Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling

Biochemical and Biophysical Research Communications 352 (2007) 896–902 www.elsevier.com/locate/ybbrc

Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling Koji Taniguchi a,b, Toranoshin Ayada a, Kenji Ichiyama a, Ri-ichiro Kohno c, Yoshikazu Yonemitsu c,d, Yasuhiro Minami e, Akira Kikuchi f, Yoshihiko Maehara b, Akihiko Yoshimura a,* a

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Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan b Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Division of Pathophysiological and Experimental Pathology, Department of Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan d Department of Gene Therapy, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan e Department of Genome Sciences, Faculty of Medical Sciences, Kobe University, Kobe 650-0017, Japan f Departments of Biochemistry and Medicine and Molecular Science, Hiroshima University, Hiroshima 734-8551, Japan Received 7 November 2006 Available online 1 December 2006

Abstract Sprouty genes encode cytoplasmic membrane-associated proteins that inhibit receptor tyrosine kinase signaling. Four orthologs of Drosophila Sprouty (dSpry) (Sprouty1–4) have been identified in mammals. Physiological function of Sprouty1 and Sprouty2 has been investigated using gene targeting approaches, however to date detailed examination of Sprouty4 knockout (KO) mice has not been reported. In this study, Sprouty4 KO mice were generated and characterized. Although a significant fraction of Sprouty4 KO mice died shortly after birth due to mandible defects, the remainder were viable and fertile. Growth retardation was observed for most Sprouty4deficient mice, with nearly all Sprouty4 KO mice having polysyndactyly. ERK activation was sustained in Sprouty4 KO mouse embryonic fibroblasts (MEFs) in response to FGF, but not to EGF. Sprouty2 and Sprouty4 double KO (DKO) mice were embryonic lethal and showed severe defects in craniofacial, limb, and lung morphogenesis. These findings suggest both redundant and non-redundant functions for Sprouty2 and Sprouty4 on embryonic development and FGF signaling. Ó 2006 Elsevier Inc. All rights reserved. Keywords: FGF; Signal transduction; Knockout mouse; Embryonic development; Sprouty; Negative regulation

Sprouty was identified in mutational screening for genes that modulate FGF signaling during tracheal and eye development in Drosophila [1,2]. In Drosophila, Sprouty has been genetically identified as an antagonist of fibroblast growth factor receptor (FGFR) in lung development, and epidermal growth factor receptor (EGFR) in eye and wing development via inhibition of the Ras-Raf-ERK pathway [1,2].

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Corresponding author. Fax: +81 92 642 6825. E-mail address: [email protected] (A. Yoshimura).

0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.11.107

Four mammalian genes with sequence similarity to Drosophila Sprouty (Sprouty1–4) have been identified [2]. The highest degree of similarity between mammalian and Drosophila Sprouty lies in the C-terminal cysteine-rich domain that localizes Sprouty to the plasma membrane. Three Sprouty-related proteins, Spred-1, Spred-2, and Spred-3, containing similar C-terminal cysteine-rich domains to Sprouty proteins (Sproutys) that inhibit growth factor and cytokine-induced ERK activation were identified [3,4]. While Spred proteins (Spreds) inhibit ERK activation from any growth factor stimuli, Sproutys inhibit FGF- and VEGF (vascular endothelial growth factor) -induced ERK

