Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. AmongDrosophila melanogaster Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and otherdNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3′-5′ cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whetherdNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1^st and 2^nd chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of thedNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicateddAlk tyrosine kinase, its activating ligandjelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in thedNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show thatdAlk,jeb andCCKLR-17D1 are among mutants that also suppress a recently identifieddNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore thedNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that humanALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man.

Neurofibromatosis type 1 (NF1) is a genetic disease that affects 1 in 3,000 and that is caused by loss of a protein that inactivates Ras oncoproteins. NF1 is a characteristically variable disease that predisposes patients to several symptoms, the most common of which include benign and malignant tumors, reduced growth and learning problems. We and others previously found that fruit fly mutants that lack a highly conserveddNf1 gene are reduced in size and exhibit impaired learning and memory, and that both defects appear due to abnormal Ras and cyclic-AMP (cAMP) signaling. The former was unremarkable, but how loss ofdNf1 affects cAMP signaling remains poorly understood. Here we report results of a genetic screen for dominant modifiers of thedNf1 growth defect. This screen and follow-up functional studies support a model in which synaptic defects and reduced cAMP signaling in specific parts of the neuroendocrine ring gland contribute to thedNf1 growth defect. Beyond these results, we show that human ALK is expressed in cells that give rise to NF1 tumors, and that NF1 regulated ALK/RAS/ERK signaling is evolutionary conserved.

Loss ofdNf1 Does Not Phenocopy Starvation or Alter Developmental Timing

Animals use elaborate hormonal mechanisms to coordinate nutrient availability and feeding with changes in metabolism and overall growth. Since starvation or crowding during the larval phase of the Drosophila life cycle reduces systemic growth[17], we first examined whether the small size ofdNf1 mutants reflected reduced feeding. Arguing against this hypothesis, wild-type anddNf1 larvae ingested similar amounts of dye-stained food throughout their development (Figure 1A). Unlike apumpless (ppl) mutant[18],dNf1 larvae also showed no tendency to move away from a food source (Figure 1B). Analysis of the expression of the starvation-induciblePepck andLip3 genes[18] provided further evidence that loss ofdNf1 does not phenocopy starvation (Figure 1C).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Loss ofdNf1 does not phenocopy starvation or alter developmental timing.

(A) Wild-type (w¹¹¹⁸) anddNf1 larvae ingest similar amounts of food. Larvae at different stages of development were photographed after 25 minutes of feeding on dye-colored yeast paste. (B) As opposed toppl mutants, wild-type anddNf1 larvae do not wander from a food source (fraction of wandering larvae: WT 3.5% (SD 0.007),dNf1 2.5% (SD 0.007) andppl 65% (SD 0.057)). In a similar assay,dNf1 larvae also showed no abnormality in moving towards a food source (not shown). (C) RNA blot analysis of the starvation-sensitive genes, PEPCK andLip3 shows thatdNf1 larvae do not show elevated levels of either mRNA under normal feeding conditions. (D) Wild-type anddNf1 larvae show no significant differences in developmental timing, as assessed by time of pupariation after egg deposition (AED). (E) ThedNf1 growth rate, as assessed by larval weight, is reduced throughout larval development when compared to wild-type or aRas2>UAS-dNf1 control. (F) Two hypomorphic insulin receptor alleles,InR⁰⁵⁵⁴⁵ andInR³²⁷, do not modifydNf1 pupal size. (G) ILP mRNA expression is not obviously reduced indNf1 larvae. H)dNf1 adult flies show no altered longevity compared to wild-type controls. (I) Over-expression ofIlp2 from ahs-Ilp2 transgene indNf1 larvae results in a similar increase in size as in wild-type flies.

https://doi.org/10.1371/journal.pgen.1003958.g001

Mechanisms that control Drosophila growth have been the topic of intense study and much has been learned about how an interplay between insulin-like peptide (ILP) controlled growth rate and ecdysone controlled growth duration determines overall growth (see[19] and[20] for reviews). Arguing against an important role for ecdysone or other factors that control the length of the larval growth period, no differences in the expression of canonical ecdysone-regulated genes was found (results not shown) and no difference in developmental timing between wild-type anddNf1 mutants was detected (Figure 1D andS1). Rather, a reduced growth rate throughout larval development results in an approximately 25% weight reduction ofdNf1 pupae relative to isogenic controls (Figure 1E andS1).

Drosophila ILPs control systemic growth, metabolism, longevity, and female fecundity[21]–[24]. Among the eight Drosophila ILP genes,Ilp2,Ilp3 andIlp5 are co-expressed in bilateral clusters of seven insulin-producing neurosecretory cells (IPCs) in the larval brain[21]. Ablation of these cells causes a severe reduction in overall size, which is rescued by inducing the expression of ahsp70-Ilp2 transgene[22],[23]. However, several results argue against a role for ILPs in thedNf1 growth defect. Firstly, two hypomorphic insulin receptor alleles,InR⁰⁵⁵⁴⁵ andInR³²⁷, did not affectdNf1 pupal size (Figure 1F). Secondly, qRT-PCR analysis of RNA extracted from wandering wild-type anddNf1 third instar larvae detected no major differences in the expression ofIlp1 (not shown),Ilp2,Ilp3,Ilp5,Ilp6 andIlp7 in fed larvae. Among the three IPC expressed ILP genes, the expression ofIlp3 andIlp5 is reduced in response to starvation[21]. Starved wild-type anddNf1 larvae showed a similar reduction inIlp5 expression, whereasIlp3 showed a less pronounced response (Figure 1G). Thirdly, while certain insulin receptor or insulin receptor substrate (chico) mutants have an up to 85% increased life span[25],[26], the lifespan ofdNf1 mutants and isogenic controls was comparable (Figure 1H). We note that others previously reported a reduced life span for the originally identifieddNf1 p-element alleles, generated in a different genetic background[27]. Finally, we previously showed thatIlp2-GAL4 drivenUAS-dNf1 expression in IPCs did not rescue thedNf1 size defect[5]. Although daily heat shocking ofhsp70-ilp2 carrying larvae increased the size ofdNf1 pupae, indicating that mutants do not lack the ability to respond to insulin, similar induction of this transgene, as previously noted[21], also substantially increased the size of wild-type controls (Figure 1I). Thus, reduced insulin signaling does not provide an obvious explanation for the slowerdNf1 growth rate, prompting us to perform a screen to identify other genes involved indNf1-mediated systemic growth control.

Screen for Dominant Modifiers ofdNf1 Systemic Growth Phenotype

While mostdNf1 defects are poorly suited for use in modifier screens, the postembryonic growth defect is robust and readily quantified during the pupal stage[4]. However, using this phenotype in a screen is complicated by the fact that organismal size is sexually dimorphic (females are larger than males) and affected by population density, feeding, environmental factors and genetic background differences. With these confounding factors in mind, we used the crossing schemes outlined inFigure 2 to test collections of isogenic 1^st and 2^nd chromosome deficiencies fordNf1^E2 pupal size modifier effects or synthetic lethal interactions. For each of 139 1^st and 347 2^nd chromosome deficiencies from the Exelixis[28], DrosDel[29] or Bloomington Stock Center (BSC) collections, we generatedDf(1)/+; Nf1^E2/Nf1^E2 (Figure 2A) orDf(2)/+; Nf1^E2/Nf1^E2 (Figure 2B) stocks, respectively. Notably, our work identified only few synthetic lethal interactions, and in all cases tested the synthetic lethality has been specific to the chromosome carrying theNf1^E2 allele, and not observed when the same deficiency was tested inNf1^E2/Nf1^E1 null trans-heterozygotes[5]. To guard against size differences caused by inadvertent differences in population density or environmental conditions, each deficiency was scored at least twice using an initial rough caliper measurement of pupae attached to the side of culture vials. For each candidate modifying deficiency thus identified, microscopy combined with image analysis was used to determine the precise head-to-tail length of at least 40 pupae, which were then allowed to individually eclose in order to establish their sex. Several controls were next performed to eliminate non-specific modifiers or artifactual results. First, for all suppressors the continued presence of theNf1^E2 nonsense mutation was confirmed by a PCR assay (Figure S2). Secondly, as a critical specificity control, all modifying deficiencies were analyzed in a wild-type background to eliminate those that affect pupal size irrespective ofdNf1 genotype. Further analysis of some of these non-specific modifiers demonstrated that loss ofAct57B dominantly increases pupal size, whereas heterozygous loss of the glutamate transporterEaat1 has the opposite effect. Thirdly, because pupal size is a function of larval growth rate and duration, modifying deficiencies were monitored for obvious changes in developmental timing.Table 1 shows the number of screened chromosome 1, 2L and 2R deficiencies, the fraction of genes uncovered and the number ofdNf1 and wild-type pupal size modifying deficiencies and loci identified.Figure 2C shows the magnitude of the pupal size modification of typical enhancers and suppressors. The number of modifying deficiencies exceeds the number of identified loci, because many modifying deficiencies uncover overlapping genomic segments (Figure 3). Not unexpectedly, individual modifying deficiencies increase or decreasedNf1 pupal size to different extents (Figure 4).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Deficiency screen for dominant modifiers of thedNf1 growth defect.

