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Functional cooperation of spns2 and fibronectin in cardiac and lower jaw development

Functional cooperation of spns2 and fibronectin in cardiac and lower jaw development Research Article 789 Functional cooperation of spns2 and fibronectin in cardiac and lower jaw development 1 1 2,3 1, Yu Hisano , Satoshi Ota , Shinji Takada and Atsuo Kawahara * Laboratory for Cardiovascular Molecular Dynamics, Riken Quantitative Biology Center, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan Okazaki Institute for Integrative Bioscience, National Institute of Natural Sciences, Okazaki, Aichi 444-8787, Japan Department of Basic Biology, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi 444-8787, Japan *Author for correspondence ([email protected]) Biology Open 2, 789–794 doi: 10.1242/bio.20134994 Received 7th April 2013 Accepted 29th May 2013 Summary The lipid mediator sphingosine-1-phosphate (S1P) is a knockdown of endothelin receptor A (ednra), which was regulator of cardiac development in zebrafish, as disruption downregulated in the spns2 mutant, caused pharyngeal of its receptor s1pr2 or transporter spns2 causes migration defects resembling those in the fn;spns2 mutant. These defects in cardiac progenitors. To examine the genetic results strongly suggest that Spns2-S1PR2 signaling and interaction of S1P signaling and the cell adhesion molecule fibronectin cooperatively regulate both cardiac and lower jaw fibronectin, we have established a fn;spns2 double mutant. development in zebrafish. Cardiac migration defects in fn;spns2 mutants were more severe than those in fn or spns2 mutants. We further found  2013. Published by The Company of Biologists Ltd. This is an that the lower jaw morphology was disorganized in the Open Access article distributed under the terms of the Creative fn;spns2 mutant, while it had a slightly shortened anterior– Commons Attribution License (http://creativecommons.org/ posterior distance in the ventral pharyngeal arch in fn and licenses/by/3.0), which permits unrestricted use, distribution spns2 mutants relative to wild type. Knockdown of fn in the and reproduction in any medium provided that the original s1pr2 mutant, but not in the s1pr1 mutant, resulted in severe work is properly attributed. defects in cardiac migration and ventral pharyngeal arch arrangement. Further, in the background of the fn mutant, Key words: Sphingosine-1-phosphate, spns2, fibronectin, Jaw, Heart Introduction treatment of anti-fibronectin antibody in chick embryos inhibits The bioactive lipid mediator sphingosine-1-phosphate (S1P) plays myocardial migration (Linask and Lash, 1986). In mice, important roles in various types of biological processes including myocardial specification is normally observed in fibronectin knockout mice, whereas myocardial migration is inhibited angiogenesis, inflammation and immunity (Skoura and Hla, 2009; (George et al., 1997). It has also been shown that fibronectin is Spiegel and Milstien, 2011; Hisano et al., 2012). In zebrafish, S1P is involved in cardiac development by regulating the myocardial required for adherens junction formation between cardiac migration, as evidenced by the migration defects of cardiac progenitors in zebrafish (Trinh and Stainier, 2004). These studies progenitors in an S1P receptor (S1PR) and an S1P transporter all suggest an important role for fibronectin in vertebrate cardiac mutant, the s1pr2 and spns2 mutants (Kupperman et al., 2000; development. Nevertheless, there is much uncertainty on how Osborne et al., 2008; Kawahara et al., 2009). S1PRs consist of at fibronectin cooperates with other signaling molecule(s) when regulating cardiac development and other organogenesis. least five G protein-couple receptors (S1PR1–S1PR5) that show differential expression patterns during mouse embryogenesis In this study, we established a double mutant, fn;spns2,to (Ohuchi et al., 2008; Meng and Lee, 2009). Intercellular S1P investigate the genetic interaction of fibronectin and S1P signaling signaling through S1PRs activates various downstream signaling in zebrafish. We found the two separated hearts phenotype in the pathways (Takuwa et al., 2012), leading to diverse cellular fn;spns2 double mutant was more severe than that in either fn responses including cell proliferation, differentiation and cell mutant or spns2 mutants. Further, the anterior–posterior distance migration. However, the developmental function of S1P signaling of the lower jaw was shorter in the fn and spns2 mutants, while the through the S1P-S1PR axis remains largely unclear. ventral pharyngeal arch structure was significantly impaired in the fn;spns2 double mutant. Our results genetically reveal a functional The cell adhesion molecule fibronectin is a major component of cooperation between S1P signaling and fibronectin for the the extracellular matrix (ECM) and is involved in various cellular processes including cytoskeletal organization and cell migration regulation of myocardial migration and lower jaw formation. (Wierzbicka-Patynowski and Schwarzbauer, 2003). The fibronectin (fn) mutant, which shows a loss of fibronectin Results and Discussion function in zebrafish, has defective boundary formation in its Cardiac progenitor migration regulated by Spns2 and fibronectin anterior somites (Koshida et al., 2005), suggesting fibronectin The heart tube develops from bilateral cardiac progenitors in the contributes to the epithelialization of somites. Additionally, anterior lateral plate mesoderm in all vertebrate (Miura and Biology Open Roles of spns2 and fibronectin 790 Yelon, 2011) and laterally positioned cardiac progenitors in fn;spns2 mutant (amhc, n57; vmhc, n59) at 30 hpf were coordinately move toward the midline and fuse to form the located at lateral distances greater than those of the spns2 (amhc, n58; vmhc, n57) or fn mutants (amhc, n58; vmhc, n58) heart tube. In zebrafish, disrupting s1pr2 (S1P receptor) or spns2 (S1P transporter) results in defective migration of these cardiac (Fig. 1). Further, we observed beating by the two separated hearts progenitors and in cardia bifida (two separated hearts), indicating in the fn;spns2 mutant (supplementary material Movies 1, 2). S1P signaling regulates myocardial migration (Kupperman et al., Thus, the cardiac progenitor migration, but not the cardiac 2000; Osborne et al., 2008; Kawahara et al., 2009). It has been differentiation is predominantly impaired in fn;spns2 double shown that the cell adhesion molecule fibronectin also mutants, suggesting that Spns2 and fibronectin synergize to contributes to the cardiac morphogenesis, as fn mutants promote cardiac progenitor migration. partially penetrate the cardia bifida phenotype (Trinh and kt259 Stainier, 2004). Our established fn mutant, a null mutant Lower jaw development is cooperatively regulated by Spns2 with a premature termination at codon 241, predominantly and fibronectin presented a straight heart tube phenotype (31/133 embryos The facial skeleton is formed from mutual interactions between kt259 obtained from the crossing of fn heterozygous fish) at cranial neural crest cells and both the pharyngeal endoderm and 25 hours post-fertilization (hpf), but also had a minor population ectoderm of zebrafish. Additionally, secreted proteins such as showing cardia bifida (3/133 embryos), a low penetration result Endothelin1, BMPs and Fgfs are