K. Taniguchi et al. / Biochemical and Biophysical Research Communications 352 (2007) 896–902

activation but do not inhibit EGF-induced ERK activation [1]. The role of vertebrate Sproutys and Spreds in growth factor signals, plus their physiological function was investigated using ectopic overexpression and gene-targeting studies. Using gain- and loss-of-function experiments, Sivak et al. showed that Spred and Sprouty proteins in Xenopus tropicalis modulate ERK and calcium-signaling pathways downstream of the FGF receptor, respectively, consequently regulating mesoderm formation and cell movement during gastrulation [5]. A role for Sprouty2 in murine lung development was demonstrated using antisense oligonucleotide strategies, as well as overexpression studies in transgenic mice [2]. Overexpression of Sprouty4 in mouse fetal lung causes severe defects in lobulation and lung hypoplasia [6], suggesting that, analogous to Drosophila Sprouty, Sprouty4 inhibits branching morphogenesis of the respiratory system. Sproutys have also been implicated in limb bud formation by modulation of FGF signals [7] and in angiogenesis through regulation VEGF signals [2]. Knockout (KO) mouse models were produced in an attempt to elucidate mammalian Sprouty (mSprouty) in vivo function. Sprouty1/ mice displayed multiple ureteric and kidney failure during kidney development due to increased sensitivity of the Wolffian duct to glial cell linederived neurotrophic factor (GDNF)/RET signaling [8]. Sprouty2/ mice suffered from enteric nerve hyperplasia and hypergangliosis resulting in esophageal achalasia, dilated esophagus, intestinal pseudo-obstruction, and abnormal physiology of the digestive motility system [9]. This was dependent on GDNF, suggesting that Sprouty2 also negatively regulates GDNF/RET signaling. Another study of Sprouty2/ mice indicated that they also had severe hearing loss with an abnormal organ of Corti and abnormal cochlear hair cell morphology [10]. This phenotype was rescued by reducing FGF8 gene dosage, indicating that Sprouty2 functions as a negative regulator of FGF8 signaling. Unlike Sprouty1 and Sprouty2, little is known about Sprouty4, with no detailed reports on Sprouty4-deficient mice, although two groups have suggested limb deformation [11] and tooth abnormalities [12]. Overlapping functions between the different Sproutys have also not been characterized. In this study, Sprouty4 KO and Sprouty2/Sprouty4 double knockout (DKO) mice were generated and characterized. This study demonstrates important redundant and non-redundant functions for Sprouty2 and Sprouty4 in embryonic development and FGF signaling. Materials and methods Targeting vector construction and genotyping. The targeting vector was constructed by replacing exon 2 of the Sprouty4 gene with a PGK-Neo cassette, preserving the 5.0 kb (left arm) and 1.7 kb (right arm) of the homologous flanking sequences (Fig. S1A). Genotyping was done by

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using genomic PCR with primers (WT: 5 0 -GCAAGGAGTCAGG TAGCTGTAAGTGGAGTG-3 0 /5 0 -CCAAAGTGCAACCTGCCACTG GGTCAGCTG-3 0 and Sprouty4 KO: 5 0 -GACCCTGGGTGAAG CTCCCAGGCTTCTTAG-3 0 /5 0 -TGCTAAAGCGCATGCTCCAGACT GCCTTGG-3 0 ). Mice were backcrossed to C57BL/6J mice at least seven times. In most experiments, F7 generation was used. Sprouty2 KO mice and mouse embryonic fibroblasts (MEFs) have been described previously [9]. Lung explant culture was done as described [6]. All experiments using these mice were approved by and performed according to the guidelines of the Animal Ethics Committee of Kyushu University, Fukuoka, Japan. Immunoprecipitation and Western blot analysis. Mouse liver was isolated and lysed in 1 mL lysis buffer (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, and 1 mM sodium vanadate) supplemented with protease inhibitors (Nacalai tesque). Tissue lysates were incubated for 2 h at 4 °C with 20 lL (50% v/v) protein G-Sepharose (Amersham Biosciences) plus 10 lL rabbit anti-Sprouty4 serum [13], then washed five times with lysis buffer. For immunoblotting, samples were separated on 10% SDS–polyacrylamide gel. Proteins were transferred then probed with antiSprouty4 (L-17) (Santa Cruz Biotechnology). Analysis of ERK and Akt activation using MEFs was performed as described previously [9]. bFGF, EGF, and Wnt-3a were purchased from PeproTech.