Isogenic 1^st and 2^nd chromosomes deficiencies from the Exelixis, DrosDel and Bloomington Stock Center collections were tested for their ability to alterdNf1 female pupal size. Crossing schemes to generateDf(1)/+; dNf1^E2 (A) andDf(2)/CyO; dNf1^E2 (B) screening stocks. Thetubby-markedTM6B 3^rd chromosome balancer allowed the selection ofdNf1^E2 homozygotes for measurements. (C) Examples of deficiencies that suppress or enhance thedNf1 size defect. Scale bar = 1 mm.

https://doi.org/10.1371/journal.pgen.1003958.g002

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Cytogenetic locations ofdNf1 modifying deficiencies.

Locations of modifying deficiencies (drawn to scale) on the 1^st and 2^nd (2L and 2R) chromosomes. Deficiencies that enhance or suppress are shown in red and green, respectively. Non-specific deficiencies that dominantly affect the size of wild-type pupae are in blue. Many modifying deficiencies uncover overlapping genomic segments.

https://doi.org/10.1371/journal.pgen.1003958.g003

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Identified deficiencies increase or decrease pupal size to different extents.

Female pupal lengths for the indicated 1, 2L and 2R deficiencies. Control measurements fordNf1^E2 and wild-type (w¹¹¹⁸) are in black. Colors for enhancing, suppressing and non-specific deficiencies are as inFigure 2. Pupal lengths are shown in mm, error bars denote standard deviations and are based on measurements described inTable S2. All shown deficiencies modifydNf1 female pupal size withp-values<0.01.

https://doi.org/10.1371/journal.pgen.1003958.g004

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Deficiency screen summary.

https://doi.org/10.1371/journal.pgen.1003958.t001

Some large non-modifying deficiencies identified in our screen completely overlapped with smaller modifying ones. In such cases, stocks were re-ordered and reanalyzed. If these tests replicated the original results, genetic complementation analysis or PCR amplification using transposon and flanking sequence-specific primers was used to confirm the mapping of the deficiencies in question. This procedure identified several mismapped or mislabeled deficiencies, most of which have since been withdrawn by stock centers. Any suspect or recessive modifying deficiency, or any deficiency that uncovers genes with non-specific size phenotypes, such asMinute loci[30],[31], were eliminated from further analysis.Table S1 lists these deficiencies and the reason for their exclusion.

During work to identify genes responsible for observed effects, we prioritized genes uncovered by suppressing deficiencies over those uncovered by enhancers. We also prioritized modifying loci uncovered by more than one deficiency, strong modifiers over weak ones, and genes uncovered by smaller deficiencies over those uncovered by larger ones, reasoning that effects of smaller deficiencies are more likely due to the loss of single genes. Validating the screen, suppressingDf(2R)Exel7144 uncoversdAlk and partially overlapping suppressingDf(2R)BSC199 andDf(2R)BSC699 each uncover the gene for its activating ligand,jeb, both previously identified as dominant suppressors ofdNf1 size, learning, and neuronal ERK over-activation phenotypes[15]. Other uncovered candidate modifiers, such as PKA catalytic and regulatory subunit genes, were tested in crosses with loss-of-function alleles and/or by tissue-specific knockdown using at least two independent UAS-RNAi transgenes, most of which were obtained from the Vienna Drosophila Stock Center (VDRC)[32]. For deficiencies that lacked obvious candidate modifiers, we used the UAS-RNAi approach to more broadly screen uncovered genes.Figure S3 shows examples of modifiers identified by this latter approach. Although the nutrient sensing fat body and other tissues outside of the CNS play important roles in Drosophila growth control[33],[34], candidate modifiers have only been tested by RNAi knockdown in neurons or glial cells. We focused on these cell types, because neuronalUAS-dNf1 expression sufficed to suppress the growth phenotype[5].

ThedNf1 pupal size modifiers identified to date can be classified into three non-exclusive categories, the first of which consists of the previously implicateddAlk/jeb receptor/ligand pair and two not previously implicated other genes involved in Ras-mediated signal transduction. Another expected category includes genes involved in cAMP/PKA signaling, including the previously reporteddnc cAMP phosphodiesterase suppressor[35], and the newly identified PKA catalytic subunit gene,PKA-C1, which acts as an enhancer. This group also includes theCCKLR-17D1 drosulfakinin receptor, recently implicated as a cAMP-coupled promoter of synaptic growth[36], which is particularly interesting given the recent identification of adNf1 larval NMJ overgrowth phenotype[16]. Finally, our screen also identified multiple genes whose roles indNf1 growth control had not been anticipated and whose functional relevance remains to be established. Several genes in this group are predominantly expressed in brain or have known neuronal functions, including genes coding for the aforementioned CCKLR-17D1 receptor, the synaptic scaffold protein Dap160, the neuronal RNA binding protein elav, the neuronal Na,K ATPase interacting protein NKAIN[37], and the larval brain and alimentary channel expressed amino acid transporter NAAT1[38]. Other genes in this group includeCKIIbeta2, encoding a casein kinase regulatory subunit, the endosomal trafficking proteinsdeep-orange andcarnation, theNotch modifier heparan sulfate 3-O sulfotransferaseHs3st-B[39], and the ubiquitin E3 ligasesHERC2,which acts as a suppressor, and CUL3, which has the opposite effect.Table 2 lists deficiencies that modifydNf1 but not wild-type pupal size, limited to those for which the responsible gene has been identified.Table S2 identifies all analyzed deficiencies, indicates which modifieddNf1 pupal size (providing female pupal sizes as a gauge of modification strength), which also altered wild-type pupal size, and which deficiencies altered developmental timing.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Modifying deficiencies and identification of responsible genes.

https://doi.org/10.1371/journal.pgen.1003958.t002

dNf1 Pupal Size Modifiers Involved in Jeb/dAlk/Ras/ERK Signaling

We previously reported that thedAlk receptor tyrosine kinase[40] acts as a rate-limiting activator of neuronal Ras/ERK pathways responsible fordNf1 size and learning defects[15]. Therefore, the fact that thedAlk andjeb genes are uncovered by one and two suppressing deficiencies, respectively (Table 2), validates our screen. Others recently reported that Jeb/dAlk signaling allows brain growth to be spared at the expense of other tissues in nutrient restricted Drosophila, and identified a glial cell niche around neuroblasts as the source of Jeb under these conditions[41]. To determine whether glial cells also produce Jeb involved in overall growth control under normal conditions, we used glial and neuronal Gal4 drivers to test the effect of tissue-specificjeb anddAlk knockdown. Arguing that neurons are the main source of Jeb involved in systemic growth control under non-starvation conditions,jeb knockdown with theRas2-Gal4, C23-Gal4, and n-syb-Gal4 neuronal drivers[5] increaseddNf1^E2 pupal size (Figure 5A), whereas theNrv2-Gal4, Eaat1-Gal4 and Gli-Gal4 glial drivers had no effect (data not shown). The only glial driver that gave rise to partial rescue was the pan-glialrepo-Gal4 line, although this effect was not enhanced by co-expressingUAS-Dcr2. Control experiments showed that any driver used in these and other experiments had no effect on pupal size in the absence of UAS transgenes or vice-versa, that UAS transgenes had no effect in the absence of Gal4 drivers (Figure 5A and data not shown). Finally, extending previous findings and further confirming a role forjeb as a dominantdNf1 size defect suppressor, thejeb^weli loss-of-function allele[42] dominantly increaseddNf1 pupal size (Figure 5B)

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 5. Validation ofdNf1 modifiers involved in Jeb/dAlk/Ras/ERK and cAMP signaling.

