Restricted Expression of Fgf16 Within the Developing Chick Inner Ear

DEVELOPMENTAL DYNAMICS 235:2276–2281, 2006

PATTERNS & PHENOTYPES

Susan C. Chapman,1* Qin Cai,1 Steven B. Bleyl,2 and Gary C. Schoenwolf1

Fibroblast growth factor (FGF) signaling is required for otic placode induction and patterning of the developing inner ear. We have cloned the chick ortholog of Fgf16 and analyzed its expression pattern in the early chick embryo. Expression is restricted to the otic placode and developing inner ear through all the stages examined. By the closed otocyst stage, expression has resolved to anterior and posterior domains that partially overlap with those of bone morphogenetic protein 4 (Bmp4), a marker of the developing sensory patches, the cristae of the anterior and posterior semicircular canals. Platelet-derived growth factor alpha (PDGFA), another growth factor with restricted otic expression, also overlaps with Fgf16 expression. The restricted expression pattern of Fgf16 suggests a role for FGF signaling in the patterning of the sensory cristae, together with BMP signaling. Developmental Dynamics 235:2276–2281, 2006. © 2006 Wiley-Liss, Inc.

Key words: Fgf; inner ear; chick; otic development; patterning

Accepted 17 May 2006

INTRODUCTION pattern by in situ hybridization within ditory cochlea, including the auditory the early embryo, finding specific ex- sensory organ (basilar papilla) and Inner ear patterning involves a com- pression only within the otic placode seven vestibular organs (sensory patch- plex, interrelated series of signaling and developing inner ear. The inner ear es): one lagena (organ of Corti), two events that include members of the fi- originates from the otic placode, an ec- maculae (sacculus, utriculus), one mac- broblast growth factor (FGF) family of secreted signaling factors (Noramly and todermal thickening adjacent to rhom- ula neglecta and three cristae ampullae Grainger, 2002; Wright and Mansour, bomeres 5 and 6, which becomes appar- (superior, posterior, and lateral; Wu 2003). FGFs have important roles in ent at Hamburger and Hamilton stage and Oh, 1996). proliferation, migration, and differenti- (HH) 9 (Hamburger and Hamilton, Fgf16 orthologues have been identi- ation (Ornitz and Itoh, 2001), especially 1951). By HH11 the placode begins to fied in human and rat (Miyake et al., in directional signaling across epitheli- invaginate, forming the otic pit/cup. 1998), mouse (Sontag and Cattini, al–mesenchymal boundaries (Hogan, The rims of the otic cup make contact, 2003), and zebrafish and chimpanzee 1999), reminiscent of the situation forming the closed otocyst at around (Katoh and Katoh, 2005), with Ensembl within the developing chick inner ear, HH16, with the endolymphatic duct reporting further orthologues in Canis where mesodermal FGF19 in synergy and sac arising from a dorsomedial pro- familiaris, Monodelphis domestica, and with WNT8c signals from the hindbrain jection of the otocyst, visible by HH21 Xenopus tropicalis, among others (Bir- are sufficient for otic placode induction (Barald and Kelley, 2004). Further com- ney et al., 2006). Fgf16 is a member of (Ladher et al., 2000). We have cloned plex developmental steps lead to emer- the FGF subfamily consisting of Fgf9/ chick Fgf16 and analyzed its expression gence of the vestibular system and au- 16/20, none of which possess the stan-

1University of Utah, School of Medicine, Department of Neurobiology and Anatomy, and Children’s Health Research Center, Salt Lake City, Utah 2University of Utah, Department of Pediatrics, Division of Medical Genetics, Salt Lake City, Utah Grant sponsor: NIH; Grant numbers: DC04185; DK066445; 1 K08 HL084559-01; Grant sponsor: American Heart Association. *Correspondence to: Susan C. Chapman, University of Utah, School of Medicine, Department of Neurobiology and Anatomy, and Children’s Health Research Center, 2R066 SOM, 30 North 1900 East, Salt Lake City, UT 84132-3401. E-mail: [email protected] DOI 10.1002/dvdy.20872 Published online 19 June 2006 in Wiley InterScience (www.interscience.wiley.com).