Results and discussion Generation of Sprouty4 KO mice To determine the function of Sprouty4 in the physiological state, mice lacking Sprouty4 gene were generated by homologous recombination. Disruption of Sprouty4 in embryonic stem cells was achieved by replacing exon 2 of the Sprouty4 gene with a PGK-Neo cassette (Fig. S1A). Successful recombination was ascertained by Southern blotting of murine tails using the indicated probe (Fig. S1B). The complete lack of functional transcripts and proteins in the Sprouty4 KO mice was confirmed by RT-PCR and Western blotting using Sprouty4 KO mouse liver (Fig. S1C and D). Dwarfism and Polysyndactyly in Sprouty4 KO mice Sprouty4-deficient offspring from F2 generation (129XC57BL/6J mixed background) were born at the expected Mendelian ratio from intercrosses of heterozygotes (n = 21/84; 25%) and were fertile. Sprouty4+/ mice were then backcrossed six times into a C57BL/6J background. Number of homozygous Sprouty4 KO mice from crossing with the heterozygous F7 generation was significantly lower than expected (eight KO mice out of 63 pups alive at P7). Body weight and postnatal growth of surviving homozygous pups from the F7 generation were significantly reduced compared to wild-type littermates (Fig. 1A, Fig. S2A). This growth retardation was not apparent in F2 generation Sprouty4 KO mice (data not shown), suggesting that dwarfism is dependent on genetic background. However, these F7 Sprouty4 KO mice survived at least 12 months after birth and were fertile. No gross abnormalities of major organs, such as lung, kidney, heart, liver, spleen, and brain, were observed in homozygous Sprouty4 KO mice from the F7 generation. However, Sprouty4 KO mice

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Fig. 1. Body weight and phenotypes of Sprouty4 KO mice. (A) Growth curves for wild-type (WT) and Sprouty4 KO mice. Data indicate average pup weight ± SD. Left, male; right, female. n = 10–12. *P < 0.05. (B) Forelimbs of Sprouty4 KO adult mice. Fusion and duplication of digits is apparent in mutants. (C) Abnormal tooth development in some Sprouty4 knockouts. Middle panel and right panel show malocclusion and incisor increase, respectively. (D) Gross appearance of wild-type and Sprouty4 KO embryos at E18.5. Arrows indicate mandible defects in Sprouty4 KO embryo.

exhibited polysyndactyly, an almost fully penetrative phenotype characterized by fusion and duplication of digits at the forelimbs (Fig. 1B, Fig. S2B). This is consistent with a previous report of a large mutagenesis screening describing Sprouty4 gene as a candidate regulator of normal limb formation [11]. Recently, it has been shown that loss of Sprouty4 function results in diastema tooth formation [12]. The current study also found that some Sprouty4 KO mice from that F7 generation had incisor increase and/or malocclusion, but at low frequency (three out of 14 KO mice) (Fig. 1C). Malocclusion was considered to be one cause of reduction in postnatal growth. However, growth retardation was also observed in mice without dental defects. Similar dwarfism was reported for Spred-2 KO mice [14]. Cause of death in F7 generation Sprouty4 KO mice was investigated by examining embryos. Four out of seven Sprouty4 KO embryos at E13.5 or E18.5 showed mandible defects (Fig. 1D, Fig. S2C). These mice died shortly after birth, probably as they could not breathe and/or suckle properly. Some of Sprouty4 KO embryos at E13.5 showed eyelid closure (Fig. S2C). This may have been due to stronger FGF10 signaling, as it has been reported that FGF10 promotes eyelid closure through activation of activin and TGFa-EGFR signaling [15]. These findings suggest that

Sprouty4 plays important roles in morphogenesis and body size determination, especially on a C57BL/6J genetic background. Deficiency of Sprouty4 does not enhance lung branching It has been reported that Sprouty2 and Sprouty4 are expressed in developing lung [16], and overexpression of Sprouty4 blocks branching morphogenesis of the lung [6]. Treatment of embryonic mouse lungs in culture with Sprouty2 antisense oligonucleotides stimulates branching of the developing tracheal tree and increases epithelial proliferation [17]. Thus, effect of loss of Sprouty4 gene on developmental lung morphogenesis was investigated using explant culture. Lungs derived from E10.5 WT and Sprouty4 KO embryos were isolated and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Fig. 2A). A slight delay in lung branching was observed in Sprouty4-deficient lung explants under these conditions. However, almost complete branching was observed in Sprouty4 KO embryonic lung on day 3. Histological examination indicated no abnormalities in 8week-old Sprouty4-deficient lung (Fig. 2B). Thus, loss of Sprouty4 may be compensated by other genes in this family.