key regulators involved in the that may be explained by the genetic background. To examine the craniofacial development (Alexander et al., 2011; Yamauchi genetic interactions between S1P signaling and fibronectin, we et al., 2011). It has been shown that the spns2 mutant displays a kt259 ko157 generated double mutant zebrafish fn ;spns2 by the disorganized anterior pharyngeal endoderm (Osborne et al., ko157 kt259 crossing spns2 and fn mutants. As shown in Fig. 1, the 2008). We noticed that our fn;spns2 double mutant exhibits fn:spns2 double mutant displayed two widely separated hearts severe defects in ventral facial morphology. Therefore, we kt259 ko157 (19/322 embryos obtained from the crossing of fn ;spns2 examined the pharyngeal arch structure of individual mutants by heterozygous fish). The distances between hearts were much Alcian Blue staining at 4 days post-fertilization (dpf). The greater than those of the spns2 (cardia bifida; 30/145 embryos anterior–posterior distance of the ventral pharyngeal arches ko157 obtained from the crossing of spns2 heterogeneous fish) or fn (Meckel’s, palatoquadrate and ceratohyal cartilages) in the mutants (straight heart tube; 31/133 embryos obtained from the spns2 or fn mutants was shorter than that of wild type (Fig. 2; kt259 crossing of fn heterozygous fish) (supplementary material supplementary material Table S2). On the other hand, the Table S1). The genotypes of individual mutants were confirmed number and morphology of the ceratobranchial arch were by the direct sequencing of individual spns2 and fn genomic loci. relatively normal. The pharyngeal defects are consistent with a Whole-mount in situ hybridization analysis revealed that the recent report that demonstrated morphological defects of the expressions of the cardiac differentiation markers amhc (atrial lower jaw in both s1pr2 and spns2 mutants (Balczerski et al., myosin heavy chain) and vmhc (ventricular myosin heavy chain) 2012). We also found that the cell adhesion molecule Fig. 1. Cardiac morphology. Heart positions are indicated by the arrowheads. (A–D) Cardiac morphology visualized by mRFP expression derived from Tg(cmlc2:mRFP). All images show ventral views at 28 hpf. (E–L) Whole-mount in situ hybridization with amhc and vmhc RNA probes. All images show ventral views at 30 hpf except for panel L (dorsal view). Genotyping was performed by genomic sequencing after taking pictures, wt (A,E,I), fn mutant (B,F,J), spns2 mutant (C,G,K) and fn;spns2 double mutant (D,H,L). Scale bars: 200 mm. (M) Average distances between two hearts from multiple experiments; error bars represent standard deviations. Biology Open Roles of spns2 and fibronectin 791 Fig. 2. Lower jaw morphology. (A–D) Lower jaw morphology at 4 dpf was visualized by Alcian Blue staining (ventral view). Anterior–posterior distances of the ventral pharyngeal arch (*) is indicated by the length of the double-headed arrows. The dorsal pharyngeal structure is identified by the crosses (+). (E–P) Whole- mount in situ hybridization using hand2, dlx2 and nkx2.3 RNA probes. The anteroventral position of these markers is marked by the arrowheads. All images show lateral views at 30 hpf. Genotyping was performed by genomic sequencing after taking pictures. wt (A,E,I,M), fn mutant (B,F,J,N), spns2 mutant (C,G,K,O), and fn;spns2 double mutant (D,H,L,P). Scale bars: 200 mm. (Q) Average anterior–posterior distances of the ventral pharyngeal arch from multiple experiments; error bars represent standard deviations. fibronectin is also required for the proper lower jaw develop- S1PR2, but not S1PR1, cooperates with fibronectin in cardiac ment (Fig. 2; supplementary material Table S2). In clear and lower jaw development contrast, the structure of the ventral pharyngeal arch (Fig. 2D, Spns2 functions as an S1P transporter (Kawahara et al., 2009), asterisks) was disorganized in the fn;spns2 double mutant, suggesting that some S1PRs contribute to cardiac and jaw whereas that of the dorsal pharyngeal structure (trabecular development. The s1pr2 mutant has been found to present the cartilage) appeared normal (Fig. 2D, cross). Jaw development is cardia bifida phenotype in zebrafish (Kupperman et al., 2000), regulated by the cooperation of several transcriptional factors whereas three independent groups recently reported that the including the hand2, dlx and nkx family genes (Miller et al., knockdown of s1pr1 causes severe defects in cardiac and 2003; Trinh et al., 2005; Talbot et al., 2010). Whole-mount in vascular development (Gaengel et al., 2012; Tobia et al., 2012; situ hybridization using the pharyngeal markers, hand2, dlx2 Mendelson et al., 2013), with the circulation of blood cells being and nkx2.3 at 30 hpf revealed that their anteroventral expres- particularly impaired in s1pr1-depleted embryos. We therefore sions of hand2, dlx2 and nkx2.3 were reduced in fn;spns2 examined the functional interaction of S1PR1/2 and fibronectin. mutants (hand2, n57; dlx2, n57; nkx2.3, n57) compared to the Using TALEN (transcription activator-like effector nuclease) their posterior expressions, whereas the expression patterns and technology, we recently established s1pr1 and s1pr2 knockout intensities of these markers were normal in fn mutants (hand2, fish (Hisano et al., 2013; Ota et al., 2013). We confirmed that n57; dlx2, n55; nkx2.3, n57) and spns2 mutants (hand2, n57; s1pr2 knockout mutants show cardia bifida phenotype (Fig. 3E). dlx2, n58; nkx2.3, n58). The genotypes of individual mutants However, no obvious cardiac or vascular defects in s1pr1 were confirmed by the direct sequencing of spns2 and fn knockout mutants were seen during early embryogenesis genomic loci after taking pictures. Because the first pouch (Fig. 3C; supplementary material Movies 3, 4). Thus, we endoderm is required for the pharyngeal arch formation conclude that zygotic s1pr1 mutants showed normal blood (Alexander et al., 2011), our results suggest that Spns2 and circulation and intersegmental vessel angiogenesis, which fibronectin contribute to the formation of the ventral pharyngeal disagrees with the aforementioned reports. Those studies all arch by regulating the anteroventral expression of various used identical S1PR1-morpholino. It is possible that the S1PR1- pharyngeal markers. morpholino caused off-target effects or also affected the maternal Biology Open Roles of spns2 and fibronectin 792 Fig. 3. Knockdown phenotype of fibronectin in S1PRs mutant. (A) Membrane topology of S1P receptors and their mutants. The region of frameshift-mediated amino acids compared with the S1PR2 wild type (WT) is shown by the dashed line. (B,C,D) Fluorescence microscopy of intersegmental vessels of wt (B), s1pr1 mutant (C) and s1pr2 mutnat (D) at 2 dpf. Endothelial cells are visualized by EGFP expression derived from Tg(fli1a:EGFP). (E,F) Cardiac morphology visualized by mRFP expression derived from Tg(cmlc2:mRFP). All images show ventral views at 28 hpf. (G) Average distances between two hearts from multiple experiments; error bars represent standard deviations. (H,I) Lower jaw morphology at 4 dpf was visualized by Alcian Blue staining (ventral view). (J) Average anterior–posterior distances of the ventral pharyngeal arch from multiple experiments; error bars represent standard deviations. Genotyping was performed by genomic sequencing or heteroduplex mobility assays after taking pictures. Scale bars: 200 mm. message