 mutant alleles enhance Smo loss-of-function and suppress Hh gain-of-function in vivo, suggesting a positive functional role for the small G protein in Hh signaling. Consistent with it being activated to its GTP-bound form, Rap1 relocalizes from the cytoplasm to the nucleus in response to Hh stimulation. Knockdown of either Rap1 or Cno attenuates ligand-induced pathway activation and target gene induction. We therefore postulate that Hh-mediated induction of Rap1 in wing disc cells enhances downstream pathway activity, and integrates Hh-mediated fate specification with Rap1-signaling during wing morphogenesis.

Experimental Procedures Fly stocks and crosses

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RalaEE1, Rheb4L1, Ras85De2f, RaprvB3, Rap11 and hhMrt alleles and all GAL4 drivers were obtained from the Bloomington Stock Center. RalaEE1 harbors an EMS-induced S154L mutation (Eun et al., 2007); Rheb4L1, an EMS-induced substitution K101@ (Stocker et al., 2003); Ras85De2f is a hypomorphic allele with a D38N substitution (Simon et al., 1991); RaprvB3 is an EMS-induced Q76@ mutation (Asha et al., 1999; Hariharan et al., 1991); Rap11 is a neomorphic allele harboring a F157L mutation (Hariharan et al., 1991). Rap1CD3 and Rap1CD5 were obtained from J. Curtiss (O’Keefe et al., 2009), cnoR2 from M. Peifer (Sawyer et al., 2009), Ric1 from D. Harrison (Cai et al., 2011) and Su(fu)LP from D. Kalderon. UAS-Rap1 and UAS-RapG12V were obtained from U. Gaul (Boettner et al., 2003). All fly crosses were carried out at 22°C or 25°C on Jazz Drosophila Food (Fisher Scientific). Crosses were performed a minimum of three times and multiple progeny analyzed. For phenotype classification and quantification, ~150 males across three independent crosses were examined. Approximately ~50 flies were analyzed for Rap11. Wings representative of the predominant phenotype are shown. Images were taken using a Zeiss Axiocam ICc3 and processed with Photoshop CS4. Cell culture, functional assays and biochemical analysis

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Clone 8 (Cl8) cells were cultured in Grace’s Insect Media supplemented with 2% fetal bovine serum and 2.5% flyblood using standard techniques. Reporter assays were performed as previously described (Carroll et al., 2012; Marada et al., 2013). Briefly, ~3×105 cells were plated in 12-well dishes and transfected the following day once cultures reached ~70% confluency. Cells were transfected with 100 ng ptcΔ136-luciferase, 10 ng pAc-renilla along with control or specific dsRNA (1x = 0.25 μg) or pAc-Rap1 (1x = 0.25 μg) normalized to empty vector control, as indicated. Cells were lysed and analyzed for luciferase and renilla activity ~48 hours post-transfection using the Dual Luciferase Assay Kit (Promega). mRNA


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regions targeted by dsRNA in knockdown reporter assays include: Rap1 coding, bp 1405-1940; Rap1 3′ UTR, bp 2620-3135; cno, bp 960-1966; C3G, 5377-6053; EPAC, bp 1502-2533; template for control dsRNA to Xenopus elongation factor 1α is provided in the MEGAscript kit (Lifetechnologies). Significance was determined using Student’s t-test. For biochemical analysis of Hh pathway induction and dsRNA knockdown efficiency, ~3×106 Cl8 cells were plated in 60 mm dishes and transfected the following morning. Cells were co-transfected with 5 μg pAc-hh, 5 μg pAc-rap1 or empty vector control and control or specific dsRNA using Lipofectamine2000 (Life Technologies). Forty-eight hours posttransfection, cells were lysed on ice using NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, 50 mM NaF, 1% NP-40, 0.5 mM DTT and 1X PIC (Roche), pH8.0). Lysates were centrifuged for 10 minutes at 2000 × g and supernatants were analyzed by SDS-PAGE and western blot.

