LETTER

Synergistic Activity of Sef and Sprouty Proteinsin Regulating the Expression of Gbx2 in theMid-Hindbrain RegionWei Lin,1,2,4 Naihe Jing,2 M. Albert Basson,3 Andree Dierich,4 Jonathan Licht,3 and Siew-Lan Ang1,4*1Medical Research Council, National Institute for Medical Research, London, UK2Shanghai Institutes for Biological Sciences, Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences, Shanghai, China3Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, New York4IGBMC, CNRS/INSERM/ULP, College de France, Strasbourg, France

Received 23 July 2004; Accepted 30 November 2004

Summary: Sef and Sprouty proteins function as feed-back antagonists of fibroblast growth factor (Fgf) signal-ing in zebrafish embryos. To study the role of Sef inmice, we generated Sef homozygous mutant animals.These animals are viable and show normal expression ofmid-hindbrain genes at embryonic days 8.5 and 9.5. Toinvestigate the possibility of functional synergismbetween Sef and Sprouty proteins, we electroporatedSprouty2Y55A, which functions in a dominant-negativemanner in tissue culture cells into the mid-hindbrainregion of wildtype and Sef mutant embryos. The expres-sion pattern of Gbx2, a downstream target of Fgf signal-ing, was expanded or shifted in electroporated embryos,and this effect was significantly enhanced in the Sefmutant background. Altogether, our results demonstratethat Sef and Sproutys function synergistically to regu-late Gbx2 expression in the anterior hindbrain. genesis41:110–115, 2005. �c 2005 Wiley-Liss, Inc.

Key words: Sprouty2 dominant negative; Sef; electro-poration; embryo culture; mid-hindbrain development

Cell–cell signaling by fibroblast growth factors (Fgfs) isinvolved in many processes, including control of cellproliferation, migration, differentiation, and embryonicpatterning. One developmental paradigm in which therole of Fgfs has been extensively studied is in the mid-hindbrain (MHB) region (reviewed in Liu and Joyner,2001; Wurst and Bally-Cuif, 2001). Here Fgf8 expressionmarks the position of the MHB organizer and loss offunction experiments in mouse and zebrafish haveshown that Fgf8 is required for the development of themidbrain anteriorly and cerebellum posteriorly (Meyerset al., 1998; Reifers et al., 1998; Chi et al., 2003). More-over, embryological experiments in chick and mousehave suggested that the intensity of Fgf signaling governsthe development of a cerebellar or midbrain fate (Marti-nez et al., 1999; Liu et al., 1999; Sato et al., 2001). Theseresults led to the suggestion that a high level of FGF8 sig-naling induces cerebellum development and a lowerlevel induces midbrain development. Likewise the dos-

age of Fgf8 determines whether cell survival is positivelyor negatively regulated in the forebrain (Storm et al.,2003). Hence, within the CNS the strength of Fgf signal-ing influences the type of cellular and molecular re-sponses and these findings emphasize the importance ofa tight regulation of this signaling pathway.

During the last few years, multiple feedback modula-tors of the Fgf signaling pathway have been identified,including Sef and Sproutys (Sprys). Sef encodes a pre-dicted transmembrane protein with amino acidsequence similarity to the intracellular domain of theInterleukin-17 receptor and was identified by in situhybridization screens (Furthauer et al., 2002; Tsanget al., 2002). In zebrafish embryos, Sef functions as afeedback antagonist of Ras/MAPK (mitogen-activatedprotein kinase)-mediated Fgf signaling. The exact mecha-nism of Sef action is not fully understood, but Sef inter-acts with Fgf receptors and prevents phosphorylation ofFgf receptor substrate 2, a mediator of Fgf signaling viaPI3 kinase and Ras/MAPK pathways. This suggests thatSef acts at the level of the receptor; however, overex-pression studies in zebrafish point to a role of Sef at thelevel or downstream of Mek, because the activity of con-stitutively active Mek was blocked by ectopic Sef expres-sion (reviewed in Tsang and David, 2004). Consistentwith this latter finding, human Sef inhibits the dissocia-tion of the Mek-Erk complex, and thus blocks nuclearlocalization of activated Erk without inhibiting the activ-

Current address for M.A. Basson, MRC Centre for Developmental Neuro-

biology, King’s College London, London SE1 1UL, UK.

* Correspondence to: Siew-lan Ang, Division of Developmental Neurobiol-

ogy, National Institute for Medical Research, The Ridgeway, Mill Hill,

NW7 1AA London, UK.

E-mail: sang@nimr.mrc.ac.ukGrant sponsors: MRC and by institutional funds from CNRS, INSERM, and

Hopital Universitaire de Strasbourg, Contract grant sponsor: Wellcome

Trust International Prize Traveling Fellowship, Contract grant number:

63370 (to M.A.B.)