of s1pr1. To explain the different conclusions, it would cardiac migration and lower jaw morphology were observed be best to study a maternal-zygotic s1pr1 mutant, which will be when S1PR2-MO (10 ng) was injected into fn mutants available in a future study. When Fn-MO (10 ng) was injected (supplemental material Fig. S1). These results suggest that into the s1pr2 mutant, a more severe cardia bifida phenotype was S1PR2, but not S1PR1, cooperates with fibronectin in both induced compared to the s1pr2 mutant (Fig. 3E–G). Consistent cardiac and lower jaw development. It has been reported that with these results, severe defects in cardiac migration and lower both s1pr2 and spns2 mutants lack the anterior endoderm jaw morphology were observed when S1PR2-MO (10 ng) was (Balczerski et al., 2012). Because signaling pathways through injected into fn mutants (supplemental material Fig. S1), which the Spns2-S1PR2 axis regulate cell proliferation of the anterior agrees with previous knockdown analysis that used S1PR2-MO endoderm tissue and therefore affect the positioning of the and Fn-MO (Matsui et al., 2007). Such a cooperative cardiac ventral pharyngeal arch, further analysis will be required to defect was not observed in s1pr1 embryos injected with Fn-MO clarify how S1P signaling and fibronectin together control the (supplementary material Table S1), which instead showed a movement and adhesion of anterior pharyngeal endodermal cells. straight heart tube phenotype similar to that in fn mutants. Fn- MO-injected s1pr1 mutants embryos had a slightly shorter Endothelin receptor A, a possible mediator downstream of anterior–posterior distance in their ventral pharyngeal arch Spns2-S1PR2 signaling structure, quite unlike the disorganized ventral pharyngeal arch Both Spns2 and S1PR2 are involved in cardiac and lower jaw structure observed in Fn-MO-injected s1pr2 mutants (Fig. 3H–J; development. To identify the genes regulated by the Spns2- supplementary material Table S2). Additionally, severe defects in S1PR2 axis, we performed microarray analysis. Total RNA was Biology Open Roles of spns2 and fibronectin 793 Fig. 4. Knockdown phenotype of endothelin receptor A (ednra)in fn mutants. (A,B) Whole-mount in situ hybridization using the ednra RNA probe. The expression of ednra was suppressed in the spns2 mutant. Both images show lateral views at 30 hpf. (C,D) Cardiac morphology visualized by mRFP expression derived from Tg(cmlc2:mRFP). Both images show ventral views at 28 hpf. (E,F) Lower jaw morphology at 4 dpf was visualized by Alcian Blue staining (ventral view). Genotyping was performed by genomic sequencing after taking pictures. wt (A,C,E), spns2 mutant (B) and fn mutant (D,F). Scale bars: 200 mm. (G,H) Average distances between hearts (G) and anterior– posterior distances of the ventral pharyngeal arch (H) from multiple experiments; error bars represent standard deviations. isolated from uninjected, S1PR2-MO (10 ng)-injected and S1P-S1PR2 and End1-Ednra being critical in the patterning of the Spns2-MO (10 ng)-injected embryos at 25 hpf, and their gene ventral pharyngeal arch, which agrees with our demonstrating expression profiles were compared. We found that the expression that Ednra, a possible downstream target of the Spns2-S1PR2 axis, synergizes with fibronectin to promote the lower jaw of endothelin receptor A (ednra) was downregulated in both development. Spns2-depleted and S1PR2-depleted embryos (see Materials and Methods). Consistent with this result, the expression of ednra in Conclusion the pharyngeal arches of the spns2 mutant (n510) was reduced In this study, using zebrafish genetic mutants (fn, spns2 and compared to that of wild type (n57) (Fig. 4A,B). In both fn;spns2), we demonstrated that Spns2-S1PR2 signaling and the zebrafish and mouse, disruption of endothelin1 (edn1) causes a cell adhesion molecule fibronectin cooperatively regulate the loss or transformation of the lower jaw (Nair et al., 2007; Tavares migration of cardiac progenitors. Further, Spns2-S1PR2 and et al., 2012). Because Edn1 functions through its cognate type-A fibronectin synergize to promote ventral pharyngeal cartilage receptor Ednra, Ednra can be considered a key regulator in formation. Because the expression of ednra in the pharyngeal pharyngeal development (Nair et al., 2007). However, it is not arches of spns2 mutants is reduced, we propose that Ednra clear how Ednra cooperates with other molecule(s) during jaw contributes to the lower jaw arrangement by cooperating with development. Therefore, we investigated the functional fibronectin. interaction between Ednra and fibronectin. Heart morphology seemed normal when Ednra-MO (10 ng) was injected into wild- Materials and Methods type embryos (n513), while cardiac defects in Ednra-MO Zebrafish mutants injected fn embryos (n511) were slightly more severe than kt259 kt259 ko157 Mutant alleles of fibronectin (fn ) and spns2 were used (Koshida et al., ko157 those of fn embryos (Fig. 4C,D; supplementary material Table 2005; Kawahara et al., 2009). To obtain double mutants, the spns2 allele was kt259 kt259 ko157 S1). Further, a disorganization of the ventral pharyngeal arch crossed into the fn allele. Embryos of fn ;spns2 were obtained from kt259 ko157 ko157 kt259 fn ;spns2 heterogeneous fish. Embryos of spns2 and fn were arrangement in Ednra-MO injected fn embryos (n510) was kt259 ko157 obtained from fn and spns2 heterogeneous fish, respectively. Genotyping ko157 kt259 kt259 ko157 observed relative to that of Ednra-MO injected wild-type embryo of spns2 and fn single mutants and fn ;spns2 double mutants was (n57) (Fig. 4E,F; supplementary material Table S2). The performed by direct sequencing of individual genomic loci as described below. To monitor the cardiac development, the transgenic line Tg(cmlc2:mRFP) was used. genotypes of individual mutants were confirmed by the direct sequencing of spns2 and fn genomic loci after taking pictures. Establishment of s1pr1 or s1pr2-knockout zebrafish Although in a cell culture system Edn1 increases the adhesion of TALEN constructs targeting s1pr1 or s1pr2 were described previously (Hisano amelanotic melanocytes to fibronectin (Ma et al., 2006), how et al., 2013; Ota et al., 2013). TALEN mRNAs (400 pg each) were injected into Edn1 signaling affects fibronectin function remains unclear. blastomeres at the 1–2 cell stage of zebrafish embryos. Identification of potential F0 founders and F1 embryos having mutant allele was performed by HMA Further analysis will be required to determine whether Edn1 ko311 (heteroduplex mobility assay) (Ota et al., 2013). The s1pr1 allele was deleted ko322 regulates the fibronectin-mediated cellular interaction during the from +362 to +371 of the s1pr1 coding region, while the s1pr2 allele was ventral pharyngeal arch arrangement. One clue comes from both deleted from +179 to +188 of the s1pr2 coding region. To monitor the cardiac and Biology Open Roles of spns2 and fibronectin 794 endodermal requirements for the growth but not dorsoventral patterning of jaw vascular development, the transgenic lines Tg(cmlc2:mRFP) and Tg(fli1a:EGFP) skeletal precursors. Dev. Biol. 362, 230-241. were used. s1pr1 or s1pr2 mutant embryos were obtained by the crossing Gaengel, K., Niaudet, C., Hagikura, K., Lavin˜a, B., Muhl, L., Hofmann, J. J., individual F1 heterogeneous fish. Ebarasi, L., Nystro¨m, S., Rymo, S., Chen, L. L. et al. (2012). The