(A) Neuronal expression ofdAlk RNAi usingRas2-Gal4,Ras2-Gal4+UAS-Dcr-2,c23-Gal4 orn-syb-Gal4 drivers suppresses thedNf1 size defect. Expression ofjeb RNAi with the same neuronal drivers also suppresses. Weaker suppression is observed whenjeb RNAi expression is controlled by the pan-glialrepo-Gal4 driver. Dark grey bars are control measurements of Gal4 drivers in thedNf1 background. Light grey bars are sizes of wild-type (w¹¹¹⁸) anddNf1^E2 controls. (B) Suppression of thedNf1 size defect by the indicatedjeb,cnk,Dsor1 andamx alleles. (C) Neuronalcnk,Dsor1 oramx knockdown suppressed thedNf1 size defect. In the case ofDsor1 v107276 andamx, cultures were maintained at 18°C to prevent lethality observed at 25°C. Some RNAi transgene/driver combinations were lethal (†) even at 18°C. (D) Validation ofdnc andPka-C1 asdNf1 modifiers was obtained in crosses withdnc^M14,dnc^ML,Pka-C1⁶³⁵³ andPka-C1^BG02142 loss-of-function alleles. In this and subsequent figures, * and ** denotep-values<0.05 and <0.01, respectively.

https://doi.org/10.1371/journal.pgen.1003958.g005

Previously, heterozygous mutations affecting RAF/MEK/ERK kinase cascade componentsDraf (pole hole;phl),Dsor1/dMEK, or ERK/rolled (rl), did not modifydNf1 size[5]. In agreement, twophl-uncovering deficiencies,Df(1)ED6574 andDf(1)ED11354, did not score as modifiers (Table S2). Norl uncovering deficiencies were analyzed, butDf(1)Exel9049, which is among the stronger suppressors identified, deletesDsor1 and only two other genes, the neurogenic genealmondex (amx), andCG17754, predicting a BTB and Kelch domain protein. Arguing that reduced Ras/ERK signaling upon loss ofDsor1 combined with abnormal neuronal differentiation due to loss ofamx may synergistically cause the observed strong effect,Ras2-Gal4 driven UAS-RNAi transgenes targeting either gene, while causing pupal lethality at 25°C, increaseddNf1 pupal size at lower temperatures (Figure 5C). Moreover, suppression of thedNf1 pupal size defect was also observed upon individual heterozygous loss of eitherDsor1 oramx, although at least with the tested alleles, combined loss of both genes did not have a more pronounced effect (Figure 5B). Previously, we did not observe suppression of thedNf1^E2 pupal size defect in crosses with theDsor1^S-1221 allele[5]. A potential explanation may be thatDsor1^LH110 is a null mutant[43], whereas the molecular nature ofDsor1^S-1221 is undetermined. Genetic background differences between theseDsor1 alleles are another potential explanation for the discrepant results.

Multiple screens aimed at identifying genes involved in Drosophila tyrosine kinase/Ras signaling have been performed[44]–[52]. Among the genes identified, several are uncovered by 1^st and 2^nd chromosome deficiencies that do not modifydNf1 size. SuppressingDf(2R)BSC161 uncovers 27 genes including connector enhancer of KSR (cnk), a scaffold protein that functions as a bimodal (both positive and negative) regulator of RAS/MAPK signaling[53],[54]. Supporting a role forcnk as adNf1 modifier, thecnk^XE-385 andcnk^E-2083 alleles acted as dominant suppressors (Figure 5B), and suppression was also observed upon RNAi-mediated Cnk knockdown usingRas2-Gal4 orP(GawB)C23-Gal4 neuronal drivers (Figure 5C). However,Df(2R)BSC154, which uncoverscnk and only nine other genes, did not score as a modifier (Table S2).

dNf1 Size Modifiers Involved in cAMP/PKA Signaling

ThedNf1 growth defect is suppressed by heat shock-induced expression of a constitutively active murine PKA catalytic subunit transgene, called PKA*[4], or by loss of thedunce (dnc) cAMP phosphodiesterase[35]. Further validating our screen, twodnc uncovering deficiencies and another that removes the region immediately upstream of thednc coding region, all scored as suppressors (Table 2). Moreover, thePka-R2 gene, encoding a cAMP binding regulatory PKA subunit, whose dissociation from the catalytic subunit activates the latter, is uncovered by two additional suppressing deficiencies, whereas a deficiency that uncovers the majorPka-C1 catalytic subunit gene scored as an enhancer (Table 2).Df(1)ED7261, which uncovers therutabaga (rut) adenylyl cyclase, did not score as a modifier (not shown). Confirmation ofdnc andPka-C1 as the genes responsible for the observed effects was obtained in crosses with threednc and threePka-C1 loss-of-function alleles (Table 2).Pka-R2 remains an attractive candidate suppressor, but expressionPka-R2^RNAi transgenes in neurons had no effect and its role as adNf1 modifier remains unconfirmed (results not shown).

NoveldNf1 Modifiers

Recently, the cAMP-coupled CCKLR-17D1 drosulfakinin receptor, but not its closely related CCKLR-17D3 paralog, was identified as a positive regulator of synaptic growth[36]. TheCCKLR-17D1 gene is uncovered by three suppressing deficiencies, includingDf(1)Exel9051, which uncovers only three other genes. The closely linkedCCKLR-17D3 paralog is not uncovered byDf(1)Exel9051, and whileRas2-Gal4 orP(GawB)C23-Gal4 driven neuronalCCKLR-17D1 RNAi expression strongly suppressed thedNf1 pupal size defect, similar suppression ofCCKLR-17D3 had no effect (Figure 6A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 6. Validation ofdNf1 modifiers with neuronal functions.

(A)Ras2-Gal4 orC23-Gal4 driven neuronal RNAi knockdown ofCCKLR-17D1 but notCCKLR-17D3 suppressed thedNf1 pupal size defect. (B) Identification of dynamin-associated protein 160 (Dap160) as a suppressor ofdNf1 growth. Neuronal RNAi targeting ofDap160 increaseddNf1 pupal size as did twoDap160 loss-of-function alleles. (C) Twoelav alleles dominantly suppress thedNf1 size defect. (D) Neuronal expression of a Rab9 RNAi transgene or of a dominant negative Rab9 mutant suppresses thedNf1 size defect.

