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Human Molecular Genetics Advance Access originally published online on January 6, 2006
Human Molecular Genetics 2006 15(4):543-553; doi:10.1093/hmg/ddi471
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations

Kimihiko Oishi1, Konstantin Gaengel2, Srinivasan Krishnamoorthy3, Kenichi Kamiya1, In-Kyong Kim1, Huiwen Ying1, Ursula Weber2, Lizabeth A. Perkins3, Marco Tartaglia4, Marek Mlodzik2, Leslie Pick5 and Bruce D. Gelb1,*

1Departments of Pediatrics and Human Genetics and 2Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1498, New York, NY 10029, USA, 3Pediatric Surgical Research Lab, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA, 4Laboratorio di Metabolismo e Biochimica Patologica, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy and 5Department of Entomology, University of Maryland, 4112A Plant Sciences Building, College Park, MD 20742, USA

* To whom correspondence should be addressed. Tel: +1 2126596705; Fax: +1 2128492508; Email: bruce.gelb{at}mssm.edu

Received October 7, 2005; Revised December 27, 2005; Accepted December 31, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, causes Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than the leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, we generated transgenic fruitflies with GAL4-inducible expression of wild-type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles were lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele caused ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses established that cswN308D's ectopic wing vein phenotype required intact EGF ligand and receptor, and that this transgene interacted genetically with Notch, DPP and JAK/STAT signaling. Expression of the mutant csw transgenes increased RAS-MAP kinase activation, which was necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Noonan syndrome (NS; MIM# 163950 [OMIM] ) is an autosomal dominant disorder with pleomorphic features that include congenital heart disease, facial dysmorphism, short stature, skeletal defects and hematological abnormalities (1Go,2Go). The prevalence of NS is relatively high among Mendelian disorders (1 in 1000–2500 live births), and it is the most common non-chromosomal syndrome with abnormal cardiac development (3Go,4Go). A variety of heart defects is observed in NS with pulmonary valve stenosis being the most common abnormality (5Go,6Go).

NS is genetically heterogeneous. A genome-wide scan established a genetic locus on human chromosome 12 (4Go) and our group identified PTPN11 as the NS gene in that region (7Go). NS-causing PTPN11 mutations are virtually always missense changes and account for approximately 50% of cases. In addition, it was shown that somatic mutations in PTPN11 cause several hematopoietic proliferative disorders including juvenile myelomonocytic leukemia (JMML), acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) (8Go–11Go). A small percentage of NS patients, particularly those inheriting the T73I mutation germline, also develop JMML (8Go).

PTPN11 encodes the non-membranous protein tyrosine phosphatase, SHP-2. SHP-2 is one of a family of src homology-2 (SH2) containing protein tyrosine phosphatases (PTPs) that are highly conserved between species (12Go). SHP-2 is ubiquitously expressed and regulates signaling for several receptor tyrosine kinases (RTKs) like EGFR or FGFR through the activation of the RAS/MAP kinase cascade, leading to cell proliferation, differentiation and migration (13Go). In most contexts and unlike its homologue SHP-1, SHP-2 plays a positive role in signal transduction. SHP-2 comprises two SH2 domains (N-SH2 and C-SH2) in the N-terminal half of the protein and a PTP domain in the C-terminal half. SHP-2 has two conformations, active and inactive (14Go–16Go). In the inactive basal state, the backside of the N-SH2 domain forms a loop and is wedged into the PTP domain, blocking the catalytic site. After engagement of proteins with phosphotyrosyl residues at binding sites in the N- and C-SH2 domains, there is a molecular switching to the active state that is initiated by a conformational change in the N-SH2 domain, which then releases from the PTP domain.

The vast majority of NS-causing PTPN11 mutations alter residues at the interface between the N-SH2 and PTP domains (7Go,17Go–19Go). Similarly, the somatic mutations associated with leukemia also affect the interface although they cluster primarily in the N-SH2 domain and tend to result in different amino acid substitutions than the NS mutations. This interface clustering of NS- and leukemia-associated mutations suggests that these amino acid substitutions alter the molecular switching of SHP-2 from its inactive to active state, resulting in the gain-of-function effects (GOF). In vitro biochemical studies have supported this, documenting increased phosphatase activity and RAS/MAP kinase signaling (8Go,17Go,20Go–22Go). In animal systems, expression of a NS-causing mutant in Xenopus animal caps resulted in the induction of dorsolateral mesoderm in an Fgf-independent manner (21Go), and introduction of an NS-causing PTPN11 mutation in mice resulted in increased Ras activity (23Go).