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Protein expression and antibodies

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To generate expression plasmids (pAc-rap1-FLAG, pAc-HA-cno, pAc-C3G-V5 and pAc-HAEPAC), cDNAs were obtained from the DGRC and cloned into the pAc expression vector (ThermoFisher). Epitope tags were inserted using the Quickchange site directed mutagenesis kit (Stratagene). For recombinant Rap1 protein expression, Rap1 cDNA was cloned into the pET-28A expression vector (Novagen). Recombinant Rap1-His was generated in BL21 PLysS cells and affinity purified on nickel agarose using standard techniques (Promega). Recombinant full length Rap1 protein was used to generate polyclonal antisera in rabbits using the Covance custom antibody service. All other antibodies have previously been described: anti-Hh (SCBT), anti-Fu (Ascano et al., 2002; Ascano and Robbins, 2004), antiCi (Motzny and Holmgren, 1995), anti-Ptc (Capdevila et al., 1994), anti-Cos2 (Stegman et al., 2004), anti-FLAG (Sigma), anti-V5 (SCBT), anti-HA (Covance) and anti-Kinesin (Cytoskeleton). Immunofluorescence microscopy

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Analysis of Rap1 subcellular localization in Cl8 cells was performed using antisera specific to the FLAG epitope tag (Sigma) and Alexa fluor secondary antibodies (Lifetechnologies) as previously described for Smo (Carroll et al., 2012). Images were acquired on a Zeiss LSM780 confocal microscope and processed using Photoshop CS4. Localization analysis was performed three times and representative cells are shown. Quantification represents the percentage of Rap1-expressing cells showing nuclear signal out of ~100 cells counted across three independent experiments. Wing imaginal discs were dissected from third instar larvae, stained with Rap1 Ci and Ptc antisera and imaged exactly as previously described (Carroll et al., 2012). Multiple discs of each genotype from two independent crosses were analyzed. Representative discs are shown.

Results To determine whether members of the Ras subbranch of GTPases play a functional role in Drosophila Hh signaling, a targeted modifier test was performed against the C765>smo5A genetic background (Collins and Cohen, 2005). These flies overexpress a Smo mutant


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harboring S/T to A mutations at five essential PKA phosphorylation sites, which triggers LV3/LV4 vein fusions and tissue loss of varying severity. We categorized phenotypic severity into five classes, with class 5 being most severe (Fig. 1A–B and Fig. 1 in (Marada et al.)). Loss-of-function alleles for representative Ras subbranch members were introduced into C765>smo5A flies, and adult wings were monitored for enhancement or suppression of the phenotype. Rheb, Rala, Ric and Ras alleles failed to significantly modify C765>smo5A (Fig. 1C–F compared to B). However, a potential interaction was identified for the Rap1rvB3 truncation mutant, which triggered a phenotypic enhancement of more pronounced proximal and distal LV3/LV4 fusions (Fig. 1G).

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To investigate the putative genetic interaction between Rap1 and the Hh pathway, additional Rap1 alleles were tested for their ability to modify C765>smo5A. Rap11 is a neomorphic gain of function allele resulting from an F157L substitution. It was identified as the mutation responsible for the rough eye phenotype of R flies (Hariharan et al., 1991). Rap11/+ flies showed an erect wing posture, but did not demonstrate a classical Hh gain-of-function wing phenotype (Fig. 2B). However, in the sensitized smo5A background, Rap11 suppressed smo5A-induced wing vein fusions, shifting the bulk of the population to the lowest severity class 1 (Fig. 2B′ compared to 2A and plotted with error bars in 2A of (Marada et al.)). Although Rap11 did not fully restore LV3/LV4 vein spacing to wild type, it suppressed wing phenotypes as effectively as a loss-of-function allele of the negative pathway regulator Suppressor of Fused (Su(fu)LP) (Fig. 2B–C and 2B in (Marada et al.)).

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Rap1CD3 and Rap1CD5 loss-of-function alleles harbor coding region deletions (Boettner et al., 2003; O’Keefe et al., 2009). Both mutations are homozygous lethal and heterozygous flies are phenotypically normal (Asha et al., 1999). However, like Rap1rvB3, introduction of Rap1CD3 or Rap1CD5 into the C765>smo5A background enhanced the phenotype, in both cases resulting in loss of LV3 and/or LV4 vein tissue and emergence of high severity phenotypic classes 4 and 5 in the population (Fig. 2D–E compared to A and Fig. 2C in (Marada et al.)). Rap1 plays a role in the cell-cell contacts that are necessary for vein integrity in developing and adult wing tissue (O’Keefe et al., 2012). We therefore tested whether the enhanced vein disruption phenotype resulted from the alleles interacting genetically with the Hh pathway or from disruption of Rap1-mediated cell contacts in vein tissue. The hhMrt gain-of-function allele triggers ectopic Hh signaling in the anterior region of the wing imaginal disc due to misexpression of hh along the dorsal/ventral (D/V) border of the wing pouch (Tabata and Kornberg, 1994). This manifests as variable anterior wing overgrowth and ectopic vein formation in the anterior wing blade (Fig. 3A compared to 1A). Like C765smo5A, did not as potently shift the hhMrt population toward low severity classes 1 and 2 (Fig. 5 in (Marada et al.)). We therefore focused all further analysis on Rap1.