Published online in

Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/gene.20103

' 2005 Wiley-Liss, Inc. genesis 41:110–115 (2005)

ity of Erk in the cytoplasm in cultured cells (Torii et al.,2004).

Spry was initially cloned as an inhibitor of receptortyrosine kinases in fruit flies (Hacohen et al., 1998). Sub-sequently, four mammalian genes (Spry1-4) encodingprotein homologs of Drosophila Spry have been identi-fied (Minowada et al., 1999. In addition to Spry, SPREDproteins (Spry-related EVH1-domain-containing proteins)have also recently been cloned. Spry and Spred proteinsact by interfering with the Ras/MAPK pathway down-stream of receptor tyrosine kinase signaling (reviewed inChristofori, 2003; Dikic and Giordano, 2003).

In order to determine a role for Sef during embryonicdevelopment, we generated Sefmutant mice. Sef homozy-gous embryos are viable and fertile and do not show anyobvious morphological phenotype during embryonicdevelopment. Since Sef, Spry1, and Spry2 show overlap-ping expression patterns in the MHB region (Minowadaet al., 1999), a dominant-negative approach involvingforced expression of Sprouty2Y55A (Sasaki et al., 2001;Hanafusa et al., 2002) into the midbrain of wildtype andSef homozygous embryos via electroporation was carriedout to test the possibility of functional synergism among

these proteins. Alteration of Gbx2 expression by electro-poration of Sprouty2Y55A into wildtype embryos supportsa role for Spry1 and Spry2 proteins in regulating MHBdevelopment. In addition, the significant enhancementof the effect of Sprouty2Y55A expression in a Sef mutantcompared to a wildtype background also strongly sug-gests that Sef and Sprys functionally cooperate to patternthe midbrain and anterior hindbrain.

RESULTS AND DISCUSSION

We previously reported the cloning of a mouse Sefgene that was expressed in very similar patterns withSpry2 and Fgf8 and suggested that these genes mightact in the same genetic pathway (Lin et al., 2002).To study the function of Sef in mouse embryos, weinserted PGK-neomycin into this gene by standardgene targeting technology in ES cells (Fig. 1), and calledthis allele Sef neo. This allele is likely to be inactive, asit contains a deletion of exons 5–12 which encodethe functional cytoplasmic domain determined in bio-logical assays in Xenopus and zebrafish embryos (Tsanget al., 2002; Torii et al., 2004). Heterozygous and

FIG. 1. Homologous recombination between the targeting construct and Sef genomic locus. The region from exon5 to exon12 of the Sefgene was replaced by a PGK-neomycin cassette. The PGK-thymidine kinase gene was simultaneously lost after recombination. Theseevents can be detected by Southern blot with both 50 and 30 probes. The pictures on the left-bottom show the bandshift before and afterrecombination. E, EcoRI; H, HindIII; K, KpnI; S, SpeI; X, XhoI.

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homozygous Sef mutant animals were obtained accord-ing to Mendelian ratios (51% and 21.5%, respectively;n ¼ 112) and are viable and fertile. Sef neo/neo embryoswere also harvested at E7.5–E9.5 (embryonic day),E12.5, and E15.5 and showed no obvious morphologicalphenotype (data not shown).

Given the importance of Fgf signaling in MHB devel-opment in vertebrate embryos, we therefore determinedwhether Sef homozygous embryos showed any transientalteration in the expression of genes involved in the pat-terning of this region of the brain. Expression of Fgf8(Fig. 2a,b) (Crossley and Martin, 1995) and Gbx2 (Fig.2c,d) (Wassarman et al., 1997), a downstream target ofFgf8 signaling is normally expressed in Sef homozygousembryos. Likewise, expression of other targets of FGFsignaling such as Spry1, Spry2, and Spry4 genes is simi-lar in Sef mutant and wildtype embryos (Fig. 2e–j). Insummary, Sef homozygous embryos showed no obviousphenotype at E8.5 and E9.5, according to the expressionof some MHB patterning genes.