Many aspects of the disease pathogenesis for NS and the hematopoietic disorders associated with PTPN11 mutation remains to be elucidated. As the biology of SHP-2 is quite complex because of interactions with several signaling pathways and a multitude of proteins, we took advantage of the existence of the closely related Drosophila orthologue, corkscrew (csw) (12Go,24Go), to model these diseases and perform epistatic studies. In this study, we generated transgenic Drosophila melanogaster expressing wild-type or mutant forms of CSW under control of the GAL4/UAS system (25Go). Ubiquitous expression of wild-type CSW resulted in no phenotype, whereas expression of GOF CSW proteins corresponding to mutations causing NS or leukemia resulted in phenotypes that differed with the strength of the alleles.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of GOF csw transgenic flies
To characterize the effects of disease-associated GOF mutants in signal transduction, we generated transgenic flies carrying wild-type csw or modified csw-bearing mutations that correspond to the mutations in PTPN11 found to be associated with NS or leukemia. Each is a point mutation that results in a change in a single amino acid: A72S, E76K, and N308D (see Introduction). Each csw allele was inserted into the pUASp vector and multiple independent transgenic lines were established for each construct (Table 1). In in vitro biochemical studies, E76K, which is associated with leukemia, is the strongest allele; A72S is a moderately strong NS allele and N308D is the weakest but commonest NS allele.


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  		Table 1. Transgenic csw alleles
 
To evaluate the CSW protein levels from the transgene expression, we performed immunoprecipitation and western analysis using anti-CSW antibodies. In stage 8–10 embryos, the levels of tub-GAL4 induced CSW were similar between the wild-type and mutant csw transgenic lines (Fig. 1).


Figure 4711
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  		Figure 1. Similar levels of CSW protein were induced in wild-type and NS-transgene expressing embryos by GAL4. Western blot of immunoprecipitated protein with anti-CSW antibody from stage 8–10 embryos. Although samples from non-GAL4 induced embryos (w1118 and transgene homozygotes) showed low or undetectable levels of CSW protein, tub-GAL4 induced CSW levels were higher and reasonably similar between the samples from wild-type and the mutant csw transgenes. Lysates from tub-GAL4/TM3 embryos were used as a negative control.

 
GOF csw transgenes rescue the CSW hypomorphic phenotype
To test the function of the wild-type and mutant csw transgenes, we performed a rescue study using a hypomorphic csw allele, cswlf. This allele produces viable homozygous female and hemizygous male adult flies that exhibit small, rough eyes (Fig. 2B and H) with loss of photoreceptors, most commonly R7 but occasionally outer photoreceptors as well (26Go). Expression of transgenic wild-type CSW with a GMR-GAL4 driver in the eyes of male cswlf hemizygotes, significantly rescued the rough eye phenotype (Fig. 2C and I). Similarly, expression of any of the mutant transgenes, E76K, A72S or N308D, partially rescued the eye phenotype (Fig. 2D–F and J–L). In all cases, the cswlf/Y;GMR-GAL4/UAS-cswtg eyes displayed normal size and the arrangement of ommatidia was notably improved (although not completely normalized). Sections of those eyes revealed significant rescue in the number of R7 and outer photoreceptors (Table 2) as well as ommatidial structure (data not shown). There was no significant difference in the degree of rescue between the transgenic alleles (Table 2). Of note, expression of the wild-type and mutant csw transgenes, driven by a variety of GAL4 eye-specific promoters, in otherwise wild-type flies failed to induce a rough eye phenotype (data not shown). The results of the rescue experiment indicated that the NS transgenes retain function and do not appear to behave as loss of function (LOF) mutations, consistent with biochemical analyses of the human mutant SHP-2 proteins (see Introduction).


Figure 4712
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  		Figure 2. The hypomorphic csw rough eye phenotype can be rescued by NS-associated csw transgenes. Images of scanning electron microscopy of Drosophila eyes are shown. (A and G) Normal size of eye and ommatidia organization of control, +/Y;GMR-GAL4/+. (B and H) The cswlf/Y;GMR-GAL4/+ eyes are small and ommatidia are disorganized. (C and I) cswlf/Y;GMR-GAL4/UAS-cswWT. (D and J) cswlf/Y; GMR-GAL4/UAS-cswE76K (E and K) cswlf/Y;GMR-GAL4/UAS-cswA72S. (F and L) cswlf/Y;GMR-GAL4/UAS-cswN308D. Expression of the wild-type and three different NS-like csw transgenes rescues the rough eye phenotype with more normal eye size and ommatidial organization. Scale bar: 100 µm.

 

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  		Table 2. Rescue of hypomorphic csw allele's eye phenotype
 
Ubiquitous expression of stronger mutant csw alleles causes lethality
Expression of the csw transgenes ubiquitously using a tub-GAL4 driver resulted in different phenotypes. Flies expressing the cswWT allele were viable, appearing in the expected Mendelian ratio with no apparent abnormalities. When UAS-cswA72S and UAS-cswE76K homozygotes were crossed to tub-Gal4/TM3,Sb, no adult fly of the genotype UAS-cswtg;tub-Gal4 was recovered (Table 3). Lethality occurred primarily during the larval or pupal stages for cswA72S and during embryonic stages for cswE76K.