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If Rap1 functions as an Hh effector, its loss-of-function should impact Hh target gene activation in vivo. We attempted to assess target gene expression in wing imaginal discs by generating Rap1 mutant clones by FRT-mediated recombination. Unfortunately, we were unable to obtain Rap1CD3 or Rap1CD5 null clones large enough to analyze, a likely result of Rap1 null cells dissociating from each other in imaginal disc tissue (Knox and Brown, 2002; O’Keefe et al., 2012). We therefore switched to an in vitro cell culture system in which Rap1 gene function could be reduced by dsRNA treatment, and effects on Hh-induced reporter gene activation could be easily monitored. Wing imaginal disc-derived Clone 8 (Cl8) cells were co-transfected with the ptcΔ136-luciferase reporter gene, pAc-renilla normalization control and pAc-hh along with control or Rap1-specific dsRNA (Fig. 4A). Hh expression induced reporter gene activity in cells treated with control dsRNA (black vs. gray bar, control dsRNA). This activation was attenuated, in a dose-dependent manner, by dsRNAs specific to both coding and UTR Rap1 sequence, indicating that Rap1 is important for maximal Hh-dependent reporter gene induction.

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Hh expression in Cl8 cells triggers a number of biochemical events that can be monitored as endogenous readouts of pathway activity. These include phosphorylation of the protein kinase Fused (Fu) (Therond et al., 1996), accumulation of the full-length, unprocessed species of the transcription factor Ci (Aza-Blanc et al., 1997) and accumulation of the Hh receptor Patched (Ptc) (Chen and Struhl, 1996). To interrogate how Rap1 reduction impacted these events, cellular lysates prepared from Cl8 cells co-transfected with control or Rap1 dsRNA in the presence or absence of pAc-hh were examined by western blot. Consistent with the pathway being highly activated by Hh, we observed Fu phosphorylation, full-length Ci stabilization and accumulation of endogenous Ptc in control dsRNA-treated cells (Fig. 4B, lanes 1–2). Rap1 coding sequence dsRNA reduced steady state levels of endogenous Rap1 protein, as evidenced by western blot with polyclonal antisera raised against the full length Rap1 protein (lanes 3–6 compared to 1–2). Hh-mediated effects on Fu, Ci and Ptc were ablated in these cells, further supporting that Rap1 is required for maximal ligand-induced pathway activation in vitro (Fig. 4B, lanes 3–6 compared to 1–2).

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The data thus far suggest a positive functional role for Rap1 in transducing the Hh signal. However, active Rap1, when expressed at endogenous levels, could not trigger ectopic activity (Fig. 2B). The ability of over-expressed Rap1 to induce pathway activity in the absence of Hh ligand, or enhance signaling activity in its presence was therefore tested. Hh reporter gene induction was assessed in Cl8 cells transfected with increasing amounts of a pAc-Rap1-FLAG expression vector. Rap1 over-expression resulted in a modest, dosedependent increase in Hh reporter gene activity in both the presence (gray bars) and absence (black bars) of Hh ligand (Fig 4C and C′). Consistent with this enhancement, a modest ligand-independent increase in full length Ci and stabilization of the scaffolding protein Cos2 were evident in cellular lysates from Rap1 over-expressing cells (Fig. 4D, lane 3 Dev Biol. Author manuscript; available in PMC 2017 January 01.

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compared to 1). However, over-expression failed to induce Fu or Cos2 phosphorylation (Fig. 4D, lane 3), indicating that while over-expressed Rap1 could to attenuate Ci processing to its truncated repressor form, it could not fully activate the pathway in the absence of Hh ligand.

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To determine whether over-expressed Rap1 would induce Hh phenotypes in vivo, wild type and constitutively active GTP-bound (G12V) Rap1 proteins were expressed in the developing wing using the UAS/GAL4 system. Wild type Rap1 did not induce an obvious Hh phenotype (Fig. 5A) or markedly rescue Smo5A-induced phenotypes when expressed under control of the low-level C765-GAL4 epithelial driver (Fig. 5B, compared to F, control). Given that Rap1 must be activated to its GTP-bound form to signal, it is possible that in order to see ligand-independent Hh gain-of-function, Rap1 would need to be in this active state. Rap1G12V over-expression triggered pronounced wing blistering, but did not induce wing phenotypes indicative of ectopic Hh signaling (Fig. 5C). However, expression of constitutively active Rap1G12V partially suppressed the C765>smo5A-induced phenotype, as evidenced by modest resolution of the proximal LV3/LV4 fusion (Fig. 5D compared to B and F). Suppression was reciprocal in that C765>smo5A modestly suppressed the Rap1G12V wing blistering (Fig. 5D compared to C). Phenotypic suppression was not likely the result of the additional transgene squelching GAL4 because Smo5A and Rap1G12V phenotypes were not affected by co-expression of a UAS-GFP transgene (Fig. 5F compared to B and E compared to C).