https://doi.org/10.1371/journal.pgen.1003958.g006

BeyondCCKLR-17D1, severaldNf1 size modifiers are expressed in brain and/or have neuronal functions. Among these, dynamin-associated protein 160 (Dap160) is an intersectin-related scaffold implicated in synaptic vesicle exocytosis and neuroblast proliferation[55]–[58].Dap160 is uncovered by suppressing deficienciesDf(2L)Exel6047 andDf(2L)BSC302, whose region of overlap encompasses ten genes. We note thatDf(2L)Exel6047 also uncovers the DrosophilaRet tyrosine kinase gene, the human ortholog of which is the receptor for glial-derived neurotrophic factor.Ret initially appeared an especially attractive candidate suppressor, because activatingRET and inactivatingNF1 mutations can both lead to human pheochromocytoma[59], and because DrosophilaRet is expressed in larval brain neurons that resemble neuroendocrine cells[60]. However, among multiple lines of evidence that argue against a role forRet in thedNf1 growth defect,UAS-dNf1 re-expression directed by a newly generatedRet-Gal4 driver that recapitulates the endogenous larval brain Ret expression pattern (Figure S4B), or RNAi-mediated Ret inhibition, did not modifydNf1 pupal size, nor did expression of aUAS-Ret K805A kinase dead transgene. Moreover,Ret-Gal4 driven expression ofUAS-Ret transgenes carrying the activating C695R mutation, which mimics a mutation found in multiple endocrine neoplasia type 2 did not phenocopy thedNf1 reduced growth phenotype, although the same transgene did produce the previously described rough eye phenotype when driven byGMR-Gal4[60];Figure S4C]. Further arguing against a role indNf1 growth control,Ret is uncovered by non-modifyingDf(2L)BSC312. By contrast,Dap160 loss-of-function alleles (Dap160^Δ1 and Dap160^Δ2;[56]), orDap160 RNAi expression driven by three neuronal Gal4 drivers, suppressed thedNf1 pupal size defect, identifying it as the responsible modifier (Figure 6B).

The gene for the neuronal RNA binding protein elav is uncovered by suppressingDf(1)Exel6221 andDf(1)ED6396 whose region of overlap includes just three other genes. Identifyingelav as the responsible modifier,elav¹ andelav^G0031 alleles strongly suppressed (Figure 6C).Rab9 is a modifier uncovered by suppressing deficiencyDf(2L)Exel8041. Neuronal but not glialRab9^RNAi expression increasesdNf1 pupal size, and the same result is seen upon neuronal expression of a Rab9 dominant negative[61] mutant (Figure 6D).

NAAT1, coding for a larval gut and brain expressed amino acid transporter with a unique affinity for D-amino acids[38], is uncovered by suppressingDf(1)Exel6290 andDf(1)BSC533 whose region of overlap includes only four other genes. IdentifyingNAAT1 as the responsible suppressor, three neuronal Gal4 lines driving the expression of threeNAAT1 targeting RNAi transgenes suppressed thedNf1 size defect, whereasRepo-Gal4 driven glial expression had no effect (Figure 7A andTable 2).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 7. Identification of modifying genes with undetermined roles indNf1 suppression.

(A) Validation ofNAAT1,HERC2 andHs3st-B asdNf1 modifiers. All three genes were identified by systematic RNAi screening of genes uncovered by suppressing deficiencies. (B) Loss-of-function alleles of Class C Vacuolar Protein Sorting complex subunitscarnation (car/Vps33A) anddeep-orange (dor/Vps18) increasedNf1 pupal size. C) RNAi-mediated neuronalcar ordor knockdown was not particularly effective, suggesting these genes may function elsewhere to modifydNf1-dependent growth.

https://doi.org/10.1371/journal.pgen.1003958.g007

Mammalian E3 ubiquitin ligase HERC2 controls the ubiquitin-dependent assembly of DNA repair proteins on damaged chromosomes[62]. DrosophilaHERC2 is uncovered by suppressing deficiencyDf(1)Exel6254, which also uncovers thesyx16, coding for syntaxin 16. NoHERC2 alleles exist, butRas2-Gal4 driven expression of aUAS-HERC2^RNAi transgene (v105374) strongly suppressed thedNf1 pupal size defect (Figure 7A), whereas similar knockdown ofSyx16 had no statistically significant effect (not shown). The gene for another E3 ligase component,Cul-3, is uncovered by three enhancing deficiencies, and aCul-3 loss-of-function allele orRas2-Gal4 driven expression of aCul-3 RNAi transgene both enhanced thedNf1 size defect, identifying it as the responsible gene (Table 2).

SuppressingDf(1)Exel9068 uncovers only four genes, including one encoding the TORC2 complex subunit Rictor. However, systematicRas2-Gal4 driven RNAi knockdown ofDf(1)Exel9068 uncovered genes identifiedHs3st-B, encoding one of two Drosophila heparan sulfate 3-O sulfotransferases, as a potentdNf1 size defect suppressor (Figure 7A), whereas knockdown of Rictor had no effect (not shown). Others previously identifiedHs3st-B as a positive regulator of Notch signaling[39]. However, the heparan sulfate proteoglycan substrates ofHs3st-B bind various growth factors and other ligands and have been implicated in a variety of biological processes. Exactly why loss ofHs3st-B suppresses thedNf1 growth defect remains to be determined.

Two functionally relateddNf1 growth defect suppressorscarnation (car/Vps33A) anddeep-orange (dor/Vps18), encode subunits of the Class C Vacuolar Protein Sorting (VPS) complex, required for the delivery of endosomal vesicles to lysosomes[63];Figure 7B]. TheVps16A gene encodes a third member of this complex[64], but whetherVps16A located on the 3^rd chromosome also acts as adNf1 suppressor, or whether pharmacological inhibition of lysosomal degradation affectsdNf1 pupal size are questions that remain to be answered.

B4/Susi is a coiled-coil protein without obvious orthologs outside of insects. It functions as a negative regulator of Drosophila class I phosphatidylinositol-3 kinase Pi3K92E/Dp110 by binding to its Pi3K21B/dP60 regulatory subunit. HomozygousB4 mutants have an increased body size[65], which may explain whyRas2-Gal4-driven RNAi-mediated suppression ofB4, uncovered by suppressing deficiencyDf(2L)BSC147, increaseddNf1 pupal size (not shown). However, whetherB4 is the responsible dominant modifier is doubtful, given that it is also uncovered byDf(2L)BSC692, a non-modifying deficiency. Moreover, we previously found that heterozygous loss ofPi3K21B, or neuronal expression of a dominant negativePi3K92E transgene, did not modifydNf1 pupal size[5]. BeyondB4,dNf1 size modifying deficiencies uncovered no genes involved in the canonical growth regulating pathways mediated by insulin and ecdysone. Indeed, several such genes were uncovered by non-modifying deficiencies. Among these genes, fat body expressed insulin-like growth factorIlp6, which regulates larval growth in the post-feeding phase[66],[67], is uncovered by two non-modifying deficiencies. A single non-modifying deficiency,Df(2L)BSC206, uncovers both thechico andpten genes, whose products antagonistically control insulin-stimulated Pi3K92E/Dp110 activity, leading to changes in body, organ, and cell size[68],[69]. Among subunits of the cell growth regulating mTORC1 complex,raptor is uncovered by three andTor by one non-modifying deficiency. Among genes implicated in ecdysone signaling, the ecdysone co-receptorultraspiracle and the ecdysone-induced growth regulatingDHR4 nuclear receptor[70] are each uncovered by non-modifying deficiencies, and two such deficiencies uncoverPtth, coding for prothoracicotropic hormone, which provides developmental timing cues by stimulating the production of ecdysone[71],[72]. These results reinforce our conclusion that the canonical growth regulating pathways involving insulin and ecdysone play no obvious roles indNf1 growth control.

Manipulating cAMP/PKA Signaling in the Ring Gland AffectsdNf1 Systemic Growth Non-Cell-Autonomously

Several results argue that defects in Ras/ERK and cAMP/PKA signaling responsible for thedNf1 growth defect involve non-overlapping cell populations. Firstly, heat shock-inducedhsp70-PKA*, orRas2-Gal4 induced attenuatedUAS-PKA* transgene (see below) expression rescued thedNf1 pupal size defect, but failed to reduce the elevated larval brain phospho-ERK level (Figure 8A). Moreover, several neuronal RNAi drivers that increasedNf1 pupal size when drivingUAS-dNf1[5], failed to modify this phenotype when drivingdnc^RNAi transgenes, even in the presence of theUAS-Dcr-2 RNAi enhancer (Table 3). This prompted us to investigate whether genetic manipulation of cAMP/PKA signaling in cells other thandNf1 requiring neurons was more effective.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 8.dNf1 systemic growth related RAS/ERK and cAMP/PKA signals appear functionally and topographically distinct.