sphingosine-1- phosphate receptor S1PR1 restricts sprouting angiogenesis by regulating the interplay Preparation of genomic DNA and genotyping of spns2 and fn between VE-cadherin and VEGFR2. Dev. Cell 23, 587-599. George, E. L., Baldwin, H. S. and Hynes, R. O. (1997). Fibronectins are essential for mutants heart and blood vessel morphogenesis but are dispensable for initial specification of Genomic DNA was isolated using the Gentra Puregene Tissue Kit (Qiagen) precursor cells. Blood 90, 3073-3081. according to the manufacturer’s protocol. For the genotyping of the spns2 mutant, Hanaoka, R., Katayama, S., Dawid, I. B. and Kawahara, A. (2006). Characterization the spns2 genomic locus was amplified by PCR using the following primers: of the heme synthesis enzyme coproporphyrinogen oxidase (CPO) in zebrafish spns2-S, 59-TCAAGGAATGTGAGCCATGT-39; and spns2-AS, 59-GGATGCCA- erythrogenesis. Genes Cells 11, 293-303. GGTAGAAGACA-39. For genotyping of the fn mutant, the fn genomic locus was Hisano, Y., Nishi, T. and Kawahara, A. (2012). The functional roles of S1P in amplified by PCR using the following primers: fn-S, 59-CTTACTCAAGC- immunity. J. Biochem. 152, 305-311. TTAACTGG-39; and fn-AS, 59-ACCAAGACTAGTAGTGTGCAG-39. In the case Hisano, Y., Ota, S., Arakawa, K., Muraki, M., Kono, N., Oshita, K., Sakuma, of embryos stained with Alcian Blue or analyzed by whole-mount in situ T., Tomita, M., Yamamoto, T., Okada, Y. et al. (2013). Quantitative assay for hybridization, nested PCR was performed using the following primers: fn-S2, 59- TALEN activity at endogenous genomic loci. Biol. Open 2, 363-367. GGTTCTAATGGGAAACATCTGC-39; and fn-AS2, 59-GAGAAGCATGC- Kawahara, A., Nishi, T., Hisano, Y., Fukui, H., Yamaguchi, A. and Mochizuki, CTCTCACAC-39. The genotypes of spns2 and fn were determined by direct N. (2009). 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Precardiac cell migration: fibronectin localization formamide, 56 SSC, 5 mM EDTA, 0.1% Tween 20, 50 mg/ml heparin and 1 mg/ml at mesoderm-endoderm interface during directional movement. Dev. Biol. 114, 87-101. RNA torula). Then, embryos were washed twice at 65 C for 30 min with washing Ma, H. J., Zhu, W. Y., Wang, D. G., Yue, X. Z. and Li, C. R. (2006). Endothelin-1 buffer I (50% formamide, 26 SSC and 0.1% Tween 20), twice at 65 C for 30 min combined with extracellular matrix proteins promotes the adhesion and chemotaxis of amelanotic melanocytes from human hair follicles in vitro. Cell Biol. Int. 30, 999-1006. with washing buffer II (26 SSC and 0.1% Tween 20), twice at 65 C for 30 min with Matsui, T., Raya, A., Callol-Massot, C., Kawakami, Y., Oishi, I., Rodriguez- washing buffer III (0.26SSC and 0.1% Tween 20) and once at room temperature for Esteban, C. and Izpisu´a Belmonte, J. C. 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(2003). Two endothelin 1 Embryos at 4 dpf were fixed overnight by 4% paraformaldehyde in PBST. effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Embryos were washed with acid alcohol buffer (0.37% HCl and 70% ethanol) and Development 130, 1353-1365. incubated with 0.1% Alcian Blue (Sigma) in acid alcohol buffer. After 3 times Miura, G. I. and Yelon, D. (2011). A guide to analysis of cardiac phenotypes in the washing with acid alcohol buffer, embryos were incubated for 10 min with zebrafish embryo. Methods Cell Biol. 101, 161-180. bleaching buffer (1% H O and 1% KOH). 2 2 Nair, S., Li, W., Cornell, R. and Schilling, T. F. (2007). Requirements for Endothelin type-A receptors and Endothelin-1 signaling in the facial ectoderm for the patterning Knockdown analysis using antisense morpholinos and microarray of skeletogenic neural crest cells in zebrafish. Development 134, 335-345. 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Total RNA was isolated with TRIzol reagent (Invitrogen) from Okada, Y. and Kawahara, A. (2013). Efficient identification of TALEN-mediated individual embryos at 25 hpf. Microarray analysis was performed using a genome modifications using heteroduplex mobility assays. Genes Cells 18, 450-458. zebrafish-specific Affymetrix chip (15617 zebrafish probes). The expression of Skoura, A. and Hla, T. (2009). Lysophospholipid receptors in vertebrate development, ednra was downregulated in Spns2-MO-injected (Log2 Ratio: 21.06) and S1PR2- physiology, and pathology. J. Lipid Res. 50 Suppl.:, S293-S298. Spiegel, S. and Milstien, S. (2011). The outs and the ins of sphingosine-1-phosphate in MO-injected embryos (Log2 Ratio: 21.06). immunity. Nat. Rev. Immunol. 11, 403-415. Takuwa, Y., Okamoto, Y., Yoshioka, K. and Takuwa, N. (2012). Sphingosine-1- Acknowledgements phosphate signaling in physiology and diseases. Biofactors 38, 329-337. Talbot, J. C., Johnson, S. L. and Kimmel, C. B. (2010). hand2 and Dlx genes specify The authors thank R. Fukuoka, M. Komeno, M. Hayashi and S. Ohara dorsal, intermediate and ventral domains within zebrafish pharyngeal arches. for zebrafish maintenance and technical assistance, and P. Karagiannis Development 137, 2507-2517. for valuable comments. This work was supported by the Funding Tavares, A. L., Garcia, E. L., Kuhn, K., Woods, C. M., Williams, T. and Clouthier, Program for Next Generation World-Leading Researchers (NEXT D. E. (2012). Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch. Dev. Biol. 371, 47-56. Program) and by the Japan Society for the Promotion of Science. Tobia, C., Chiodelli, P., Nicoli, S., Dell’era, P., Buraschi, S., Mitola, S., Foglia, E., van Loenen, P. B., Alewijnse, A. E. and Presta, M. (2012). Sphingosine-1- Competing Interests phosphate receptor-1 controls venous endothelial barrier integrity in zebrafish. Arterioscler. Thromb. Vasc. Biol. 32, e104-e116. The authors have no competing interests to declare. Trinh, L. A. and Stainier, D. Y. (2004). Fibronectin regulates epithelial organization during myocardial migration in zebrafish. Dev. Cell 6, 371-382. References Trinh, L. A., Yelon, D. and Stainier, D. Y. (2005). Hand2 regulates epithelial Alexander, C., Zuniga, E., Blitz, I. L., Wada, N., Le Pabic, P., Javidan, Y., Zhang, formation during myocardial diferentiation. Curr. Biol. 15, 441-446. T., Cho, K. W., Crump, J. G. and Schilling, T. F. (2011). Combinatorial roles for Wierzbicka-Patynowski, I. and Schwarzbauer, J. E. (2003). The ins and outs of BMPs and Endothelin 1 in patterning the dorsal–ventral axis of the craniofacial fibronectin matrix assembly. J. Cell Sci. 116, 3269-3276. skeleton. Development 138, 5135-5146. Yamauchi, H., Goto, M., Katayama, M., Miyake, A. and Itoh, N. (2011). Fgf20b is Balczerski, B., Matsutani, M., Castillo, P., Osborne, N., Stainier, D. Y. and Crump, required for the ectomesenchymal fate establishment of cranial neural crest cells in J. G. (2012). Analysis of sphingosine-1-phosphate signaling mutants reveals zebrafish. Biochem. Biophys. Res. Commun. 409, 705-710. Biology Open http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology Open The Company of Biologists

Functional cooperation of spns2 and fibronectin in cardiac and lower jaw development