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  		Table 3. Viability of adult flies with ubiquitous expression of csw transgenes
 
Next, we sought to determine whether these strong GOF alleles were perturbing RTK signaling pathways that rely upon CSW as occurs with csw null and hypomorphic alleles (24Go,26Go). Specifically, we examined signaling in the Torso pathway, which regulates anterior/posterior patterning in the early embryo, using the expression of tailless (tll) as the read out. Morphologic assessments using immunohistochemistry were made for the Breathless (FGF) pathway in tracheal system formation and the Heartless pathway for the development of the Drosophila larval musculature and dorsal tube, the fly equivalent of the vertebrate heart. Analysis of these three RTK pathways revealed no abnormalities when the A72S and E76K transgenes were expressed ubiquitously, a notable contrast to the effects of LOF alleles (data not shown). Combined with the data showing that expression of these transgenes using a variety of GAL4 eye drivers failed to engender a rough eye phenotype, our results documented that these GOF alleles do not universally perturb development for signaling pathways dependent upon CSW.

Expression of the N308D csw transgene causes a wing vein phenotype
Flies expressing the cswN308D transgene ubiquitously were viable, appearing in the expected Mendelian ratio (Table 3). The viability of the N308 allele contrasted to the A72S and E76K alleles and was consistent with previous evidence that the former has the mildest GOF (8Go,20Go). Of note, doubling the dose of N308D through homozygosity resulted in lethality (n.b., cswN308D homozygotes without a GAL4 driver were normal). This showed that the lethality depended on the level of the GOF effect and not on the position of the mutation per se (i.e. N-SH2 versus PTP domain).

Although the N308D transgene did not appear to alter early development when expressed ubiquitously, the majority of the resulting adult flies displayed wing vein abnormalities (Fig. 3C). Specifically, N308D wings exhibited ectopic veins in the peripheral areas of L2 and the posterior cross vein. This wing phenotype was the opposite of that observed in cswlf hypomorphic flies (26Go), which have interrupted wing veins that failed to reach the distal wing margin (Fig. 3D). This finding confirmed that the N308 allele behaves as a GOF allele. In fact, the N308 wing phenotype closely resembled that observed with an Egfr GOF allele, EgfrE1 (also known as EgfrEllipse) (Fig. 3E) (27Go). As CSW (and SHP-2) has a known role in EGFR signaling, a pathway that is critical for wing vein development, this result implied that the N308D allele was engendering increased signaling through EGFR during wing vein formation.


Figure 4713
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  		Figure 3. Ubiquitous N308D CSW expression causes ectopic wing veins. Panels show adult wings from (A) w1118, (B) UAS-cswWT;tub-GAL4, (C) UAS-cswN308D;tub-GAL4, (D) cswlf and (E) EgfrE1. (B and C) Ubiquitous expression of cswN308D engenders ectopic veins, mainly in the peripheral areas of L2 and posterior cross vein (arrows), whereas wild-type csw transgene does not. (D) cswlf flies show abnormal wings with the shortened L2 and L5 vein (arrows) not properly reaching the margin of the wing. (C and E) The N308D wing phenotype resembles closely that observed with an Egfr GOF allele, EgfrE1.

 
Overexpression of CSW enhances MAP kinase signaling
As CSW is a positive regulator of signaling from a variety of growth factor receptors flowing through the RAS/MAP kinase cascade, the activation of this cascade was assessed immunohistochemically using an antibody specific for the diphosphorylated (activated) form of the MAP kinase, ERK (dpERK). Compared with wild-type flies (w1118), ubiquitous expression of wild-type or mutant CSW resulted in a significant increase in dpERK levels (Fig. 4A). The pattern of dpERK in those transgenic embryos, however, revealed no apparent ectopic foci (28Go,29Go). Of particular interest, the increase in activated ERK was equivalent between the mutant and wild-type csw transgenic embryos.


Figure 4714
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  		Figure 4. MAP kinase activation in ubiquitous NS-transgene expressing embryos. (A) Immunohistochemistry. Stage 10 embryos labeled with anti-dpERK antibody (detects activated, diphosphorylated MAP kinase) and anti-ERK antibody (detects all forms of MAP kinase). ERK is expressed ubiquitously at approximately equal levels in all fly lines. The expression pattern of dpERK is tissue-specific in all lines as previously described (28Go,29Go). The expression level of dpERK is greater in the transgenic lines compared to the wild-type embryo (w1118). The increase in dpERK is equivalent between the wild-type and mutant transgenic embryos. Similar findings were observed when MAP kinase activation was assessed in stage 7 and 8 embryos (data not shown). (B) Immunoblotting. Immunoblots of protein lysates from stage 8–10 embryos using antibodies against dpERK (activated MAP kinase) and ERK (all forms of MAP kinase). ERK is expressed at approximately equal levels in all fly lines. The expression of dpERK is increased in the wild-type and mutant transgenic embryos when compared with the wild-type embryo (w1118) showing an undetectable level of dpERK.