(A) The elevated larval CNS pERK level ofdNf1 mutants is reduced by neuronal expression ofdNf1, but not by neuronal or heat-shock induced ubiquitous expression of PKA*. Western blot of pERK levels in larval CNS of the indicated genotypes. In lane 6, larvae received a daily 20 min 37°C heat shock throughout development, a protocol that suppresses thedNf1 growth defect[4]. (B) Structure ofUAS-PKA* transgenes with 1 to 5 UAS elements. The lethality of these transgenes when driven with eitherAc5C-Gal4 orelav-Gal4 is indicated by † whereas (−) indicates viable offspring. (C) Western blot of adult head lysates showing relative expression ofGMR-Gal4-driven transgenic PKA*. Tubulin is used as a loading control. (D) Expression of PKA* or knockdown ofdnc by shRNAi in the ring gland rescues thedNf1 pupal size defect. In contrast,UAS-dNf1 expression with the same ring gland drivers fails to restore systemic growth. (E–H) Expression pattern ofAkh-Gal4 drivingUAS-GFP, co-stained with DAPI and anti-dNF1. GFP expression in the corpora cardiaca (CC) is indicated. Scale bar = 50 µm. As previously noted[74], anti-dNf1 staining is strong in the CNS, whereas staining in the ring gland is close to background.

https://doi.org/10.1371/journal.pgen.1003958.g008

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 3. Restoration of systemic growth bydNf1 and cAMP/PKA involves different tissues.

https://doi.org/10.1371/journal.pgen.1003958.t003

To manipulate cAMP/PKA signaling tissue-specifically we used threeUAS-dnc^RNAi transgenes. We also generated a series of attenuatedUAS-PKA* transgenes using vectors with modified Gal4-inducible promoters harboring just 2, 3 or 4 Gal4-binding UAS elements (Figure 8B and C). We made the latter transgenes because aUAS-PKA* expression using the five UAS element containing standard UAS-T vector is lethal in combination with most Gal4 drivers[73]. As reported previously[74], drivingUAS-dNf1 ubiquitously withAct5C-Gal4, or broadly in neurons withelav-Gal4,Ras2-Gal4,c23-Gal4, or386Y-Gal4 restoreddNf1 pupal size, whereas driving the same transgene with more restricted neuronal or non-neuronal drivers had no effect (Figure 8D andTable 3). By contrast, driving the expression ofUAS-dnc^RNAi or attenuatedUAS-PKA* transgenes with the same set of broadly expressed neuronal drivers was ineffective (Tables 3 andS5). We note that expression of the2×UAS-PKA* and3×-UAS-PKA* transgenes was generally well tolerated, whereas the4×UAS-PKA* and the5×UAS-PKA* transgenes exhibited increasing levels of lethality (Tables 3 andS5). Arguing that rescue of thedNf1 growth defect by manipulating cAMP/PKA signaling ordNf1 expression involves different cells, strong pupal size rescue was observed by increasing cAMP/PKA signaling in adipokinetic hormone-producing cells at the base of the neuroendocrine ring gland using theAkh-Gal4 driver (Figure 8D). Rescue was also observed with theFeb36-Gal4 andAug21-Gal4 ring gland drivers (Figure 8D), which give rise to expression in the corpora allata, the source of juvenile hormone, but not with the P0206-Gal4 or Mai60-Gal4 drivers, which express predominantly in the prothoracic gland (Table 3). The tissue specificity of all Gal4 drivers used in this and other experiments was verified by microscopic observation of dissectedUAS-GFP expressing larvae (Table S4 andFigures 8E–H andS5).

dAlk,Jeb,Cnk andCCKLR-17D1 Suppress adNf1 NMJ Architectural Defect

During larval development, significant expansion of the NMJ arbor must occur, reflecting the steady muscle growth that takes place during larval life. As the NMJ grows, additional branches and boutons are added to the initial synaptic arbor that forms during late embryonic stages upon motor axon contact with its target muscle. As a result, at the wandering third instar stage, wild-type NMJs contain a highly stereotyped, segment specific number of synaptic boutons[75]. Recently, it was reported thatdNf1 functions presynaptically to constrain NMJ synaptic growth and neurotransmission[16]. IndNf1 null mutant wandering third instar larvae, while the distribution of major presynaptic proteins is unaffected, increased overall size and synaptic bouton number is apparent at multiple NMJs, supporting a specific role fordNf1 in restricting NMJ expansion[16]. SeveraldNf1 suppressors that emerged in the current screen have also been linked to synapse morphogenesis, including CCKLR-17D1, which functions as a promoter of NMJ growth[36]. As our screen identifiedCCKLR-17D1 as a dominantdNf1 size defect suppressor, we wanted to confirm thedNf1 NMJ phenotype and test whetherCCKLR-17D1 and other suppressors affected this defect.

By quantifying bouton number at the NMJ on muscles 6 and 7, we confirmed thatdNf1 mutants have a significant increase in mean bouton number (Figure 9A and B). In addition, this analysis confirmed previously published phenotypes fordAlk,jeb andCCKLR-17D1[36],[76]. Importantly, thedNf1 synaptic overgrowth phenotype is dominantly suppressed byCCKLR-17D1,dAlk,jeb, andcnk alleles (Figure 9B), arguing that all four genes are epistatic todNf1. As a control we analyzed an allele ofspitz (spi), which encodes an EGF-like growth factor and is uncovered by suppressingDf(2L)Exel8041. However,spi shows no genetic interaction withdNf1, as loss ofspi modified neither the pupal size nor the NMJ overgrowth phenotypes (Figure 9B and data not shown).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 9. SeveraldNf1 pupal size defect suppressors also suppress a NMJ synaptic overgrowth phenotype.

(A–E) Representative micrographs of larval muscle 6/7 NMJs of the indicated genotypes. F: Mean bouton number per NMJ normalized to wild-type control. Compared to wild-type (w¹¹¹⁸; A),dNf1 mutants (dNf1^E2; B) have an increased bouton number. While acnk loss-of-function allele had no obvious NMJ phenotype, it dominantly suppressed thedNf1 NMJ defect (C). Similarly, thedNf1 NMJ phenotype was suppressed inDf(1)Exel9051 males that lack CCKLR-17D1 (D), while females heterozygous for CCKLR-17D1 (E) showed a lower level of suppression.Spitz (spi) is uncovered by a modifying deficiency but does not affectdNf1 size and was used as a negative control. In panels A–E, scale bars represent 5 µm. In panel F, error bars denote standard error of the mean.

https://doi.org/10.1371/journal.pgen.1003958.g009

Human ALK Is Expressed in Schwann Cells and May Serve as a Therapeutic Target in NF1

The identification ofdAlk as a suppressor of all hitherto analyzeddNf1 defects prompted us to explore whether human ALK represents a therapeutic target in NF1. Given our hypothesis that NF1 negatively regulates ALK stimulated Ras/ERK signaling, in order to play such a role, ALK and NF1 must be co-expressed in cells that give rise to symptoms. We previously found thatdNf1 anddAlk expression overlaps extensively in Drosophila larval and adult CNS[15], and the expression of orthologs of both genes also overlaps in the murine CNS[77],[78]. While overlapping CNS expression is compatible with a role for ALK in NF1-associated cognitive dysfunction, a causative role in another hallmark NF1 symptom, peripheral nerve-associated tumors, is less obvious. Among the near universal symptoms on NF1, benign neurofibromas consist of Schwann cells, perineurial fibroblasts, infiltrating mast cells, and nerve elements, with the Schwann cells sustaining the secondNF1 hit[79]. To test whether increased ALK signaling in the absence of NF1 might play a role in the development of neurofibromas, we used reverse transcription/PCR to detect the presence or absence ofALK mRNA in neurofibroma-derivedNF1^−/− Schwann cells andNF1^+/− fibroblasts, using RNAs kindly provided by Drs. Eric Legius and Eline Beert. In these experiments, two different primer sets readily detected ALK mRNA inNF1^−/− Schwann cells, but not inNF1^+/− fibroblasts derived from the same tumors (Figure S6).