Since Spry1, Spry2, and Sef interfere with Fgf/Ras/MAPK signaling and have similar expression patterns inthe MHB region, we hypothesized that these differentantagonists might have synergistic and/or redundantroles. We used Sprouty2Y55A (Spry2Y55A), which func-tions as a dominant-negative mutation of Sprys in culturedcells (Sasaki et al., 2001; Hanafusa et al., 2002) to over-come redundant activities of Spry proteins. The domi-nant-negative approach was also chosen becauseSef;Spry1 double homozygous mutants showed noobvious phenotype at E9.5 (our unpubl. results). In orderto interfere with Spry1 and Spry2 activity in the MHBregion prior to their expression at E8.5, we electropo-rated an expression construct of Spry2Y55A into the MHBregion of wildtype embryos at E7.75. After 1 day of

embryo culture, electroporated embryos were examinedfor the expression of Gbx2, a downstream target gene ofFgf8. The Spry2Y55A construct also contains an iresGFPcassette, so that the electroporation efficiency and theregion electroporated can be easily traced (Fig. 3a-e). Inwildtype embryos, electroporation of 1 mg/ml ofSpry2Y55A lead to 23% of embryos with expansion or shiftin the domain of Gbx2 expression (Fig. 3a,f,k; Table 1,and data not shown), while embryos electroporated witha control GFP construct showed normal expression ofGbx2within the MHB region (Fig. 3e,j,o). Consistent withthis finding, electroporation of 1 mg/ml of Spry2Y55A alsoresulted in a reduction in the domain of Otx2 expression(data not shown), possibly due to the repressive effects ofGbx2 on Otx2 expression (Wassarman et al., 1997). Alto-gether, these results demonstrate that Spry2Y55A is effec-tive in upregulating Gbx2 expression in the MHB regionand suggest that Spry1 and Spry2 function normally toregulate MHB development.

To determine whether Sef and Sprys also share similarroles in regulating Fgf signaling, we determined whethera Sef mutant background is able to modulate the effect ofSpry2Y55A on Gbx2 expression. We therefore electropo-rated 1 mg/ml of the Spry2Y55A construct into Sef homozy-gous embryos. Electroporated Sef mutant embryosshowed similar changes in Gbx2 expression in the MHBregion (Fig. 3b,g,l). Gbx2 expression was also observedin the r3 to r5 region in electroporated Sef mutantembryos (arrows in Fig. 3l,n), but not in electroporatedwildtype embryos (Fig. 3k,m). Importantly, a statisticallysignificant increase in the percentage of embryos show-ing alteration in Gbx2 expression was observed inthe Sef mutant background compared to the wildtypebackground (Table 1). In addition, electroporation of0.6 mg/ml of Spry2Y55A lead to changes in Gbx2 expres-

FIG. 2. Comparison of some MHB gene expression patterns in mouse embryo at E9.5 (a–d), E8.75 (e–h) and E8.5 (i,j). Analysis by whole-mount in situ hybridization for (a,b) Fgf8, (c,d) Gbx2, (e,f) Spry1, (g,h) Spry2, and (i,j) Spry4 in wildtype (WT) and Sef neo/neo mutant (MT)embryos.

112 LIN ET AL.

sion in Sef mutant embryos (Fig. 3d,n). In contrast,no change in Gbx2 expression was observed when0.6 mg/ml of Spry2Y55A was introduced into the MHBregion of wildtype embryos (Fig. 3c,m). Altogether,these results indicate that the effect of the Spry2Y55A

construct on Gbx2 expression is enhanced in the Sefhomozygous mutant background (Table 1). The ability ofSef to increase the effect of Spry proteins on Gbx2expression suggests that these molecules synergize tomodulate Fgf signaling in mouse embryos.

CONCLUSIONS AND PERSPECTIVES

Our results indicate that the Spry2Y55A construct iseffective in upregulating the expression of Gbx2, anFgf8 target gene in mouse embryos. This is the firstdemonstration of a functional role for the conservedamino acid residue tyrosine 55 of the Spry gene familyin mouse embryos. Our results also predict thatSpry2Y55A might be an effective tool to overcome func-tional redundancies among Spry family members dur-ing embryonic development. This work has thereforeprovided the impetus for generating transgenic miceexpressing Spry2Y55A that can be used to study therole of Spry genes in different organ systems, includingin the MHB region. In this article, we have also demon-strated that Sef and Spry function synergistically toregulate Gbx2 expression in the MHB region, likelyvia regulating FGF signaling, by performing electropo-ration studies on wildtype and mutant embryos. Thecombination of genetic and embryological methods,

exemplified here, therefore represents an alternativeapproach to overcome functional redundancies ofgenetic networks inherent to tightly regulated negativefeedback systems. Finally, although our phenotypicstudies have so far not revealed any obvious phenotypefor Sef mutants, future studies will focus on analysis atlater embryological and adult stages using molecularand histological approaches.