 
To confirm the increase in activated ERK quantitatively, we performed western analysis using antibodies against ERK and dpERK. As observed in immunohistochemical staining, ubiquitous expression of wild-type or mutant CSW resulted in a significant increase in dpERK levels when compared with wild-type flies (w1118) (Fig. 4B). The expression level of in the N308D transgenic embryo is slightly lower than those observed in the wild-type and the other transgenic lines.

As the wild-type transgene did not cause the phenotypes observed with the NS transgenes, we concluded that the increased activation of the RAS-MAP kinase cascade induced by GOF CSW is not sufficient to cause lethality or wing vein abnormalities at the stage of development we analyzed.

NS allele N308D interacts with EGFR signaling pathways
Given the fact that the wing phenotype observed in N308D transgenic flies closely resembled that of the flies carrying a GOF Egfr allele, we speculated that N308D increased EGFR signaling during wing vein formation. To test that, we crossed flies expressing cswN308D ubiquitously with a number of LOF and GOF alleles for genes in the EGFR signaling cascade (Table 4). The fly stock with the genotype of UAS-cswN308D/CyO;tub-GAL4/TM2 was crossed with a variety of fly lines carrying hypomorphic, LOF or GOF alleles for genes of interest. The wing vein phenotypes of progeny flies, carrying UAS-cswN308D;tub-GAL4 and a mutant allele are as shown in Figure 5B. As the majority of the wings with the genotype of UAS-cswN308D/+;tub-GAL4/+ were categorized into subclasses 3–5 (Table 4, Fig. 5A), we designated these subclasses as class B, the baseline phenotype of the cswN308D transgene. If wings had no or small ectopic veins (subclasses 0–2), they were scored as class A (suppression) and if wings have longer and complex vein formation, they were scored as class C (enhancement) (Fig. 5A). Figure 5B shows some examples of such wings from this study.


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  		Table 4. Genetic interactions with the cswN308D ectopic wing vein phenotype
 

Figure 4715
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  		Figure 5. Evaluation of the wing vein phenotype and wings of flies with ubiquitous N308D CSW expression combined with mutant alleles for genes from various signaling pathways. (A) Evaluation of the wing veins was performed according to the classification as illustrated. Each wing phenotype was assigned to one of the subclasses between 0 (no ectopic vein) and 6 (severe ectopic vein formation) according to the pattern of ectopic veins. Subclasses were grouped into classes A, B or C as shown. Class B was designated as the baseline phenotype associated with the N308D transgene. Assignment to classes A and C were considered to be suppression and enhancement, respectively. (B) Examples of ectopic wing vein phenotypes observed in flies of different genotypes. All flies had the UAS-cswN308D;tub-GAL4 genotype and were heterozygous for the indicated mutant alleles. Null or LOF alleles for vein, Ras85D, rolled, decapentaplegic and Mad (vn10567, Ras85DN17.UAS, rl10A, dppd6 and Mad8-2, respectively) suppressed the ectopic vein phenotype compared with the baseline N308D CSW phenotype. In contrast, null or LOF alleles for Gap1, sprouty, Delta, Enhancer of split and Stat92E (Gap1B2, sty226, Dl6B, E(spl)R1 and Stat92E06346, respectively) enhanced it.

 
Crosses to alleles affecting RAS signaling downstream of RTKs revealed that LOF alleles of positive regulators (downstream of receptor kinase (drk), son of sevenless (sos), Ras85D, Downstream of raf1 (Dsor1), rolled (Mapk), pointed (pnt), Hsp83) resulted in statistically significant suppression of ectopic vein formation (Table 4, Fig. 5B). Most wings showed no or minimal ectopic vein formation in the anterior part of the wing, which were consistently observed in the UAS-cswN308D/+;tub-GAL4/+ control wings. On the other hand, LOF alleles of negative regulators (sprouty (sty), Gap1) enhanced the phenotype with longer ectopic veins in the anterior part of the wings and multiple and complex ectopic vein formation in the posterior part (Table 4, Fig. 5B). LOF alleles of Egfr, its ligand (vein (vn)) and positive extracellular regulators (Star (S), rhomboid (rho)) suppressed the wing phenotype, whereas LOF alleles for argos (aos), a negative ligand regulator, enhanced it. LOF of Draf, daughter of sevenless (dos) and the Egfr ligand Spitz (spi) did not alter the ectopic wing vein phenotype; this could indicate that partial reductions of these signaling components was not rate-limiting. The results with the alleles that suppressed or enhanced the phenotype are consistent with the N308D allele causing an increase in EGFR signaling during wing vein formation. As the wing vein formation in Drosophila is tightly controlled by expression of ligands for EGFR (30Go–32Go) and the ectopic wing vein phenotype of N308D was suppressed with the reduced dosage of EGFR ligands, these studies documented the necessity of ligand stimulation of the receptor to engender the phenotype.