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Research Article 789 Functional cooperation of spns2 and fibronectin in cardiac and lower jaw development 1 1 2,3 1, Yu Hisano , Satoshi Ota , Shinji Takada and Atsuo Kawahara * Laboratory for Cardiovascular Molecular Dynamics, Riken Quantitative Biology Center, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan Okazaki Institute for Integrative Bioscience, National Institute of Natural Sciences, Okazaki, Aichi 444-8787, Japan Department of Basic Biology, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi 444-8787, Japan *Author for correspondence ([email protected]) Biology Open 2, 789–794 doi: 10.1242/bio.20134994 Received 7th April 2013 Accepted 29th May 2013 Summary The lipid mediator sphingosine-1-phosphate (S1P) is a knockdown of endothelin receptor A (ednra), which was regulator of cardiac development in zebrafish, as disruption downregulated in the spns2 mutant, caused pharyngeal of its receptor s1pr2 or transporter spns2 causes migration defects resembling those in the fn;spns2 mutant. These defects in cardiac progenitors. To examine the genetic results strongly suggest that Spns2-S1PR2 signaling and interaction of S1P signaling and the cell adhesion molecule fibronectin cooperatively regulate both cardiac and lower jaw fibronectin, we have established a fn;spns2 double mutant. development in zebrafish. Cardiac migration defects in fn;spns2 mutants were more severe than those in fn or spns2 mutants. We further found  2013. Published by The Company of Biologists Ltd. This is an that the lower jaw morphology was disorganized in the Open Access article distributed under the terms of the Creative fn;spns2 mutant, while it had a slightly shortened anterior– Commons Attribution License (http://creativecommons.org/ posterior distance in the ventral pharyngeal arch in fn and licenses/by/3.0), which permits unrestricted use, distribution spns2 mutants relative to wild type. Knockdown of fn in the and reproduction in any medium provided that the original s1pr2 mutant, but not in the s1pr1 mutant, resulted in severe work is properly attributed. defects in cardiac migration and ventral pharyngeal arch arrangement. Further, in the background of the fn mutant, Key words: Sphingosine-1-phosphate, spns2, fibronectin, Jaw, Heart Introduction treatment of anti-fibronectin antibody in chick embryos inhibits The bioactive lipid mediator sphingosine-1-phosphate (S1P) plays myocardial migration (Linask and Lash, 1986). In mice, important roles in various types of biological processes including myocardial specification is normally observed in fibronectin knockout mice, whereas myocardial migration is inhibited angiogenesis, inflammation and immunity (Skoura and Hla, 2009; (George et al., 1997). It has also been shown that fibronectin is Spiegel and Milstien, 2011; Hisano et al., 2012). In zebrafish, S1P is involved in cardiac development by regulating the myocardial required for adherens junction formation between cardiac migration, as evidenced by the migration defects of cardiac progenitors in zebrafish (Trinh and Stainier, 2004). These studies progenitors in an S1P receptor (S1PR) and an S1P transporter all suggest an important role for fibronectin in vertebrate cardiac mutant, the s1pr2 and spns2 mutants (Kupperman et al., 2000; development. Nevertheless, there is much uncertainty on how Osborne et al., 2008; Kawahara et al., 2009). S1PRs consist of at fibronectin cooperates with other signaling molecule(s) when regulating cardiac development and other organogenesis. least five G protein-couple receptors (S1PR1–S1PR5) that show differential expression patterns during mouse embryogenesis In this study, we established a double mutant, fn;spns2,to (Ohuchi et al., 2008; Meng and Lee, 2009). Intercellular S1P investigate the genetic interaction of fibronectin and S1P signaling signaling through S1PRs activates various downstream signaling in zebrafish. We found the two separated hearts phenotype in the pathways (Takuwa et al., 2012), leading to diverse cellular fn;spns2 double mutant was more severe than that in either fn responses including cell proliferation, differentiation and cell mutant or spns2 mutants. Further, the anterior–posterior distance migration. However, the developmental function of S1P signaling of the lower jaw was shorter in the fn and spns2 mutants, while the through the S1P-S1PR axis remains largely unclear. ventral pharyngeal arch structure was significantly impaired in the fn;spns2 double mutant. Our results genetically reveal a functional The cell adhesion molecule fibronectin is a major component of cooperation between S1P signaling and fibronectin for the the extracellular matrix (ECM) and is involved in various cellular processes including cytoskeletal organization and cell migration regulation of myocardial migration and lower jaw formation. (Wierzbicka-Patynowski and Schwarzbauer, 2003). The fibronectin (fn) mutant, which shows a loss of fibronectin Results and Discussion function in zebrafish, has defective boundary formation in its Cardiac progenitor migration regulated by Spns2 and fibronectin anterior somites (Koshida et al., 2005), suggesting fibronectin The heart tube develops from bilateral cardiac progenitors in the contributes to the epithelialization of somites. Additionally, anterior lateral plate mesoderm in all vertebrate (Miura and Biology Open Roles of spns2 and fibronectin 790 Yelon, 2011) and laterally positioned cardiac progenitors in fn;spns2 mutant (amhc, n57; vmhc, n59) at 30 hpf were coordinately move toward the midline and fuse to form the located at lateral distances greater than those of the spns2 (amhc, n58; vmhc, n57) or fn mutants (amhc, n58; vmhc, n58) heart tube. In zebrafish, disrupting s1pr2 (S1P receptor) or spns2 (S1P transporter) results in defective migration of these cardiac (Fig. 1). Further, we observed beating by the two separated hearts progenitors and in cardia bifida (two separated hearts), indicating in the fn;spns2 mutant (supplementary material Movies 1, 2). S1P signaling regulates myocardial migration (Kupperman et al., Thus, the cardiac progenitor migration, but not the cardiac 2000; Osborne et al., 2008; Kawahara et al., 2009). It has been differentiation is predominantly impaired in fn;spns2 double shown that the cell adhesion molecule fibronectin also mutants, suggesting that Spns2 and fibronectin synergize to contributes to the cardiac morphogenesis, as fn mutants promote cardiac progenitor migration. partially penetrate the cardia bifida phenotype (Trinh and kt259 Stainier, 2004). Our established fn mutant, a null mutant Lower jaw development is cooperatively regulated by Spns2 with a premature termination at codon 241, predominantly and fibronectin presented a straight heart tube phenotype (31/133 embryos The facial skeleton is formed from mutual interactions between kt259 obtained from the crossing of fn heterozygous fish) at cranial neural crest cells and both the pharyngeal endoderm and 25 hours post-fertilization (hpf), but also had a minor population ectoderm of zebrafish. Additionally, secreted proteins such as showing cardia bifida (3/133 embryos), a low penetration result Endothelin1, BMPs and Fgfs are key regulators involved in the that may be explained by the genetic background. To examine the craniofacial development (Alexander et al., 2011; Yamauchi genetic interactions between S1P signaling and fibronectin, we et al., 2011). It has been shown that the spns2 mutant displays a kt259 ko157 generated double mutant zebrafish fn ;spns2 by the disorganized anterior pharyngeal endoderm (Osborne et al., ko157 kt259 crossing spns2 and fn mutants. As shown in Fig. 1, the 2008). We noticed that our fn;spns2 double mutant exhibits fn:spns2 double