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To test for effects on Ci stabilization and target gene expression, Rap1 was expressed in the dorsal (D) compartment using the high-level apterous (ap)-GAL4 driver. This GAL4 driver allows direct comparison of transgene-induced effects in the D compartment to a control condition in the ventral (V) compartment. There was no obvious effect on Ci or Ptc levels when one UAS-Rap1 or UAS-Rap1G12V transgene was expressed (Fig. 5G–H). Whereas expression of two UAS-Rap1G12V transgenes induced lethality, expression of two copies of wild type UAS-Rap1 modestly, but consistently, enhanced Ci stabilization and Ptc induction (Figs. 5I–J, D compartment compared to V control). These results are consistent with the modest Ci stabilization and ptc reporter enhancement observed following Rap1 overexpression in vitro (Fig. 4C–D). Taken together, these results support a functional interaction between Hh and Rap1 signaling in which Rap1 operates as a positive modulator of the Hh pathway. However, although Rap1 can modestly enhance Hh signal output when over-expressed, its signaling is insufficient to induce robust ectopic Hh pathway activity in vivo.

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If Rap1 is activated downstream of Hh to enhance signaling, it should be converted from its inactive GDP-bound form to its active GTP-bound form in response to Hh ligand. Unfortunately, available Rap1 activation assays are designed for vertebrate cell culture systems, and have not been optimized for fly cells. Rap1 subcellular localization was therefore monitored as a surrogate read-out for GTP binding. This is because in some cell types, active Rap1 shuttles to the nucleus and/or nuclear membrane following GTP exchange (Goitre et al., 2014; Lafuente et al., 2007; Mitra et al., 2003). To determine whether this would be the case in Cl8 cells, subcellular localization of the constitutively GTP-bound Rap1G12V mutant was tested. Rap1G12V-FLAG showed a predominantly nuclear localization pattern (~80%), indicating that subcellular localization can be used as a Dev Biol. Author manuscript; available in PMC 2017 January 01.

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reporter for Rap1-GTP binding in Cl8 cells (Fig. 6A, D). To test the effect of active Hh signaling on Rap1 subcellular localization, wild type Rap1-FLAG was expressed in the presence of pAc-hh or empty vector control, and its localization was determined by immunofluorescence microscopy (Fig. 6B–C). Whereas ~30% of unstimulated Rap1-FLAG expressing cells showed nuclear localization of the wild type protein, the majority (~70%) had a diffuse cytoplasmic signal (Fig. 6B, D). Co-expression of Hh triggered a significant increase in the percentage of cells showing nuclear localization of Rap1-FLAG to approximately 80%, suggestive of it being activated to its GTP-bound form in the presence of Hh ligand (Fig. 6C–D).

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Activation of small G proteins is controlled by GTPase exchange factors (GEFs) (Pannekoek et al., 2009). To determine whether Rap1 activation downstream of Hh was influenced by a specific GEF, Cl8 cells were treated with control or dsRNA against two Drosophila Rap1 GEFs, Exchange Protein Activated by cAMP (EPAC) and C3G (Ishimaru et al., 1999; Pannekoek et al., 2009), and Hh reporter gene activation was examined. EPAC knockdown failed to attenuate Hh-induced reporter gene induction (Fig. 6E). This was not likely the result of poor knockdown, as the EPAC dsRNA efficiently decreased steady state levels of exogenously expressed EPAC-HA protein (Fig. 6E, lower panel). Conversely, knockdown of C3G attenuated reporter gene induction in a statistically significant, dosedependent manner, suggesting that C3G may serve as an Hh-responsive Rap1 GEF (Fig. 6F).