To look for genetic interactions between the N308D transgene and genes involved in other signal transduction pathways, we performed additional epistatic studies (Table 4). First, we tested the Notch pathway signaling. Activation of Notch signaling prevents vein formation by lateral inhibition (30Go). Among the genes involved in this signaling cascade, haploinsufficiency of the Notch ligand, Delta, and a positive regulator, enhancer of split, significantly enhanced the N308D phenotype. Decapentaplegic (dpp), the Drosophila orthologue of bone morphogenetic protein (BMP), also has an important role in wing vein differentiation (30Go,33Go). Along with hedgehog (Hh) expression, DPP signaling coordinates the expression of both vein- and intervein-specific genes along the anterior/posterior compartment boundary in the larval wing disc (30Go). In addition, maintenance of dpp expression as well as positive EGFR signaling determines vein cell differentiation in the pupal wing (30Go,31Go). Loss of one copy of the gene for the ligand, dpp, or a positively regulating SMAD homologue, Mad, caused significant loss of ectopic veins, whereas haploinsufficiency of a negative regulator, lack (Smurf1 homologue), enhanced ectopic vein formation (Table 4). These findings were consistent with the known crosstalk between EGFR signaling and the Notch and DPP pathways during wing development.

SHP-2 is required for JAK/STAT signaling, although its precise function has not been elucidated (34Go). Although no role for JAK/STAT signaling has been documented previously in the context of Drosophila wing vein development, crosses with mutant alleles for genes in this pathway revealed suppression of ectopic vein formation with haploinsufficiency for the positive regulators, hop (Jak) and Stat92E, and suppression with a GOF hop allele, hopTUM (Table 4).

Taken together, these observations indicated that the N308D transgene has GOF activity in EGFR signaling that requires the Ras-MAP kinase cascade as well as intact ligand and receptor. Furthermore, we showed that the N308D transgene had genetic interactions with other signaling pathways in wing development including DPP and Notch signaling, two pathways with known roles in wing development (henceforth referred to as canonical) and JAK/STAT signaling, which has no known role in wing development (henceforth referred to as non-canonical).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
CSW is the Drosophila orthologue of SHP-2 and works as a positive regulator of multiple RTK pathways. The amino acid sequence of SHP-2's PTP domain is 63% identical to CSW excluding an insertion of unknown consequence in the latter and is 76% similar in the SH2 domains (12Go,24Go). As the amino acids altered by the mutations in PTPN11 are conserved in the fly, we hypothesized that transgenic Drosophila expressing mutant CSW would model the GOF effects on signal transduction observed previously in cell culture (20Go) and frog animal cap (21Go) systems. In this study, we demonstrated that the mutant CSW proteins were biologically active in the eye rescue experiment and that they altered development. One NS transgene, N308D, had a GOF effect on wing vein formation, whereas two stronger GOF transgenes caused lethality when expressed ubiquitously.

Graded effects of SHP-2 and CSW GOF mutants
Among patients with NS who harbor PTPN11 mutations, no significant correlation between genotype and phenotype has been established (17Go,35Go), although the statistical power of those studies has been limited. Biochemical and cell physiologic studies, however, have documented that the GOF effects of NS-associated SHP-2 mutants vary, with the N308D allele being weakest (20Go,22Go). More strikingly, there are clinical, genetic and biochemical data showing that mutant SHP-2's causing hematopoietic proliferative disorders tend to have greater GOF effects than those associated with isolated NS. JMML occurring in the context of NS can be a milder disease than when it occurs in an otherwise normal child (36Go). NS with JMML is caused by specific germline PTPN11 mutations (8Go) (i.e. most NS-associated PTPN11 mutations including the commonest N308D allele have never been associated with JMML). In contrast, somatic PTPN11 mutations result in JMML, ALL and AML. The molecular lesions also differ from those in NS with generally less conservative amino acid substitutions that are almost entirely restricted to the N-SH2 domain. In vitro biochemical analyses revealed that the phosphatase activities of the leukemia-associated SHP-2 proteins are generally greater than the NS-associated ones (8Go,22Go,37Go). Finally, the leukemia-associated PTPN11 mutations appear to result in embryonic lethality in humans when transmitted through the germline (unpublished data). Thus, we had hypothesized previously that the GOF effects of PTPN11 mutation on signal transduction would be graded with leukemia mutants>NS/JMML mutants>NS mutants.