mutant displayed two widely separated hearts severe defects in ventral facial morphology. Therefore, we kt259 ko157 (19/322 embryos obtained from the crossing of fn ;spns2 examined the pharyngeal arch structure of individual mutants by heterozygous fish). The distances between hearts were much Alcian Blue staining at 4 days post-fertilization (dpf). The greater than those of the spns2 (cardia bifida; 30/145 embryos anterior–posterior distance of the ventral pharyngeal arches ko157 obtained from the crossing of spns2 heterogeneous fish) or fn (Meckel’s, palatoquadrate and ceratohyal cartilages) in the mutants (straight heart tube; 31/133 embryos obtained from the spns2 or fn mutants was shorter than that of wild type (Fig. 2; kt259 crossing of fn heterozygous fish) (supplementary material supplementary material Table S2). On the other hand, the Table S1). The genotypes of individual mutants were confirmed number and morphology of the ceratobranchial arch were by the direct sequencing of individual spns2 and fn genomic loci. relatively normal. The pharyngeal defects are consistent with a Whole-mount in situ hybridization analysis revealed that the recent report that demonstrated morphological defects of the expressions of the cardiac differentiation markers amhc (atrial lower jaw in both s1pr2 and spns2 mutants (Balczerski et al., myosin heavy chain) and vmhc (ventricular myosin heavy chain) 2012). We also found that the cell adhesion molecule Fig. 1. Cardiac morphology. Heart positions are indicated by the arrowheads. (A–D) Cardiac morphology visualized by mRFP expression derived from Tg(cmlc2:mRFP). All images show ventral views at 28 hpf. (E–L) Whole-mount in situ hybridization with amhc and vmhc RNA probes. All images show ventral views at 30 hpf except for panel L (dorsal view). Genotyping was performed by genomic sequencing after taking pictures, wt (A,E,I), fn mutant (B,F,J), spns2 mutant (C,G,K) and fn;spns2 double mutant (D,H,L). Scale bars: 200 mm. (M) Average distances between two hearts from multiple experiments; error bars represent standard deviations. Biology Open Roles of spns2 and fibronectin 791 Fig. 2. Lower jaw morphology. (A–D) Lower jaw morphology at 4 dpf was visualized by Alcian Blue staining (ventral view). Anterior–posterior distances of the ventral pharyngeal arch (*) is indicated by the length of the double-headed arrows. The dorsal pharyngeal structure is identified by the crosses (+). (E–P) Whole- mount in situ hybridization using hand2, dlx2 and nkx2.3 RNA probes. The anteroventral position of these markers is marked by the arrowheads. All images show lateral views at 30 hpf. Genotyping was performed by genomic sequencing after taking pictures. wt (A,E,I,M), fn mutant (B,F,J,N), spns2 mutant (C,G,K,O), and fn;spns2 double mutant (D,H,L,P). Scale bars: 200 mm. (Q) Average anterior–posterior distances of the ventral pharyngeal arch from multiple experiments; error bars represent standard deviations. fibronectin is also required for the proper lower jaw develop- S1PR2, but not S1PR1, cooperates with fibronectin in cardiac ment (Fig. 2; supplementary material Table S2). In clear and lower jaw development contrast, the structure of the ventral pharyngeal arch (Fig. 2D, Spns2 functions as an S1P transporter (Kawahara et al., 2009), asterisks) was disorganized in the fn;spns2 double mutant, suggesting that some S1PRs contribute to cardiac and jaw whereas that of the dorsal pharyngeal structure (trabecular development. The s1pr2 mutant has been found to present the cartilage) appeared normal (Fig. 2D, cross). Jaw development is cardia bifida phenotype in zebrafish (Kupperman et al., 2000), regulated by the cooperation of several transcriptional factors whereas three independent groups recently reported that the including the hand2, dlx and nkx family genes (Miller et al., knockdown of s1pr1 causes severe defects in cardiac and 2003; Trinh et al., 2005; Talbot et al., 2010). Whole-mount in vascular development (Gaengel et al., 2012; Tobia et al., 2012; situ hybridization using the pharyngeal markers, hand2, dlx2 Mendelson et al., 2013), with the circulation of blood cells being and nkx2.3 at 30 hpf revealed that their anteroventral expres- particularly impaired in s1pr1-depleted embryos. We therefore sions of hand2, dlx2 and nkx2.3 were reduced in fn;spns2 examined the functional interaction of S1PR1/2 and fibronectin. mutants (hand2, n57; dlx2, n57; nkx2.3, n57) compared to the Using TALEN (transcription activator-like effector nuclease) their posterior expressions, whereas the expression patterns and technology, we recently established s1pr1 and s1pr2 knockout intensities of these markers were normal in fn mutants (hand2, fish (Hisano et al., 2013; Ota et al., 2013). We confirmed that n57; dlx2, n55; nkx2.3, n57) and spns2 mutants (hand2, n57; s1pr2 knockout mutants show cardia bifida phenotype (Fig. 3E). dlx2, n58; nkx2.3, n58). The genotypes of individual mutants However, no obvious cardiac or vascular defects in s1pr1 were confirmed by the direct sequencing of spns2 and fn knockout mutants were seen during early embryogenesis genomic loci after taking pictures. Because the first pouch (Fig. 3C; supplementary material Movies 3, 4). Thus, we endoderm is required for the pharyngeal arch formation conclude that zygotic s1pr1 mutants showed normal blood (Alexander et al., 2011), our results suggest that Spns2 and circulation and intersegmental vessel angiogenesis, which fibronectin contribute to the formation of the ventral pharyngeal disagrees with the aforementioned reports. Those studies all arch by regulating the anteroventral expression of various used identical S1PR1-morpholino. It is possible that the S1PR1- pharyngeal markers. morpholino caused off-target effects or also affected the maternal Biology Open Roles of spns2 and fibronectin 792 Fig. 3. Knockdown phenotype of fibronectin in S1PRs mutant. (A) Membrane topology of S1P receptors and their mutants. The region of frameshift-mediated amino acids compared with the S1PR2 wild type (WT) is shown by the dashed line. (B,C,D) Fluorescence microscopy of intersegmental vessels of wt (B), s1pr1 mutant (C) and s1pr2 mutnat (D) at 2 dpf. Endothelial cells are visualized by EGFP expression derived from Tg(fli1a:EGFP). (E,F) Cardiac morphology visualized by mRFP expression derived from Tg(cmlc2:mRFP). All images show ventral views at 28 hpf. (G) Average distances between two hearts from multiple experiments; error bars represent standard deviations. (H,I) Lower jaw morphology at 4 dpf was visualized by Alcian Blue staining (ventral view). (J) Average anterior–posterior distances of the ventral pharyngeal arch from multiple experiments; error bars represent standard deviations. Genotyping was performed by genomic sequencing or heteroduplex mobility assays after taking pictures. Scale bars: 200 mm. message of s1pr1. To explain the different conclusions, it would cardiac migration and lower jaw morphology were observed be best to study a maternal-zygotic s1pr1 mutant, which will be when S1PR2-MO (10 ng) was injected into fn mutants available in a future study. When Fn-MO (10 ng) was injected (supplemental material Fig. S1). These results suggest that into the s1pr2 mutant, a more severe cardia bifida phenotype was S1PR2, but not S1PR1, cooperates with fibronectin in both induced compared to the s1pr2 mutant (Fig. 3E–G). Consistent cardiac