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ila Sprouty, that down-regulates both FGF and EGF signaling [1,2]. It was recently reported that Sprouty4 gene is the evolutionarily conserved target of the Wnt/b-catenin signaling pathway in silico, based on conservation of double Tcf/ Lef-binding sites within the 5 0 -promoter region of mammalian Sprouty4 orthologs [18]. This is consistent with studies indicating that Sprouty4 is induced by Wnt-7a in non-small cell lung cancer (NSCLC) cell lines [19]. In the current study, Sprouty4 protein was induced by Wnt-3a (Fig. 3C). Therefore, whether Sprouty4 is involved in crosstalk between Wnt and FGF pathways was investigated. Pre-treatment of cells with Wnt-3a for 6 h suppressed FGF-induced ERK and Akt activation in WT MEFs (Fig. 3D), while Wnt-3a mediated ERK suppression was not observed in Sprouty4 KO MEFs (Fig. 3E). These findings suggest that Sprouty4 is the target of Wnt-3a signaling, implicated it in the down-regulation of FGFdependent ERK activation via Wnt-3a. Sprouty2/Sprouty4 double knockout (DKO) mice are embryonic lethal and showed severe morphological abnormalities

Fig. 2. Effect of Sprouty4 deficiency during lung bud culture. (A) Lungs of E10.5 wild-type and Sprouty4 KO embryos were isolated and cultured for 3 days. Lungs were photographed at indicated periods. Representative data from three experiments are shown. (B) Sections (hematoxylin–eosin (H.E.) staining) from lung tissues of 8-week-old wild-type (left) and Sprouty4 KO (right) mice. Scale bars; 100 lm.

Sprouty4 functional effect on FGF and EGF signaling We have shown that loss of Sprouty2 resulted in enhanced FGF- but not EGF-induced ERK activation in mouse embryonic fibroblasts (MEFs) [9]. To determine the biochemical effect of loss of Sprouty4 expression on FGF and EGF signaling, MEFs derived from WT and Sprouty4 KO embryos were obtained, then stimulated with FGF and EGF. Like Sprouty2 KO MEFs, FGF induced stronger and sustained ERK activation in Sprouty4 KO than WT MEFs (Fig. 3A). EGF induced weaker ERK activation in Sprouty4 KO than WT MEFs (Fig. 3B). These results were consistent with several reports indicating that mammalian Sproutys down-regulate FGF signaling but up-regulate EGF signaling. This is in contrast to Drosoph-

To determine if there is any overlapping function between Sprouty2 and Sprouty4, Sprouty2/Sprouty4 DKO mice were generated. Sprouty2/+ Sprouty4/+ male and female mice were crossed. Sprouty2/+ Sprouty4/ mice were small and born rarely, with several postnatal mice appearing sick and most dying within a few weeks of birth for unknown reasons. No Sprouty2/ Sprouty4/ (DKO) or Sprouty2/ Sprouty4/+ pups were obtained from >200 pup births, indicating that deletion of both Sprouty2 and Sprouty4 genes resulted in embryonic lethality. Most Sprouty2/Sprouty4 DKO embryos died by E12.5 with craniofacial and limb morphogenesis abnormalities (Fig. 4A and B). Sprouty2/4 DKO embryos showed cyclopia (Fig. 4B, right panel). Histological examination suggested abnormal development of the brain and lung (Fig. 4C and D). Alobar brain development and cyclopia (Fig. 4B and C) resembled holoprosencephaly, which is mainly caused by Sonic hedgehog (Shh) mutations in human and mouse model [20]. Sprouty2/4 DKO embryos also showed abnormalities of the lung in the pattern and the extent of epithelial branching with significantly reduced number of airways (Fig. 4D). Defects of the lung were much severer in Sprouty2/4 DKO embryos than in Sprouty4 single KO embryos (cf. Fig. 2A and Fig. 4D). These findings indicate that Sprouty2 and Sprouty4 have redundant functions for embryonic morphological development. Sprouty4 KO phenotypes resemble mouse and human diseases such as Apert syndrome, relating to mutations that impact activation of FGFR2 [21]. Thus, it is likely that loss of Sprouty expression results in hyperactivation of FGF signaling. Body weight and postnatal growth of Sprouty4 KO mice from the F7 generation were significantly