The Drosophila AF6 ortholog Cno is a downstream effector of Rap1 (Boettner et al., 2003; Choi et al., 2013; Sawyer et al., 2009). Cno functions in signal transduction during morphogenesis, in some cases serving as a scaffold upon which signaling complexes assemble (Boettner et al., 2003; Choi et al., 2013; Miyamoto et al., 1995; Sawyer et al., 2009). Cno binds filamentous actin to affect cell adhesion, which is most clearly illustrated by dorsal closure defects evident in cno mutant embryos (Boettner et al., 2003; Sawyer et al., 2009). A minor pool of Cno localizes to the nucleus in Drosophila embryos and wing imaginal discs, but its nuclear role has not yet been established (O’Keefe et al., 2012; Sawyer et al., 2009). To determine whether Rap1 functions through Cno to modulate Hh target gene induction, Hh reporter assays were performed in Cl8 cells treated with control or cno dsRNA. Similar to what was observed for knockdown of Rap1 and its GEF C3G, Cno knockdown triggered a ~50–60% reduction in Hh reporter gene activity in cells transfected with the highest dsRNA dose (Fig. 7A). This result is consistent with Cno being required for maximal reporter induction. To test Cno involvement in Hh signaling in vivo, the cnoR2 loss of function allele was crossed into C765>smo5A and hhMrt backgrounds, and its ability to modify wing phenotypes was determined. Heterozygous cno mutation does not impact wing morphogenesis (Boettner et al., 2003; Boettner and Van Aelst, 2007). However, reduction of cno gene dosage in both Hh pathway sensitized backgrounds modified the mutant wing phenotypes (Figs. 7D and F and 2D and 3B in (Marada et al.)). Introduction of cnoR2 enhanced C765>smo5A, as evidenced by a decrease in the percentage of flies in class 1 and increases in the percentage showing severe classes 4 and 5 phenotypes (Fig. 6C–D and Fig. 2D in (Marada et al.)). Conversely, introduction of cnoR2 into the hhMrt background effectively suppressed the phenotype; the majority of cnoR2/hhMrt flies shifted to low


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severity classes 1 and 2 (Fig. 7E–F and Fig. 3B in (Marada et al.)). This suppression was similar to that observed in response to a loss-of-function allele of the positive pathway effector Fu (Fig. 4 in (Marada et al.)). Taken together, these results support a positive role for Cno in the Hh signaling cascade.

Discussion

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The study presented here identifies a connection between the Hh pathway and the monomeric GTPase Rap1, an established regulator of cell adhesion and polarity during morphogenesis. Our results suggest that Rap1 signaling is activated by Hh ligand to augment pathway activity. This is supported by in vivo data demonstrating that i) Rap1 overexpression enhances Ci stabilization and ptc induction, and ii) reduction in Rap1 gene dosage rescues wing phenotypes induced by ectopic Hh signaling. In addition, in vitro results revealed Rap1 to be an enhancer of Hh-induced Fu phosphorylation, Ci stabilization and Ptc induction. However, Rap1 does not appear to be an indispensable Hh effector in the wing, as its mutation or over-expression failed to trigger pronounced Hh phenotypes in vivo. It is therefore likely that Rap1 serves as modulator of Hh signaling to link Hh-directed cell fate decisions with other Rap1-regulated morphogenetic pathways during development.


The developmental processes dependent upon Hh-Rap1 crosstalk were not identified in the current study. However, the importance of Hh in governing cell affinity and the involvement of Rap1 in cell adhesion suggest that their signals could possibly converge to influence cellular compartment boundaries. During wing development, Hh signaling is required to establish and maintain the anterior/posterior (A/P) compartment boundary of the wing imaginal disc (Blair and Ralston, 1997; Dahmann and Basler, 2000; Zecca et al., 1995). This boundary divides Hh-ligand producing posterior cells from ligand-receiving anterior cells (Felsenfeld and Kennison, 1995; Tashiro et al., 1993). Although the mechanisms by which A and P cells interact with each other to establish and maintain their segregation are not clear, it is generally accepted that compartment boundaries are influenced through shape and adhesion properties of cells situated adjacent to the border (Dahmann and Basler, 1999, 2000; Landsberg et al., 2009; Rodriguez and Basler, 1997). Given the documented role for Rap1 in both of these processes (O’Keefe et al., 2012), it is tempting to speculate that its activation at the A/P boundary may integrate Hh signaling with border cell adhesion. Consistent with this notion, it was previously suggested that Hh may control cell sorting at the boundary by inducing expression of cell adhesion molecules and/or activating a small GTPase to affect the actin cytoskeleton (Dahmann and Basler, 2000). The former scenario is supported by the cadherin gene Cad99C being an Hh target gene that is activated at the A/P border (Schlichting et al., 2005). However, its loss is insufficient to disrupt cellular compartment affinity (Schlichting et al., 2005). Cad99C may therefore influence the border, but its activation is not likely the sole mechanism by which Hh establishes anteroposterior cell affinity. Rap1 is an attractive new candidate to test for involvement in this process. Activation of small GTPases commonly involves a GEF (Wennerberg et al., 2005). Accordingly, the Rap1 GEF C3G is required for maximal ptc reporter gene induction in vitro, suggesting that it may serve as the conduit through which Hh signaling influences Rap1 activity. The Rap1 effector Cno is also required for maximal in vitro reporter