and lower jaw development. It has been reported that with these results, severe defects in cardiac migration and lower both s1pr2 and spns2 mutants lack the anterior endoderm jaw morphology were observed when S1PR2-MO (10 ng) was (Balczerski et al., 2012). Because signaling pathways through injected into fn mutants (supplemental material Fig. S1), which the Spns2-S1PR2 axis regulate cell proliferation of the anterior agrees with previous knockdown analysis that used S1PR2-MO endoderm tissue and therefore affect the positioning of the and Fn-MO (Matsui et al., 2007). Such a cooperative cardiac ventral pharyngeal arch, further analysis will be required to defect was not observed in s1pr1 embryos injected with Fn-MO clarify how S1P signaling and fibronectin together control the (supplementary material Table S1), which instead showed a movement and adhesion of anterior pharyngeal endodermal cells. straight heart tube phenotype similar to that in fn mutants. Fn- MO-injected s1pr1 mutants embryos had a slightly shorter Endothelin receptor A, a possible mediator downstream of anterior–posterior distance in their ventral pharyngeal arch Spns2-S1PR2 signaling structure, quite unlike the disorganized ventral pharyngeal arch Both Spns2 and S1PR2 are involved in cardiac and lower jaw structure observed in Fn-MO-injected s1pr2 mutants (Fig. 3H–J; development. To identify the genes regulated by the Spns2- supplementary material Table S2). Additionally, severe defects in S1PR2 axis, we performed microarray analysis. Total RNA was Biology Open Roles of spns2 and fibronectin 793 Fig. 4. Knockdown phenotype of endothelin receptor A (ednra)in fn mutants. (A,B) Whole-mount in situ hybridization using the ednra RNA probe. The expression of ednra was suppressed in the spns2 mutant. Both images show lateral views at 30 hpf. (C,D) Cardiac morphology visualized by mRFP expression derived from Tg(cmlc2:mRFP). Both images show ventral views at 28 hpf. (E,F) Lower jaw morphology at 4 dpf was visualized by Alcian Blue staining (ventral view). Genotyping was performed by genomic sequencing after taking pictures. wt (A,C,E), spns2 mutant (B) and fn mutant (D,F). Scale bars: 200 mm. (G,H) Average distances between hearts (G) and anterior– posterior distances of the ventral pharyngeal arch (H) from multiple experiments; error bars represent standard deviations. isolated from uninjected, S1PR2-MO (10 ng)-injected and S1P-S1PR2 and End1-Ednra being critical in the patterning of the Spns2-MO (10 ng)-injected embryos at 25 hpf, and their gene ventral pharyngeal arch, which agrees with our demonstrating expression profiles were compared. We found that the expression that Ednra, a possible downstream target of the Spns2-S1PR2 axis, synergizes with fibronectin to promote the lower jaw of endothelin receptor A (ednra) was downregulated in both development. Spns2-depleted and S1PR2-depleted embryos (see Materials and Methods). Consistent with this result, the expression of ednra in Conclusion the pharyngeal arches of the spns2 mutant (n510) was reduced In this study, using zebrafish genetic mutants (fn, spns2 and compared to that of wild type (n57) (Fig. 4A,B). In both fn;spns2), we demonstrated that Spns2-S1PR2 signaling and the zebrafish and mouse, disruption of endothelin1 (edn1) causes a cell adhesion molecule fibronectin cooperatively regulate the loss or transformation of the lower jaw (Nair et al., 2007; Tavares migration of cardiac progenitors. Further, Spns2-S1PR2 and et al., 2012). Because Edn1 functions through its cognate type-A fibronectin synergize to promote ventral pharyngeal cartilage receptor Ednra, Ednra can be considered a key regulator in formation. Because the expression of ednra in the pharyngeal pharyngeal development (Nair et al., 2007). However, it is not arches of spns2 mutants is reduced, we propose that Ednra clear how Ednra cooperates with other molecule(s) during jaw contributes to the lower jaw arrangement by cooperating with development. Therefore, we investigated the functional fibronectin. interaction between Ednra and fibronectin. Heart morphology seemed normal when Ednra-MO (10 ng) was injected into wild- Materials and Methods type embryos (n513), while cardiac defects in Ednra-MO Zebrafish mutants injected fn embryos (n511) were slightly more severe than kt259 kt259 ko157 Mutant alleles of fibronectin (fn ) and spns2 were used (Koshida et al., ko157 those of fn embryos (Fig. 4C,D; supplementary material Table 2005; Kawahara et al., 2009). To obtain double mutants, the spns2 allele was kt259 kt259 ko157 S1). Further, a disorganization of the ventral pharyngeal arch crossed into the fn allele. Embryos of fn ;spns2 were obtained from kt259 ko157 ko157 kt259 fn ;spns2 heterogeneous fish. Embryos of spns2 and fn were arrangement in Ednra-MO injected fn embryos (n510) was kt259 ko157 obtained from fn and spns2 heterogeneous fish, respectively. Genotyping ko157 kt259 kt259 ko157 observed relative to that of Ednra-MO injected wild-type embryo of spns2 and fn single mutants and fn ;spns2 double mutants was (n57) (Fig. 4E,F; supplementary material Table S2). The performed by direct sequencing of individual genomic loci as described below. To monitor the cardiac development, the transgenic line Tg(cmlc2:mRFP) was used. genotypes of individual mutants were confirmed by the direct sequencing of spns2 and fn genomic loci after taking pictures. Establishment of s1pr1 or s1pr2-knockout zebrafish Although in a cell culture system Edn1 increases the adhesion of TALEN constructs targeting s1pr1 or s1pr2 were described previously (Hisano amelanotic melanocytes to fibronectin (Ma et al., 2006), how et al., 2013; Ota et al., 2013). TALEN mRNAs (400 pg each) were injected into Edn1 signaling affects fibronectin function remains unclear. blastomeres at the 1–2 cell stage of zebrafish embryos. Identification of potential F0 founders and F1 embryos having mutant allele was performed by HMA Further analysis will be required to determine whether Edn1 ko311 (heteroduplex mobility assay) (Ota et al., 2013). The s1pr1 allele was deleted ko322 regulates the fibronectin-mediated cellular interaction during the from +362 to +371 of the s1pr1 coding region, while the s1pr2 allele was ventral pharyngeal arch arrangement. One clue comes from both deleted from +179 to +188 of the s1pr2 coding region. To monitor the cardiac and Biology Open Roles of spns2 and fibronectin 794 endodermal requirements for the growth but not dorsoventral patterning of jaw vascular development, the transgenic lines Tg(cmlc2:mRFP) and Tg(fli1a:EGFP) skeletal precursors. Dev. Biol. 362, 230-241. were used. s1pr1 or s1pr2 mutant embryos were obtained by the crossing Gaengel, K., Niaudet, C., Hagikura, K., Lavin˜a, B., Muhl, L., Hofmann, J. J., individual F1 heterogeneous fish. Ebarasi, L., Nystro¨m, S., Rymo, S., Chen, L. L. et al. (2012). The sphingosine-1- phosphate receptor S1PR1 restricts sprouting angiogenesis by regulating the interplay Preparation of genomic DNA and genotyping of spns2 and fn between VE-cadherin and VEGFR2. Dev. Cell 23, 587-599. George, E. L., Baldwin, H. S. and Hynes, R. O. (1997). Fibronectins are essential for mutants heart and blood vessel morphogenesis but are dispensable for initial specification of Genomic DNA was isolated using the Gentra Puregene Tissue Kit (Qiagen) precursor cells. Blood 90, 3073-3081. according to the manufacturer’s protocol. For the genotyping of the spns2 mutant, Hanaoka, R., Katayama, S., Dawid, I. B. and Kawahara, A. (2006). Characterization the spns2 genomic locus was amplified by PCR using the following primers: of the heme synthesis enzyme coproporphyrinogen oxidase (CPO) in zebrafish spns2-S, 59-TCAAGGAATGTGAGCCATGT-39; and spns2-AS, 59-GGATGCCA- erythrogenesis. Genes Cells 11, 293-303. GGTAGAAGACA-39. For genotyping of the fn mutant, the fn genomic locus was Hisano, Y., Nishi, T. and Kawahara, A. (2012). The functional roles of S1P in amplified by PCR using the following primers: fn-S, 59-CTTACTCAAGC- immunity. J. Biochem. 