To test whether functional interactions between NF1 and ALK exist in human cells, we used the SK-SY5Y and Kelly neuroblastoma cells, both of which harbor constitutively active F1174LALK alleles, and both of which are highly sensitive to pharmacological ALK inhibition[80]. Compatible with a role for NF1 as a negative regulator of mitogenic ALK/RAS signals, qRT-PCR verifiedNF1 knockdown with two shRNA retroviral vectors increased the resistance of both lines to ALK inhibitors NVP-TAE684 and Crizotinib (Figures 10A, 10C andS7). Compatible with a model in which NF1 negatively regulates ALK/RAS signaling,NF1 knockdown resulted in elevated ERK and AKT activation (Figures 10B). Moreover, expression of activatedKRAS,BRAF, orMEK transgenes, but not of other Ras effector transgenes, in SH-SY5Y cells conferred similar resistance to ALK inhibition (Figure S8).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 10.NF1 suppression leads to ERK activation and confers resistance to ALK inhibitors in human neuroblastoma cells.

(A)NF1 knockdown confers resistance to ALK inhibitors in human neuroblastoma cells. SH-SY5Y cells expressing pRS and shGFP control vectors, or shNF1 vectors were grown in the absence or presence 50 nM NVP-TAE684 or 250 nM crizotinib. The cells were fixed, stained and photographed after 14 (untreated and crizotinib treated), or 21 (NVP-TAE684 treated) days. (B) Down-regulation ofNF1 results in elevated level of phosphorylated p-ERK and p-AKT. Western blot analysis of total lysates of SH-SY5Y cells expressing pRS, shGFP or shNF1 vectors. (C) The level ofNF1 knockdown by each of the RNAi vectors was measured by examining theNF1 mRNA levels by qRT-PCR. Error bars denote standard deviation.

https://doi.org/10.1371/journal.pgen.1003958.g010

Fly Stocks and Experiments

ThedNf1^E1 anddNf1^E2 alleles have been described[5]. Exelixis, DrosDel and BSC deficiencies were obtained from the Bloomington Stock Center. Transgenic RNAi lines were obtained from the ViennaDrosophila Research Center (VDRC) and the TRiP Collection at Harvard Medical School.Eaat1^SM1 andEaat1^SM2 were provided by D. van Meyel,dALK⁸ andjeb^weli by R. Palmer,cnk^XE-385 andcnk^E-2083 by M. Therrien, andcar^Δ146 by H. Kramer,ppl⁰⁶⁹¹³ by M. Pankratz,hs-Ilp2 transgenic line by E. Rulifson andUAS-Rab9 DN by R. Hiesinger. Flies were maintained on agar-oatmeal-molasses medium at 25°C, unless otherwise indicated.

To assess feeding, larvae at various stages of development were placed on blue food dye-stained yeast paste, removed after 20 min, washed and photographed. To analyze wandering behavior, 100 larvae (age 40–44 hr after egg deposition (AED)) were placed on an agar plate with a central blob of yeast paste, and their position after 24 hr was documented. To assess the expression of starvation-sensitive genes, larvae at 72 h AED were placed in vials with water for 16 hr, after which RNA was prepared and subjected to blot analysis. To determine developmental timing, L1 larvae were collected 24 hr AED using a 2 hr egg collection and reared at 140 animals per vial. The number of larvae that pupariated was scored at hourly intervals. To determine the larval weight, L1 larvae were collected 24 hr AED using a 2 hr egg collection. Larvae were reared at 140 larvae per vial and groups of 10 larvae were weighed at 8 hr intervals. Longevity was assessed by maintaining adult flies under standard conditions and counting the number of dead flies at regular intervals. In each of these assays, genotypes were tested in duplicate. To inducehs-Ilp2 transgene expression, culture vials were placed in a circulating water bath at 37°C for 10 min once or twice a day with an 8 hr interval.

Insulin-Like Protein mRNA Quantification

The 7500 Fast Real-Time PCR System from Applied Biosystems was used to determineIlp mRNA levels in RNA prepared from dissected larval brains or from whole wandering stage 3^rd instar larvae. Results were normalized toRpL32. The following primers were used:IIp2-Forward,GGCCAGCTCCACAGTGAAGT,Ilp2-Reverse,TCGCTGTCGGCACCGGGCAT,Ilp3-Forward,CCAGGCCACCATGAAGTTGT.Ilp3-Reverse,TTGAAGTTCACGGGGTCCAA,Ilp5-Forward,TCCGCCCAGGCCGCAAACTC,Ilp5-Reverse,TAATCGAATAGGCCCAAGGT,Ilp6-Forward,CGATGTATTTCCCAACAGTTTCG,Ilp6-Reverse,AAATCGGTTACGTTCTGCAAGTC,Ilp7-Forward,CAAAAAGAGGACGGGCAATG,Ilp7-Reverse,GCCATCAGGTTCCGTGGTT. Expression of the distantly relatedIlp8 and the midgut-expressedIlp4 genes[21] was not analyzed.

Genetic Screening, Validation, and Statistical Analysis

The crossing schemes inFigure 2 were used to generatedNf1^E2 mutants carrying 1^st and 2^nd chromosome deficiencies. To avoid crowding, cultures were maintained at 100–200 pupae per culture vial. Initial scoring used calipers set at the length ofdNf1 female pupae, ignoringdNf1 heterozygotes recognizable by the presence of theTM6B balancer. Next, the length of individual pupae carrying candidate modifying deficiencies was measured by determining their head-to-tail length using a microscope fitted with NIS-Elements AR 3.0 imaging software. Measured pupae were then placed in 96-well plates (Falcon) to determine their gender and, if necessary, the genotype of eclosed flies. At least 40 pupae were measured for each genotype, and only measurements of female pupae were used to calculate mean values and standard deviations. Statistical significance was assessed with a two-tailed Student'st-test. Throughout this report, single or double asterisks denotep-values<0.05 or <0.01 respectively.

To identify responsible modifiers we used specific alleles orUAS-RNAi knockdown. Alleles andUAS-RNAi lines on the 1^st and 2^nd chromosomes were crossed into thedNf1^E2 background.UAS-RNAi lines on the 3^rd chromosome were recombined withdNf1^E2.UAS-RNAi lines in thedNf1^E2 background were crossed to Gal4 drivers in the same background. The few deficiencies that gave rise to synthetic lethal interactions were backcrossed withdNf1^E1 flies to produce Df/+;dNf1^E2/dNf1^E1 progeny.

To test whether genetic suppression reflected the inadvertent introduction of a wild-typedNf1 allele, we used fly DNA prepared using DNAzol (Molecular Research Inc.) in a PCR assay withAGTCACATTAATTGATCCTG andGAGATCGTTGATAAAGAAGT primers. The second primer introduces a penultimate single nucleotide change, which together with the E2 mutation results in the introduction of an RsaI restriction site. RsaI digestion of the PCR product gives rise to 370 and 61 bp fragments for the wild-type allele, and 348, 61 and 22 bp fragments forthe dNf1^E2 allele. Digests were run on 8% acrylamide gels using both wild-type (w¹¹¹⁸) anddNf1^E2 controls.

Construction ofAkh-Gal4 and AttenuatedUAS-PKA* Transgenes

TheAkh promoter region was amplified withAkh-FORWARD (AGATCTAATCTCCTGAATGCCGCAGCG) andAkh-REVERSE (AGATCTATGCTGGTCCACTTCGATTC) primers. The resulting PCR fragment was subcloned into the BamHI site of a GAL4 coding region containing pCaSpeR derivative. The final construct was sequenced to ensure correct orientation of the Akh promoter before being used generate transgenic flies by standard protocols.