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induction, and can modify Hh phenotypes in vivo. Taken together, these results suggest that a C3G-Rap1-Cno regulatory circuit intersects the Hh pathway to modulate signaling during wing morphogenesis. Future studies are needed to define the mechanism by which Rap1 and Cno contribute to Hh pathway activity and to ascertain whether their crosstalk is conserved in vertebrates. Such knowledge will enhance our understanding of how Hh signaling directs tissue morphogenesis during development, and may reveal novel processes by which pathway activity is usurped to cause disease.

Acknowledgments

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This work was supported by SJCRH Comprehensive Cancer Center Developmental Funds from the National Cancer Institute P30CA021765, National Institute of General Medical Science grant 5R01GM101087 (S.K.O.) and ALSAC of SJCRH. We thank J. Curtiss, M. Peifer, D. Harrison, U. Gaul, D. Kalderon and the Bloomington Stock Center for fly lines. Rap1, EPAC, C3G and Cno cDNAs were obtained from the Drosophila Genomics Resource Center. Ptc and Ci hybridoma cells were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. Rap1 protein for antibody production was purified by the SJCRH Protein Production facility. Confocal microscopy was performed at the SJCRH Cell & Tissue Imaging Center which is supported by SJCRH and NCI P30CA021765. We thank P. Thimmaiah and D. Stewart for technical assistance and Y. Ahmed and F. Demontis for comments on the manuscript.

References


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Highlights •

The monomeric GTPase Rap1 is a positive modulator of Hedgehog (Hh) pathway activity.

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The Rap1 GTPase exchange factor C3G modulates Hh signaling.

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The Rap1 effector Canoe (Cno) modulates Hh signaling in vivo.

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Author Manuscript Author Manuscript Figure 1. Rap1 enhances SmoA

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A–B. Longitudinal veins (LV) 1–5 are indicated on a wild type wing (A). Expression of UAS-smo5A under control of the C765-GAL4 epithelial driver triggered a moderate Hh lossof-function phenotype as evidenced by proximal fusion of LV3 and LV4 (B). C–G. Mutant alleles of the indicated monomeric GTPases were introduced into the C765>smo5A background and assessed for their ability to enhance or suppress Smo5A-induced wing phenotypes. Only Rap1rvB3 appreciably enhanced the phenotype, as evidenced by more pronounced distal LV3/LV4 fusions (G).

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Figure 2. Rap1 genetically interacts with the Hh pathway

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A. Quantification of Smo5A phenotypic class distribution. A control cross into the w1118 background was scored. The percent of flies in each phenotypic class are indicated by the pie chart. A class 3 wing is shown. Data are shown with error bars in Fig. 2 of (Marada et al.). B–C. Rap1 gain-of-function suppresses smo5A. Wings from flies harboring a single copy of the Rap11 gain-of-function allele had phenotypically normal wings (B). Rap11 introduction into the C765>smo5A background suppressed LV3/LV4 vein fusions (B′) similarly to introduction of a mutant allele of the negative regulator SuFu (Su(fu)LP) (C). Class 1 wings are shown. Significance of the percent change from control is indicated for each class (shown with error bars in Fig. 2 of (Marada et al.)). D–E. Rap1 loss-of-function alleles enhance C765>smo5A. Introduction of CD3 and CD5 Rap1 alleles into the C765>smo5A sensitized background enhanced the phenotype, triggering pronounced LV3/LV4 fusion and loss of vein tissue. Quantification confirms the population shift toward more severe phenotypic classes 3–5 (D–E compared to A). Class 4 wings are shown. Crosses were performed at least three times with multiple progeny analyzed (~150). For Rap11, approximately 50 flies from three independent crosses were scored. Representative wings from male progeny are shown.