152, 305-311. TTAACTGG-39; and fn-AS, 59-ACCAAGACTAGTAGTGTGCAG-39. In the case Hisano, Y., Ota, S., Arakawa, K., Muraki, M., Kono, N., Oshita, K., Sakuma, of embryos stained with Alcian Blue or analyzed by whole-mount in situ T., Tomita, M., Yamamoto, T., Okada, Y. et al. (2013). Quantitative assay for hybridization, nested PCR was performed using the following primers: fn-S2, 59- TALEN activity at endogenous genomic loci. Biol. Open 2, 363-367. GGTTCTAATGGGAAACATCTGC-39; and fn-AS2, 59-GAGAAGCATGC- Kawahara, A., Nishi, T., Hisano, Y., Fukui, H., Yamaguchi, A. and Mochizuki, CTCTCACAC-39. The genotypes of spns2 and fn were determined by direct N. (2009). 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(2007). miles-apart-Mediated regulation of 15 min with maleic acid buffer (0.1 M maleic acid [pH 7.5]). Embryos were cell–fibronectin interaction and myocardial migration in zebrafish. Nat. Clin. Pract. incubated with anti-DIG alkaline phosphatase (Roche) in blocking buffer (0.1 M Cardiovasc. Med. 4 Suppl. 1:, S77-S82. maleic acid [pH 7.5], 5% sheep serum and 2% blocking reagent) for 4 hr. After Mendelson, K., Zygmunt, T., Torres-Va´zquez, J., Evans, T. and Hla, T. (2013). embryos were washed with phosphate buffer saline (PBS) containing 0.1% Tween 20 Sphingosine 1-phosphate receptor signaling regulates proper embryonic vascular (PBST), color reactions were performed using BM purple (Roche) as the substrate. patterning. J. Biol. Chem. 288, 2143-2156. Meng, H. and Lee, V. M. (2009). Differential expression of sphingosine-1-phosphate receptors 1-5 in the developing nervous system. Dev. Dyn. 238, 487-500. Alcian Blue staining Miller, C. T., Yelon, D., Stainier, D. Y. and Kimmel, C. B. (2003). Two endothelin 1 Embryos at 4 dpf were fixed overnight by 4% paraformaldehyde in PBST. effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Embryos were washed with acid alcohol buffer (0.37% HCl and 70% ethanol) and Development 130, 1353-1365. incubated with 0.1% Alcian Blue (Sigma) in acid alcohol buffer. After 3 times Miura, G. I. and Yelon, D. (2011). A guide to analysis of cardiac phenotypes in the washing with acid alcohol buffer, embryos were incubated for 10 min with zebrafish embryo. Methods Cell Biol. 101, 161-180. bleaching buffer (1% H O and 1% KOH). 2 2 Nair, S., Li, W., Cornell, R. and Schilling, T. F. (2007). Requirements for Endothelin type-A receptors and Endothelin-1 signaling in the facial ectoderm for the patterning Knockdown analysis using antisense morpholinos and microarray of skeletogenic neural crest cells in zebrafish. Development 134, 335-345. Ohuchi, H., Hamada, A., Matsuda, H., Takagi, A., Tanaka, M., Aoki, J., Arai, analysis H. and Noji, S. (2008). Expression patterns of the lysophospholipid receptor genes Antisense morpholinos for fibronectin, spns2, s1pr2 and ednra were obtained from during mouse early development. Dev. Dyn. 237, 3280-3294. Gene Tools as follows: Fn-MO, 59-TTTTTTCACAGGTGCGATTGAACAC-39; Osborne, N., Brand-Arzamendi, K., Ober, E. A., Jin, S. W., Verkade, H., Holtzman, Ednra-MO, 59- AGTGGTGTGTTCACCTGTTTGAGGT-39; Spns2-MO, 59-GGA- N. G., Yelon, D. and Stainier, D. Y. (2008). The spinster homolog, two of hearts, is GGGAATATGTGATGCTTACTTC-39; and S1PR2-MO, 59-CCGCAAACA- required for sphingosine 1-phosphate signaling in zebrafish. Curr. Biol. 18, 1882- GACGGCAAGTAGTCAT-39 (Trinh and Stainier, 2004; Nair et al., 2007). Individual morpholinos (10 ng) were injected into the yolk of 1–4 cell stage Ota, S., Hisano, Y., Muraki, M., Hoshijima, K., Dahlem, T. J., Grunwald, D. J., embryos. Total RNA was isolated with TRIzol reagent (Invitrogen) from Okada, Y. and Kawahara, A. (2013). Efficient identification of TALEN-mediated individual embryos at 25 hpf. Microarray analysis was performed using a genome modifications using heteroduplex mobility assays. Genes Cells 18, 450-458. zebrafish-specific Affymetrix chip (15617 zebrafish probes). The expression of Skoura, A. and Hla, T. (2009). Lysophospholipid receptors in vertebrate development, ednra was downregulated in Spns2-MO-injected (Log2 Ratio: 21.06) and S1PR2- physiology, and pathology. J. Lipid Res. 50 Suppl.:, S293-S298. Spiegel, S. and Milstien, S. (2011). The outs and the ins of sphingosine-1-phosphate in MO-injected embryos (Log2 Ratio: 21.06). immunity. Nat. Rev. Immunol. 11, 403-415. Takuwa, Y., Okamoto, Y., Yoshioka, K. and Takuwa, N. (2012). Sphingosine-1- Acknowledgements phosphate signaling in physiology and diseases. Biofactors 38, 329-337. Talbot, J. C., Johnson, S. L. and Kimmel, C. B. (2010). hand2 and Dlx genes specify The authors thank R. Fukuoka, M. Komeno, M. Hayashi and S. Ohara dorsal, intermediate and ventral domains within zebrafish pharyngeal arches. for zebrafish maintenance and technical assistance, and P. Karagiannis Development 137, 2507-2517. for valuable comments. This work was supported by the Funding Tavares, A. L., Garcia, E. L., Kuhn, K., Woods, C. M., Williams, T. and Clouthier, Program for Next Generation World-Leading Researchers (NEXT D. E. (2012). Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch. Dev. Biol. 371, 47-56. Program) and by the Japan Society for the Promotion of Science. Tobia, C., Chiodelli, P., Nicoli, S., Dell’era, P., Buraschi, S., Mitola, S., Foglia, E., van Loenen, P. B., Alewijnse, A. E. and Presta, M. (2012). Sphingosine-1- Competing Interests phosphate receptor-1 controls venous endothelial barrier integrity in zebrafish. Arterioscler. Thromb. Vasc. Biol. 32, e104-e116. The authors have no competing interests to declare. Trinh, L. A. and Stainier, D. Y. (2004). Fibronectin regulates epithelial organization during myocardial migration in zebrafish. Dev. Cell 6, 371-382. References Trinh, L. A., Yelon, D. and Stainier, D. Y. (2005). Hand2 regulates epithelial Alexander, C., Zuniga, E., Blitz, I. L., Wada, N., Le Pabic, P., Javidan, Y., Zhang, formation during myocardial diferentiation. Curr. Biol. 15, 441-446. T., Cho, K. W., Crump, J. G. and Schilling, T. F. (2011). Combinatorial roles for Wierzbicka-Patynowski, I. and Schwarzbauer, J. E. (2003). The ins and outs of BMPs and Endothelin 1 in patterning the dorsal–ventral axis of the craniofacial fibronectin matrix assembly. J. Cell Sci. 116, 3269-3276. skeleton. Development 138, 5135-5146. Yamauchi, H., Goto, M., Katayama, M., Miyake, A. and Itoh, N. (2011). Fgf20b is Balczerski, B., Matsutani, M., Castillo, P., Osborne, N., Stainier, D. Y. and Crump, required for the ectomesenchymal fate establishment of cranial neural crest cells in J. G. (2012). Analysis of sphingosine-1-phosphate signaling mutants reveals zebrafish. Biochem. Biophys. Res. Commun. 409, 705-710. Biology Open

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Published: Aug 15, 2013

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