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Fig. 3. Effect of Sprouty4 deficiency on FGF and EGF signaling. (A,B) WT and Sprouty4 KO primary MEFs were stimulated with 100 ng/mL bFGF (A) or 100 ng/mL EGF (B). Cell extracts were immunoblotted with indicated antibodies. (C) WT and Sprouty4 KO primary MEFs were stimulated with 50 ng/mL Wnt-3a. Cell extracts were immunoblotted with indicated antibodies. ERK2 was analyzed as loading control. (D,E) WT (D) and Sprouty4 KO (E) primary MEFs were pretreated with (+) or without () 50 ng/mL Wnt-3a for 6 h, then stimulated with 100 ng/mL bFGF.

Fig. 4. Phenotypes of Sprouty2/4 DKO embryos. (A,B) Gross appearance of wild-type and Sprouty2/4 DKO embryos at E11.5 (A) and E12.5 (B). In (B), lateral view (left panels) and front view (right panels) of E12.5 embryos are shown. (C,D) Sections (H.E. staining) of brain (C) and lung (D) tissues of control and Sprouty2/4 DKO embryos at E12.5. Scale bars; (C) 1 mm, (D) 100 lm.

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reduced. Overexpression of FGFs [22,23], activating FGFR3 mutants [24] or constitutive activation of MEK1 in chondrocytes [25] causes achondroplasia-like dwarfism in mice. These indicate that FGF signaling through the FGFR3-ERK pathway plays a major role in regulation of bone growth. Since Sprouty4 is an inhibitor of FGF-induced ERK signaling, loss of Sprouty4 may accelerate ERK signaling, thereby suppressing chondrocyte differentiation. Constitutive activation of FGFs also cause digit fusion [26]. Selective ablation of FGF8 expression during limb development can lead to hypoplasia or loss of digits [27,28]. Therefore, increased FGF8 activity may explain digit fusion and the formation of extra digits in developing Sprouty4 KO limbs. The tooth abnormalities in Sprouty4 KO mice might be also due to FGF4/FGF9 hyper-signaling, because Sprouty4 in the dental mesenchyme block signaling via FGF4/FGF9 [12]. This study demonstrated that ablation of both Sprouty2 and Sprouty4 function in the mouse results in the severe truncation of forebrain and cephalic neural crest-derived head tissues and the lung. This finding provides a direct genetic evidence that Sprouty2 and Sprouty4 play an essential role in vertebrate head and lung development. It is known that Shh and FGF8 are essential for the first pharyngeal arch and lung development and Shh mutations cause holoprosencephaly [20,29]. Since Shh negatively regulates FGF10 expression in embryonic lung mesenchymal cells [30], Sprouty2/Sprouty4 might be important for first pharyngeal arch and lung development by modulating FGF8/FGF10 signaling. In conclusion, this study demonstrates both essential and redundant functions for Sprouty4 in mammalian development and FGF signaling.

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Acknowledgments We thank T. Yoshioka, H. Fujii, N. Kinoshita, M. Ohtsu, Y. Yamada for technical assistance, and Ms. Y. Nishi for manuscript preparation. This work was supported by special Grants-in-aid from the Ministry of Education, Science, Technology, Sports, and Culture of Japan. Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc. 2006.11.107.

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