The results of the present work with the transgenic csw GOF fly models are consistent with that hypothesis. The leukemic allele, E76K, caused the earliest lethality when expressed ubiquitously. The A72S allele, which has been shown to be a relatively strong NS allele biochemically (20Go), was also lethal. The weakest NS allele, N308D, was not lethal until the dose was increased through transgene homozygosity. Similarly, exposing developing embryos to lower ambient temperatures, which decreases transgene expression, shifted the lethality of the A72S allele to later stages and permitted a few to survive to adulthood (data not shown). The molecular basis of the GOF strength differences among SHP-2 mutants has been attributed to relative effects on the molecular switching mechanism, an idea that still awaits experimental confirmation. The data presented here provide the first proof that the disease-associated PTPN11 mutations have graded effects on development.

N308D transgene expression and EGFR signaling
Ubiquitous expression of the cswN308D transgene resulted in ectopic wing vein formation. This closely resembled the phenotype resulting from an Egfr GOF allele. Consistent with the well-defined role of EGFR signaling and its activation by ligand stimulation in wing vein development (30Go–32Go), LOF alleles of nearly all extra- and intra-cellular members of the EGFR-RAS-MAP kinase signaling cascade that promote signaling suppressed the cswN308D-associated phenotype, whereas loss of negative regulators enhanced it. Despite the fact that most disease-associated SHP-2 mutant proteins including N308D have increased basal activity, our data from the fly model revealed that the N308D-induced perturbation of wing development requires an intact ligand and RTK. This is consistent with findings from our previous in vitro studies showing Egf-dependent and Gab1-mediated ERK2 prolonged kinase activity (8Go,20Go), as well as from our unpublished frog animal cap studies in which expression of a dominant-negative Fgfr quenched the induction of dorsolateral mesoderm from mutant SHP-2 expression (unpublished data). Although the precise basis for this reliance on ligand-induced RTK stimulation has not been elucidated, we speculate that there are two critical factors. First, signaling through RTKs such as EGFR requires ligand-induced phosphorylation of the receptor that then results in recruitment of numerous signaling and docking proteins including SHP-2 and CSW to the plasma membrane. In this context, basally activated SHP-2 or CSW is not physically associated with its requisite signaling partners unless and until the relevant RTK engages ligand. Second, SHP-2 is a positive regulator of signal transduction for many RTK-RAS-MAP kinase pathways, which primarily rely on phosphorylation to propagate their signals. This is true despite the fact that SHP-2 and CSW dephosphorylate RTKs such as PDGFR and Torso (38Go,39Go). Although the elucidation of the full range of bona fide SHP-2 substrates continues to be pursued, two defined SHP-2 substrates, PAG/cbp and Paxillin, negatively regulate RTK-RAS signaling (40Go,41Go). SHP-2 dephosphorylates these substrates, preventing them from recruiting Csk, which, in turn, is a negative regulator of Src family kinases. Although loss of SHP-2 activity reduces RAS signaling, SHP-2 GOF should not be sufficient to initiate it. Taken together, these two factors provide a rationale for our finding that reduction of EGFR or its ligand, vein, suppressed the formation of ectopic wing veins from N308D.

Pleomorphic effects of CSW GOF mutants on RTK signaling
Another striking finding observed with the csw GOF alleles was the pleomorphism of effects among RTK pathways in which CSW participates. Development of the trachea, dorsal tube and photoreceptor R7, structures dependent upon Breathless, Heartless and Sevenless signaling, respectively, were not altered. The domains of tailless expression in the anterior and posterior poles, which have been used as a read out of Torso signaling and diminish with loss of maternal CSW, showed no augmentation from CSW GOF. The pleomorphism was also present among structures whose development is EGFR-signaling dependent: N308D expression altered wing vein formation but did not affect photoreceptor development. Thus, we conclude that the CSW GOF mutants alter signal transduction in a selective and specific manner. This is consistent with the findings in NS, in which the GOF SHP-2 protein is ubiquitously expressed but the phenotype reflects developmental abnormalities occurring in a tissue- and stage-specific manner. In addition to that specificity, increased MAP kinase activation in the Ptpn11D61G knock-in model mice was noted only in specific tissues (23Go).