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Figure 3. Rap1 suppresses hhMrt

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A–C. The hhMrt mutation triggered anterior over-growth and ectopic vein formation (A, class 3 hhMrt compared to 1A, wild type). Introduction of Rap1CD3 or Rap1CD5 alleles into the hhMrt background effectively suppressed overgrowth and ectopic vein formation, increase in low severity classes 1 and 2, reducing the percentage of flies in classes 3 and 4 and eliminating highest severity class 5. Quantification of class shifting is shown in pie chart and in Fig. 3 of (Marada et al.). Significance of the percent change from control is indicated for each class (B–C, class 2 is shown). Approximately ~150 male progeny were classified for quantification. Representative wings are shown. D–F. Rap1 suppresses ectopic Ci and ptc. Wing imaginal discs from larvae of the indicated genotype were stained for Ci (green) and Ptc (magenta). Introduction of Rap1CD5 suppressed wing pouch overgrowth and ectopic Ci and Ptc. Scale bar = 50 μm. Crosses were performed at least three times with multiple discs (>10) analyzed per genotype.


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Figure 4. Manipulation of Rap1 expression alters Hh signaling in vitro

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A. Rap1 knockdown attenuates Hh reporter gene induction. Approximately ~7×105 Cl8 cells were transfected with 100 ng ptcΔ136-luciferase, 10 ng pAc-renilla, 100 ng pAc-hh or empty vector control in the presence of control or Rap1-specific dsRNA (1X = 250 ng). dsRNA specific to coding or UTR sequence attenuated Hh-dependent reporter gene induction in a dose dependent manner. Reporter activity is shown as percent luciferase activity normalized to renilla control, and plotted relative to the control Hh response set to 100%. Assays were repeated two or three times in duplicate and all data pooled. For all reporter assays error bars indicate standard error of the mean (s.e.m.). Significance was determined using Student’s t-test. B. Rap1 knockdown blocks high-level Hh signal transduction in vitro. Approximately ~3 × 106 Cl8 cells were transfected with 2.5 μg pAc-hh or empty vector control in the presence of control or Rap1 coding sequence dsRNA (1X=2.5 μg). Hh expression triggered activation of endogenous pathway components as evidenced by phosphorylation of Fu, stabilization of full-length Ci and induction of Ptc (lanes 1–2). Rap1 knockdown significantly blunted these responses (lanes 4 and 6 compared to 2). The experiment was repeated three times and a representative experiment is shown. C. Rap1 Dev Biol. Author manuscript; available in PMC 2017 January 01.

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overexpression modestly enhances Hh reporter gene induction. Reporter assays were performed as in A on cells transfected with empty vector control and increasing concentrations of pAc-Rap1 (1X = 250 ng). C′ shows the Hh-independent reporter activity from C. Total DNA content was normalized with empty vector. D. Rap1 overexpression stabilizes Ci in vitro. Cl8 cells were co-transfected with pAc-hh, pAc-Rap1 or empty vector control as in B. Rap1 overexpression in the absence of Hh partially stabilized Ci and Cos2 but did not induce Fu phosphorylation, suggestive of pathway de-repression, rather than robust activation (lane 3 compared to 1–2). The experiment was repeated three times and a representative experiment is shown. Total DNA content was normalized using empty pAc vector.


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Figure 5. Rap1 over-expression modulates Hh signaling in vivo

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Wild type or GTP-bound (G12V) Rap1 proteins were overexpressed in developing wings using C765- and apterous (ap)-GAL4 drivers in wild type (A, C, E, I–J) or smo sensitized (B, D) backgrounds. Wild type Rap1 expressed under control of the low-level epithelial driver C765-GAL4 did not induce an Hh wing phenotype (A) or rescue the Smo5A phenotype (B compared to F). A single UAS-Rap1 transgene expressed using the higherlevel dorsal wing disc driver ap-GAL4 did not impact Ci stabilization or Ptc induction (G, dorsal, D compared to ventral, V). Rap1G12V induced severe wing blistering, (C), but failed to appreciably induce ectopic Ci or Ptc in the wild type background (H). GTP-bound Rap1 partially rescued the Smo5A-induced LV3/LV4 proximal fusion (D compared to F), and Smo5A partially suppressed Rap1G12V-induced wing blistering (D compared to C). Introduction of the UAS-GFP transgene did not modify C765>Rap1G12V or C765>smo5A phenotypes (E–F). Crosses were performed at least three times with multiple progeny analyzed. Representative wings from male progeny are shown. I–J. Expression of two copies of UAS-Rap1 under control of ap-GAL4 induced modest Ci stabilization and enhanced ptc expression in the dorsal compartment (I). Range indicator shows increased signal intensity (J, red). All 2x UAS-Rap1 discs analyzed showed dorsal-specific signal


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enhancement. Multiple discs (>10) were analyzed. A representative disc is shown. Scale bars = 50 μm.