To reduce the toxicity associated with high-level PKA expression, we generated modified pUAS-T vectors containing 1, 2, 3 or 4, rather than 5 Gal4-binding sites. The primers used to generate these vectors were: 1×UAS-FOR:AACTGCAGAGCGGAGTACTGTCCTCCGAGCGGAGACTCTAG; 2×UAS-FOR:AACTGCAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCG; 3×UAS-FOR:AACTGCAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCG, and UAS-REV:CTAGAGGTACCCTCGAGCGCGGCCGCAAGAT. An initial PCR was performed using the 1×UAS-FOR and UAS-REV primers with the standard pUAS-T vector as a template. The resulting amplified fragment was TA subcloned into pCR2.1 to make pCR2.1-1×UAS. The 2×UAS-FOR and UAS-REV primers were then used with pCR2.1-UAS(1×) as a template to generate a UAS(2×) clone, which was subcloned to produce pCR2.1-UAS(2×). Similarly, 3×UAS-FOR and UAS-REV primers in a PCR reaction with pCR2.1-UAS(2×) as template generated pCR2.1-UAS(3×) and pCR2.1-UAS(4×)). The pCR2.1-UAS clones were sequenced, their inserts excised with PstI and subcloned into PstI-digested p-UAST. Correct insert orientation was verified by sequence analysis, after which the mutationally activated murine PKA* coding region[100] was subcloned into the modified vectors using XbaI and NotI.

Immunofluorescence and Analysis of NMJ Morphology

Wandering third instar larvae were dissected in Ca²⁺-free saline and fixed in 4% paraformaldehyde for 25 min at room temperature. Following fixation, larval pelts were washed three times in phosphate-buffered saline (PBS) and then blocked for one hour in PBT (PBS+0.1% Triton-X 100)+5% normal goat serum. Larvae were incubated in primary antibody solution for three hours at room temperature. Anti-HRP 568 (1∶1000, Invitrogen) was used to visualize neurons and Alexa Fluor 488 phalloidin (1∶500, Invitrogen) was used to visualize F-actin in the musculature. Images were collected using a Yokogawa CSU-X1 spinning-disk confocal microscope with the Spectral Applied Research (Richmond Hill, ON, Canada) Borealis modification on a Nikon (Melville, NY) Ti-E inverted microscope using a 60× Plan Apo (1.4 NA) objective. The microscope was equipped with a Prior (Rockland, MA) Proscan II motorized stage. Larval samples were excited with 488-nm (for phalloidin) and 561-nm (for HRP) 100-mW solid-state lasers from a Spectral Applied Research LMM-5 laser merge module and was selected and controlled with an acousto-optical tunable filter. Emission was collected with a Semrock (Rochester, NY) quad pass (405/491/561/642 nm) dichroic mirror and 525/50 nm (for phalloidin) and 620/60 nm (for HRP) Chroma (Bellows Falls, VT) emission filters. Images were acquired using a Hamamatsu ORCA-ER-cooled CCD camera. Hardware was controlled with MetaMorph (version 7.7.9) software (Molecular Devices, Sunnyvale, CA.). Five individual animals were imaged for subsequent morphological analysis. Motor nerve terminals of muscles 6 and 7 were imaged in abdominal segments A2 and A3 and Z-stacks (0.25 µM between images) and were captured from the top to bottom of each NMJ. Morphological analysis of the NMJ was performed using NIH Image J and was assessed by quantifying the number of synaptic boutons per square micron. The number of synaptic boutons was counted as previously described[16],[101] and muscle area covered by the NMJ was quantified by tracing a polygon connecting each terminal branch point[102].

Human NF1 Experiments

The retroviral RNAi vectors targeting humanNF1 and expression constructs of active alleles of RAS effectors were as described previously[94]. Crizotinib (S1068) and NVP-TAE648 (S1108) were purchased from Selleck Chemicals. Antibody against NF1 was from Bethyl Laboratories (A300-140A); antibodies against pAKT(S473) and ATK1/2 were from Cell Signalling; antibodies against p-ERK (E-4), ERK1 (C-16), ERK2 (C-14) and CDK4 (C-22) were from Santa Cruz Biotechnology; A mixture of ERK1 and ERK2 antibodies was used for detection of total ERK from human cell lines. Antibody against mouse PKAα-cat (A-2) SC-28315 was from Santa Cruz Biotechnology, β-Tubulin E7 from Developmental Studies Hybridoma Bank.

SH-SY5Y, Kelly and Phoenix cells were cultured in DMEM with 8% heat-inactivated fetal bovine serum, penicillin and streptomycin at 5% CO₂. Subclones of each cell line expressing the murine ecotropic receptor were generated and used for all experiments shown. Phoenix cells were used to produce retroviral supernatants as described athttp://www.stanford.edu/group/nolan/retroviral_systems/phx.html.

To measure cell proliferation, single cell suspensions were seeded into 6-well plates (1–2×10⁴ cells/well) and cultured both in the absence and presence of ALK inhibitors. At the indicated endpoints, cells were fixed, stained with crystal violet and photographed. All knockdown and overexpression experiments were done by retroviral infection as described previously[103].

The 7500 Fast Real-Time PCR System from Applied Biosystems was used to determine mRNA levels.NF1 mRNA expression levels were normalized to expression ofGAPDH. The following primers sequences were used in the SYBR Green master mix (Roche):GAPDH-Forward,AAGGTGAAGGTCGGAGTCAA;GAPDH-Reverse,AATGAAGGGGTCATTGATGG;NF1-Forward,TGTCAGTGCATAACCTCTTGC;NF1-Reverse,AGTGCCATCACTCTTTTCTGAAG.ALK mRNA levels in neurofibroma-derivedNF1^−/− Schwann cells andNF1^+/− fibroblasts were analyzed using the following two primer sets:ALK-N-Forward,GGAGTGCAGCTTTGACTTCC;ALK-N-Reverse,TGGAGTCAGCTGAGGTGTTG;ALK-C-Forward,GCAACATCAGCCTGAAGACA;ALK-C-Reverse,GCCTGTTGAGAGACCAGGAG.

Figure S1.

Loss ofdNf1 does not alter developmental timing but reduces larval growth rate. (A) Wild-type,dNf1^E1, anddNF1^E1/E2 mutants show no altered developmental timing, as judged by their rate of pupariation (also shown inFigure 1D). By contrast, larvae withphm-Gal4 drivingUAS-Ras1^V12 undergo accelerated development resulting in miniature pupae[104], whereasphm-Gal4 driving a dominant negativeUAS-PI3K^D954A transgene delayed development and produced giant pupae[71]. (B) Mouth hook length measurements (in µm) show thatdNf1 larvae grow at a reduced rate. The marker represents the mean length; the upper box represents the median to Q3 value, the lower box median to Q1 value and the error bars identify the outliers.

https://doi.org/10.1371/journal.pgen.1003958.s001

(PDF)

Figure S2.

PCR/RFLP assay fordNf1^E2 mutation. (A) To make sure that stocks with putative suppressing deficiencies preserved thedNf1^E2 C->T nonsense transition, we used a PCR/Restriction Fragment Length Polymorphism assay. The E2 mutation does not create or destroy a restriction site. Rather, we used a reverse primer with a penultimate A->C transversion to amplify a 431 genomic fragment as indicated. The mutant primer creates a GTAC RsaI restriction site when E2 genomic DNA is used as a template. (B) RsaI digestion of PCR products gives rise to 370 and 61 bp fragments for the wild-type allele, and 348, 61 and 22 bp fragments fordNf1^E2. An example of the assay is shown with both wild-type (w¹¹¹⁸) and dNf1^E2 controls (lanes 2, 3 and 4) and various deficiencies (Df) either in wild-type (Df/CyO; +; lanes 5 and 15),dNf1 homozygous (Df/CyO; dNf1^E2; lanes 6–13) or heterozygous (Df/CyO; dNf1^E2/+; lanes 14 and 16) backgrounds.

https://doi.org/10.1371/journal.pgen.1003958.s002

(PDF)

Figure S3.