What is the basis for the specificity in developmental abnormalities arising from SHP-2/CSW GOF? First, GOF SHP-2 might dephosphorylate substrates promiscuously, thereby recruiting non-canonical signaling affecting specific developmental processes. In support of this, MAP kinase activation proved necessary but not sufficient for generating the CSW GOF phenotypes. Moreover, N308D expression recruited JAK/STAT signaling in the context of wing vein development, a non-canonical interaction. Of note, a similar phenomenon was observed for Torso signaling in which the effects of a torso GOF allele required STAT activation even though the JAK/STAT pathway plays no role in Torso signaling ordinarily (42Go). Second, there may be a critical developmental window that is dependent upon the dosage of SHP-2/CSW and/or MAP kinase activity. The tolerance to variability in these activities may differ among signaling pathways and between tissues for a given pathway. A comparison of the phenotypes observed in the N308D transgenic and the GOF EgfrE1 flies provide an example of the latter. Although both alleles increase MAP kinase activation in the context of EGFR signaling, the former resulted in ectopic wing veins, whereas the latter exhibited ectopic veins and a rough eye phenotype (27Go). The rough eye phenotype in EgfrE1 flies is a LOF EGFR phenotype as a result of negative feedback caused by high expression of a negative regulator, Argos, which is induced by GOF EGFR activity (43Go). Argos inhibits EGFR signaling by binding to an activating ligand, Spitz (44Go). On the other hand, the NS GOF CSW appears to cause high, non-constitutive EGFR signal activation, which apparently does not increase the argos expression level enough to induce the EGFR LOF phenotype during photoreceptor development. It is likely that there is a distinct difference (although it may be subtle) in the levels of EGFR signaling activity between these two models and it determines the phenotypic pattern. In particular, specificity of the developmental abnormality seems to be established by the level of increased signaling activity caused by the NS alleles in a given pathway and by a specific requirement of a fine-tuned signaling activity for normal development in a given tissue and/or cell type. This idea is supported by the work of Lesokhin et al. (43Go) using mutants with different levels of EGFR activity, which demonstrated that there are multiple distinct thresholds of required EGFR signaling activity in different cell types during retinal development. In contrast, EGFR signaling in wing vein specification predominantly uses the ligand, Vein, a neuregulin-like molecule. Vein is a relatively weak ligand, which is proposed to be required for the tight basal control of the level of EGFR/RAS/MAP kinase pathway signaling during wing vein specification (31Go). Thus, the more modest increased EGFR signaling induced by CSW GOF alleles is still sufficient to induce ectopic veins.

Epistatic studies and variability in the NS phenotype
The N308D allele interacted genetically with several genes from the EGFR/RAS/MAP kinase pathway as well as some from the Notch, DPP (BMP) and JAK/STAT pathways. The degree of the suppression or enhancement of the interacting genes varied. Although some of this variability could be attributed to allelism (e.g. null versus hypomorphic alleles for a single gene), it was apparent that there were differences in the intensity of the epistatic effects between interacting genes. A fuller elucidation of these epistatic genes might provide clues for understanding the clinical variability in NS. The phenotype of NS is variable among patients sharing the same PTPN11 mutation, even within families (35Go,45Go). Phenotypic variability was also observed in Ptpn11D61G knock-in mice, in which the cardiac phenotype was dependent upon the genetic background (23Go). Although stochastic effects may explain some of the variability in NS patients, the results of our epistatic studies underscore the fact that the PTPN11 mutant alleles are interacting with a variety of signal pathways, some finely balanced, such that relatively minor differences in gene expression or protein activities might significantly alter the phenotype.

Future directions for elucidating NS pathogenesis with animal models
Congenital heart disease is a prominent feature of NS. Pulmonary valve stenosis is the most common cardiac defect and is particularly prevalent among NS patients with PTPN11 mutations (17Go,35Go). To date, the specific molecular mechanism underlying pulmonary stenosis in NS is unknown. Recent cardiac embryologic studies have shown that a delicate regulation of a variety of signaling pathways including VEGF, Notch, Wnt/ß-catenin, TGFß, BMP and EGFR are important for valvulogenesis (46Go–49Go). Altered signaling leads to aberrant endothelial–mesenchymal transformation during cardiac valve cushion formation or remodeling of those cushions. Interestingly, the critical pathways for valvulogenesis are conserved in Drosophila and play essential roles for wing vein development (30Go,32Go). The utility of the Drosophila models was the high-throughput fashion in which epistasis between the N308D transgene and genes regulating the EGFR, Notch and DPP (BMP) pathways could be performed. As a result, a manageable set of testable hypotheses has emerged, which can be addressed experimentally in the existing mouse model. A sensitized screen with these CSW GOF flies, being unbiased and genome-wide, may provide additional insights. An alternative use of these flies will be to perform high throughput screening of therapeutic compounds as a first step towards the development of novel treatment strategies for NS and PTPN11-related cancers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Drosophila stocks
Unless otherwise specified, fly stocks are as described in FlyBase (http://flybase.bio.indiana.edu/), and were provided by the Bloomington Stock Center (http://flystocks.bio.indiana.edu/). w1118 was used as the wild-type control. Flies were cultured in standard medium and crosses were performed at 25°C if not otherwise noted.