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Author Manuscript Author Manuscript Figure 6. Hh signaling induces Rap1-GTP exchange


A–C. Hh induces a change in Rap1 subcellular localization. Cl8 cells were transfected with pAc-Rap1G12V-Flag (A) or pAc-Rap1-Flag plus empty vector control (B) or pAc-hh (C). GTP-bound Rap1G12V localized to the nucleus/nuclear membrane in the majority of cells (A, D). In the absence of Hh, the majority of Rap1-Flag showed a diffuse cytoplasmic localization (B, D). Co-expression of Hh caused Rap1 to localize similarly to Rap1G12V (C– D). The experiment was repeated three times and multiple fields of cells analyzed. Representative images are shown. Data from three independent experiments (~100 cells total) were used for quantification shown in D. Error bars indicate s.e.m. E–F. The GEF C3G is required for maximal Hh reporter gene activation. Hh reporter assays were performed in Cl8 cells treated with dsRNA specific to the Rap1 GEFs EPAC and C3G as in 4A. EPAC and C3G dsRNAs were validated by monitoring steady state levels of exogenously-expressed EPAC-HA or C3G-V5 proteins (lower panels). Whereas EPAC dsRNA had no significant effect on Hh-induced reporter gene induction, C3G dsRNA attenuated activation in a dose dependent manner (1X = 250 ng). For EPAC knockdown lysates, protein concentration was determined by BCA assay (Bio-rad), and 25 μg total protein added per lane. For C3G lysates, kinesin (Kin) serves as loading control. The experiments were performed two or three times in duplicate and all data points pooled. Error bars indicate s.e.m.


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Figure 7. The Rap1 effector Cno is a positive modulator of Hh pathway activity

A–B. Cno knockdown attenuates Hh reporter gene induction. Hh reporter assays were performed as in 4A. Cno knockdown attenuated Hh reporter gene induction in a dose dependent manner (1X = 250 ng). Knockdown was confirmed by monitoring steady state levels of exogenously expressed HA-Cno (B). C–F. The loss of function cnoR2 allele modifies Hh phenotypes in vivo. Reducing cno gene dosage enhanced C765>smo5A and suppressed hhMrt. Representative wings from male progeny are shown: C, class 2 smo5A; D, class 3 smo5A; E, class 3 hhMrt; F class 1 hhMrt. Approximately 150 male flies across 3 independent crosses were scored for quantification. Percent change is shown with error bars in Fig. 3 of (Marada et al.). Significance of the percent change from control is indicated for each class.

Author Manuscript Author Manuscript Dev Biol. Author manuscript; available in PMC 2017 January 01.

Bidirectional Synaptic Structural Plasticity after Chronic Cocaine Administration Occurs through Rap1 Small GTPase Signaling.

Small GTPase Rap1 Is Essential for Mouse Development and Formation of Functional Vasculature.

RAP1 GTPase overexpression is associated with cervical intraepithelial neoplasia.

Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1.

A rapid genome-wide microRNA screen identifies miR-14 as a modulator of Hedgehog signaling.

Rnd3 is a critical regulator of Notch1 signaling.

Trif pathway in TLR4 signaling.

RasGRP3 limits Toll-like receptor-triggered inflammatory response in macrophages by activating Rap1 small GTPase.

The dynamic of the apical ectoplasmic specialization between spermatids and Sertoli cells: the case of the small GTPase Rap1.

IQGAP1 is a key node within the small GTPase network.

The small GTPase Rac1 is required for smooth muscle contraction.

A large Rab GTPase family in a small GTPase world.

Hedgehog lipoprotein(a) is a modulator of activation of plasminogen at the fibrin surface. An in vitro study.

Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells.

Multiple Roles of the Small GTPase Rab7.

Hedgehog signaling and steroidogenesis.

The role of glypicans in Hedgehog signaling.

Sonic hedgehog signaling in the postnatal brain.

Ral small GTPase signaling and oncogenesis: More than just 15minutes of fame.

SOX18 Is a Novel Target Gene of Hedgehog Signaling in Cervical Carcinoma Cell Lines.

Tyrosine phosphorylation of Dbl regulates GTPase signaling.

Na,K-ATPase β1-subunit is a target of sonic hedgehog signaling and enhances medulloblastoma tumorigenicity.

Activation of hedgehog signaling pathway in human non-small cell lung cancers.

Cytotoxic-T-lymphocyte antigen 4 receptor signaling for lymphocyte adhesion is mediated by C3G and Rap1.