MATERIAL AND METHODS

Generation of the Targeting Construct, MouseProduction, and Genotyping

A 3-kb mouse Sef full-length cDNA probe was used toisolate two genomic clones, containing both 30 and 50Sef genomic sequences from a 129SV/J genomic library(IGBMC, Strasbourg). To construct the targeting vector,pPNT-Sef, a 2.4-kb EcoRI-HindIII piece of 50 homologywas first subcloned into the EcoRI-KpnI site of pPNT(Tybulewicz et al., 1991). The 30 4-kb homology XhoI-EcoRI fragment was subsequently cloned into the XhoI-NotI site of the above vector. The NotI linearized target-ing construct was electroporated into R1 ES cells andneomycin-resistant ES clones were screened by genomicSouthern blot using 50 and 30 probes (Fig. 1). Thetargeted allele, referred to as Sef neo, was introduced intomice by blastocyst injection. Sef neo/þ animals weremaintained in the SV129 � C57BL/6 genetic back-ground. Animal genotypes were accessed by PCR usingthe following conditions: The primer sets for the wild-type allele is forward primer: GAT TCT AAA GGA CCC

FIG. 3. Comparison of the effect of Spry2Y55A on Gbx2 expression in wildtype and Sef homozygous mutant embryos. k–o: Flat-mounts ofdissected neural tubes; brackets indicate the Gbx2 expression domain. Arrows in l and n indicate ectopic Gbx2 expression in the caudalhindbrain region that was only observed in Sef mutant embryos. All the pictures were taken in dorsal views with anterior to the left (a,f) oranterior to the left-hand corner (b–e,g–j). EP, electroporation; Spry2DN, Spry2 dominant negative, i.e., Spry2Y55A; WT, wildtype embryos;Sef, Sef neo/neo mutant embryos.

113ACTIVITY OF SEF AND SPROUTY PROTEINS

CAA AC and reverse primer: GGA AGG GAA AGG GACAAT CT. The primer sets for mutant is forward primer:GAC AAT CGG CTG CTC TGA and reverse primer: CAAGCA GGC ATC GCC ATG. The PCR condition for geno-typing is 948C 1 min, 558C 30 s, 728C 30 s for 30 cycles.

Whole-Mount In Situ Hybridization

In situ hybridization to whole embryos was performedas described by Conlon and Herrmann (1993). The fol-lowing probes were used: Fgf8 cDNA (Crossley et al.,1995) and Spry1, 2 and 4 (Minowada et al., 1999), Gbx2(Wassarman et al., 1997). After in situ hybridization, theembryo was postfixed in 4% paraformaldehyde, washed,and progressively immersed into 100% glycerol. Theanterior neural tube of the embryo was then dissectedout by removing the underlying mesoderm and cuttingfrom the midbrain to the hindbrain region for flat-mountpreparation.

In Vitro Culture, Microinjection, and Electropora-tion of Mouse Embryos

Wildtype mouse embryos were obtained from F1female (C57BL/10xCBA/Ca) at E7.75 (late-streak to earlyheadfold stage); Sef mutant embryos at the same stagewere collected from Sef homozygous intercrosses.Mouse Spry2Y55A cDNA (Mason et al., 2004) was subcl-oned into pMES-GFP expression vector (Swartz et al.,2001). Electroporation of plasmid DNA into mouse

embryos and embryo culture were performed asdescribed in Mellitzer et al. (2002). After culture for 16–18 h, embryos were examined for GFP expression. Mildto strong GFP-expressing embryos were picked up andfixed in 4% paraformaldehyde for 45 min on ice, washedand stored in methanol before processing for whole-mount in situ hybridization.

Quantification of the Data

The higher dose electroporation data came from fiveparallel experiments between wildtype and Sef neo/neo

embryos, while the lower dose data came from four par-allel experiments in the same condition (data notshown). After analysis by whole-mount in situ hybridiza-tion, only flat-mounted embryos with a shift or expan-sion of Gbx2 expression were counted as positives(Fig. 3k,i, and data not shown). The standard deviationapplied here used formulas from Excel (Microsoft), andthe T stringent test from a website (http://home.clara.net/sisa/). Results of these calculations were then incor-porated into the chart generated by Excel automatically(Table 1).

ACKNOWLEDGMENTS

We thank Dr. Nobue Itasaki for showing the flat-mounttechnique and Dr. Gail Martin for in situ probes. Wethank all the members of lab for helpful discussions andcomments on the manuscript.

Table 1Frequencies of Embryos Showing Expansion or Shift of Gbx2 Expression on the Electroporated Side with Spry2Y55A Construct*

*Expansion or shift of Gbx2 expression scored in the electroporated side with Spry2Y55A construct. Only embryos showing a moderate tostrong expression of the GFP were analyzed. n ¼ Total number of embryos analyzed. WT; wild type, MT; Sef neo/neo mutant embryos. Thenumber under each bar indicates amount of DNA being electroporated into mouse embryo. The error bar represents the standard deviation;the P value is less than 0.001 between MTand WT groups under the T-stringent test.

114 LIN ET AL.

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