Systematic identification fordNf1 modifiers. For deficiencies that did not uncover obvious candidate modifier genes, a systematic RNAi approach was used. UAS-RNAi lines targeting genes uncovered by a modifying deficiency were driven byRas2-Gal4 in thedNf1^E2 background and the effect on pupal size determined. (A) Identification ofcarnation as adNf1 modifier uncovered by suppressingDf(1)BSC275. (B) Identification ofNAAT1 as the responsible gene uncovered by suppressing deficienciesDf(1)BSC533 andDf(1)Exel6290. RNAi-induced lethality is denoted by †. Error bars show standard deviations and * indicates ap-value of <0.05. As part of the systematic identification of modifiers 385 RNAi lines were tested.

https://doi.org/10.1371/journal.pgen.1003958.s003

(PDF)

Figure S4.

TheRet tyrosine kinase is not involved indNf1 growth control. (A) Reagents generated to analyze the involvement of Ret includeRet-Gal4 transgenic lines made by inserting a 957-bp genomic segment representing theRet promoter region into the pChs-Gal4 vector. Other reagents includeUAS-Ret transgenes harboring kinase-dead (K805A) and constitutively active (C695R) mutations made by site-directed mutagenesis. (B)Ret-Gal4 drivenUAS-GFP expression recapitulates the endogenous larval brainRet expression pattern[60]. (C)GMR-Gal4 drivenUAS-Ret with a constitutively active C695R mutation produces a rough eye phenotype as previously reported[60]. (D)Ret-Gal4 drivenUAS-dNf1 re-expression, RNAi-mediatedRet inhibition or expression of aUAS-Ret kinase dead transgene, all failed to modifydNf1 pupal size. Moreover,Ret-Gal4 driven expression ofUAS-Ret with constitutively active C695R mutation failed to phenocopy thedNf1 size defect. By contrast, a small pupal size phenocopy was observed when Ret C695R was driven ectopically withRas2- andelav-Gal4, likely reflecting Ret-mediated activation of Ras/ERK signaling.

https://doi.org/10.1371/journal.pgen.1003958.s004

(PDF)

Figure S5.

Expression pattern of ring gland drivers. Ring gland driversP0206-Gal4,Feb36-Gal4,Aug21-Gal4 andAkh-Gal4 were crossed toUAS-GFP. The CNS and ring glands were dissected from third instar larvae, stained with DAPI and imaged using confocal microscopy. The prothoracic gland (PG), corpora allatum (CA) and corpora cardiaca (CC) are indicated. Specimens are orientated such that the base of the brain hemispheres is at the top, indicated by a dotted line. Scale bar = 50 µm.

https://doi.org/10.1371/journal.pgen.1003958.s005

(PDF)

Figure S6.

ALK mRNA expression in neurofibroma-derived Schwann cells. Reverse transcription/PCR was used to analyze ALK expression in neurofibroma-derivedNF1^−/− Schwann cells andNF1^+/− fibroblasts. Two primer sets, (A) ALK-N and (B) ALK-C, designed to amplify N-terminal and C-terminalALK mRNA segments, detectedALK expression inNF1^−/− Schwann cells, but not inNF1^+/− fibroblasts.GAPDH primers were used as a control. To guard against positive signals due to contaminating genomic DNA, each PCR reaction was set up either with (+RT) or without (−RT) reverse transcriptase.

https://doi.org/10.1371/journal.pgen.1003958.s006

(PDF)

Figure S7.

NF1 suppression confers resistance to ALK inhibitors in human neuroblastoma cells. (A) Kelly cells expressing pRS and shGFP controls or shNF1 vectors were grown in the absence or presence 200 nM NVP-TAE684 or 500 nM crizotinib. Cells were fixed, stained and photographed after 14 (untreated) or 17 (NVP-TAE684 or crizotinib-treated) days. (B) Level ofNF1 knockdown assayed by qRT-PCR. Error bars denote standard deviation.

https://doi.org/10.1371/journal.pgen.1003958.s007

(PDF)

Figure S8.

Activation of RAS-RAF-MEK cascade confers resistance to ALK inhibitors in neuroblastoma cells. (A) Constitutively activeKRAS^V12,BRAF^V600E orMEK1^S218D,S222D mutants confer resistance to ALK inhibitors. SH-SY5Y neuroblastoma cells expressing pBabe vector control or the indicated active RAS effector mutants were grown in the absence or presence 50 nM NVP-TAE684 or 350 nM crizotinib. The cells were fixed, stained and photographed after 12 (untreated) or 19 (NVP-TAE684 and crizotinib-treated) days. (B) Level of phosphorylated ERK and AKT in the SH-SY5Y cells described above.

https://doi.org/10.1371/journal.pgen.1003958.s008

(PDF)

Table S1.

Excluded deficiencies. Listed deficiencies were excluded for the reasons indicated. Deficiencies that failed to produce screening stocks are labeled ‘Impossible’. Unhealthy (sick) deficiencies or those that uncoveredMinute mutations were also excluded.

https://doi.org/10.1371/journal.pgen.1003958.s009

(PDF)

Table S2.

dNf1 modifier deficiency screen results. All deficiencies analyzed are listed according to their relative chromosomal position. The cytological location, molecular coordinates and the dominant effect ondNf1 pupal size (NO – no interaction, SUP - suppressor, ENH - enhancer) of each deficiency is given. Female pupal length measurements for deficiencies in thedNf1 mutant background are provided, together with standard deviations andp-values. Modifying deficiencies that were subsequently found to have an effect on wild-type pupal size are indicated (Yes – indicates that a deficiency has a non-specific effect; No – no observed effect on wild-type size; No* - has an effect on wild-type size, but in the opposite direction from the effect ondNf1 mutants). Where determined, the responsible gene identified under each modifying deficiency is shown. The final column contains notes such as deficiencies that result in altered developmental timing.

https://doi.org/10.1371/journal.pgen.1003958.s010

(PDF)

Table S3.

Growth related genes uncovered by screened deficiencies. 18 cell, tissue, or systemic growth implicated genes uncovered by analyzed 1^st and 2^nd chromosome deficiencies. Among the deficiencies listed, only those that uncovereddAlk orjeb modifieddNf1 pupal size.

https://doi.org/10.1371/journal.pgen.1003958.s011

(PDF)

Table S4.

Larval tissue expression patterns of Gal4 drivers. List of Gal4 driver lines used in this study and their expression patterns in third instar larvae as determined by crossing Gal4 drivers toUAS-GFP, or from published data. Abbreviations: Ring gland (RG), central nervous system (CNS), mushroom body (MB), prothoracic gland (PG), corpora allata (CA), corpora cardiaca (CC), neurosecretory neurons (NSNs), pars intercerebralis neurons (PI), corpora cardiaca innervating neurosecretory neuron of the medial subesophageal ganglion 2 (CC-MS 2), proventriculus (PV), fat body (FB), salivary glands (SG), imaginal discs (IDs), first instar (L1).

https://doi.org/10.1371/journal.pgen.1003958.s012

(PDF)

Table S5.

Identification of tissues that requiredNf1 or cAMP/PKA signaling for growth regulation. Various Gal4 drivers in thedNf1 background were crossed todNf1 mutants bearing attenuatedUAS-PKA* transgenes ordnc RNAi lines. Rescue was assessed by measuring pupae, followed by genotyping adult flies upon eclosion. All crosses produced viable adults unless otherwise stated. † denotes lethality; NR non-rescue; NR* denotes non-rescue with adult eclosers with unfurled wings; n/a not applicable; n/d not determined.

https://doi.org/10.1371/journal.pgen.1003958.s013

(PDF)