Generation of transgenic flies
Site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) was used to introduce three mutations into csw that corresponded to the A72S, E76K and N308D mutations in PTPN11 (Table 1). The cDNAs were sequenced to document the presence of the desired mutation. The wild-type and mutated csw cDNAs were inserted into a pUASp vector, and P-element mediated transformation was performed to obtain NS transgenic flies (50Go,51Go). Multiple fly lines were generated for each construct, designated generally as UAS-cswtg and specific alleles as UAS-cswWT, UAS-cswA72S, UAS-cswE76K and UAS-cswN308D. Transgenic lines expressing protein at similar levels in the presence of the same GAL4 driver as assessed with immunoblots were chosen and used for further analyses (Fig. 1).

Genetic analyses
A rescue study of the hypomorphic csw allele, cswlf (26Go), was performed to establish activity of the respective transgenes: cswlf/FM6;GMR-GAL4/CyO females were crossed to UAS-cswtg homozygous males. The eye phenotype of the male progeny with the cswlf/Y;GMR-GAL4/UAS-cswtg genotype was analyzed. Hemizygous males of the cswlf/Y;GMR-GAL4/+ genotype served as controls. Images of adult eyes were obtained using light and scanning electron microscopy. For the analysis of tangential eye sections, eyes were embedded and sectioned as described (52Go). At least 200 ommatidia from three eyes were analyzed for each genotype. The number of photoreceptors in each ommatidium was counted and the percentage of the abnormal ommatidia was calculated. For statistical analysis, t-test was performed with a significance threshold of P<0.05.

For epistasis experiments, we generated a stable stock of the genotype UAS-cswN308D/CyO;tub-GAL4/TM2. This stock was then crossed to a variety of mutant flies with hypomorphic, LOF or GOF alleles for genes of interest. Using the ectopic vein phenotype as a read out, we graded changes in vein patterning using a classification system (Fig. 5A) to assess whether the presence of a mutant allele for the gene of interest suppressed or enhanced the ectopic wing veins. The N308D baseline ectopic vein phenotype was evaluated with UAS-cswN308D/+;tub-GAL4/+ flies and designated as class B (Table 4, Fig. 5A). Classes A and C were categorized as suppression and enhancement, respectively. For statistical analysis, female wings were scored and {chi}2-test was performed. Significant suppression or enhancement was declared with a threshold of P<0.05.

Immunobloting, immunoprecipitation and immunostaining
For immunobloting, protein lysates were obtained by homogenizing stage 8–10 embryos in a protein extraction buffer (20 mM Tris (pH 8.0), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml benzamidin, 5 mM sodium vanadate, and 1 mM NaF). Total protein of 40 µg was separated with SDS-PAGE and transferred to PVDF membrane. dpERK and ERK levels were analyzed using anti-dpERK and ERK antibodies (Cell Signaling Technology) and HRP-conjugated secondary antibodies. Signal was detected using a SuperSignal West Pico Chemiluminiscent Substrate (PIERCE). To evaluate the CSW protein level in embryos, 300 µg of total protein was incubated with F1088, a rabbit anti-CSW polyclonal antibody (53Go) and immobilized on protein A beads. Proteins of the immunoprecipitates were used for immunobloting as described earlier. For the CSW protein detection, FRat1, a rat polyclonal antibody was used (53Go).

For immunohistochemical or immunofluorescent staining, embryos were collected, fixed and stained as described (54Go,55Go). The primary antibodies used were: rabbit anti-ßGal (Cappel), mouse anti-dpERK (SIGMA) (28Go,29Go), rabbit anti-ERK2 (Santa Cruz), rabbit anti-Tinman (gift from M. Frasch) and mouse anti-trachea luminal antigen (2A12: Developmental Studies Hybridoma Bank). Biotin or FITC-conjugated secondary antibodies (Roche) were used. For some antibodies, the signal was enhanced using the VECTASTAIN ABC and DAB Substrate Kits (Vector).

In situ hybridization was performed as described (56Go). The tll cDNA was cloned into the pCRII-TOPO vector (Invitrogen), and cRNA probe was synthesized using the DIG RNA Labeling Kit (Roche).

Genotyping selection of embryos was performed using balancer chromosomes carrying either an eve-lacZ or GFP markers.


   ACKNOWLEDGEMENTS
 
We thank Cheryl C. Tan and Atsushi Sato for discussion with the experiments and Manfred Frasch for the anti-tinman antibody. This work was supported by grants from the National Institutes of Health to B.D.G. (HL71207), L.A.P. (GM61707 and HD39942, Project 2) and M.M. (EY14597) as well as from Telethon-Italy (GGP04172) and Programma di Collaborazione Italia-USA/Malattie Rare to M.T. The Uehara Memorial Foundation Fellowship supported K.O.

Conflict of Interest statement. Two of the authors (M.T. and B.D.G.) have a pending patent application concerning PTPN11 mutations in Noonan syndrome.


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