Fig. 1. Predicted amino acid sequence comparison of chick fibroblast growth factor (FGF) 16 with human FGF16 and mouse FGF16. The colon indicates identical amino acid residues of the compared sequences. The 187/207 residues are identical to human (90.3%), and 185/207 are identical to mouse (89.3%). Mouse and human are 99.0% identical, with a two amino acid difference. dard cleavable signal sequence. How- 2003). Otic expression was restricted head mesoderm, neural crest, surface ever, all are secreted due to a to a single region, the dorsolateral ectoderm, or branchial endoderm. combination of a unique N-terminal re- region of the posterior otocyst. Our gion and a central hydrophobic region, extensive analysis of the chick Fgf16 containing the EFISIA motif, encoding expression pattern demonstrates RESULTS AND DISCUSSION for a nonclassical mode of secretion that, in early developmental stages, Cloning of Chick Fgf16 (Miyakawa and Imamura, 2003; Popo- Fgf16 is detected solely in the otic vici et al., 2004). Although several FGF region, with no other expression in Using the known mouse Fgf16 se- receptors have been identified in the de- the developing embryo. Dynamic ex- quence, we identified a chick ex- veloping inner ear (Wright et al., 2003), pression within the otic placode was pressed sequence tag (EST) ortholog previously only FGF receptor 4 refined, until at the otocyst stage, that contained a partial chick Fgf16 (FGFR4) had been shown to bind to two defined spots of expression are fragment lacking the 5Ј end. We FGF16 in vitro (Konishi et al., 2000). detected in the patches of cells fated cloned the full-length 624-bp coding FGF receptor affinity for FGF16 has to become the anterior and posterior sequence of chick Fgf16 using 5Ј-rapid now been determined in a mammalian sensory cristae. Chick hindbrain ro- amplification of cDNA ends (RACE) cell culture system. FGF16 preferen- tation experiments suggest that sig- GeneRacer Kit (Invitrogen). The pre- tially binds to the c splice form recep- nals from the hindbrain are respon- dicted chick 207 amino acid sequence tors with affinity from strongest to sible for dorsoventral patterning of is 90.3% and 89.4% identical to that of weakest, 3c Ͼ 2c Ͼ 1c Ͼ 3b Ͼ 4, and no the inner ear but not anterior–poste- human and mouse FGF16 (Miyake et activity toward FGFR1b or FGFR2b al., 1998), respectively (Fig. 1). A frag- (Zhang et al., 2006). rior patterning (Bok et al., 2005). ment of the full-length sequence in- Expression of Fgf16 has been re- Thus, as yet unidentified intrinsic sig- cluding a portion of the 3Ј-untrans- ported previously in late embryonic nals and/or extrinsic signals originat- and adult cardiac tissues and embry- ing from tissues other than the hind- lated region was used to make an in onic brown adipocytes of mice and brain are required for anterior– situ hybridization probe, eliminating rats (Miyake et al., 1998; Konishi et posterior patterning. Based on the the possibility of cross-hybridization al., 2000; Sontag and Cattini, 2003; restricted expression pattern of Fgf16, to other Fgf family members. Analysis Lavine et al., 2005). In an evaluation we hypothesize that FGF16 is an in- of the sequence within the respective of 18 mouse Fgf gene expression pat- trinsic signal involved in patterning genomes reveals that both the mouse terns, Fgf16 was identified as a the anterior and posterior sensory and the human FGF16 genes are lo- marker within the murine otic pla- cristae. This hypothesis does not ex- cated on the X chromosome, whereas code, developing otic vesicle, pharyn- clude an extrinsic signal upstream of FGF16 in the Gallus genome is lo- geal endoderm, branchial arches, FGF16 arising from tissue surround- cated on chromosome 4. The signifi- and olfactory placode (Wright et al., ing the developing inner ear, such as cance of this difference is not clear. 2278 CHAPMAN ET AL.

Restricted Expression of Fgf16 Within the Developing Inner Ear Analysis of the gene expression pattern of Fgf16 was undertaken by whole- mount in situ hybridization (ISH), using embryos from prestreak stages, HH1 to HH20 (Hamburger and Hamil- ton, 1951). The earliest stage with detectable expression was HH8/9 in an anterior-to-posterior stripe of dorsally expressing non-neural ectoderm adjacent to the hindbrain, corresponding to the dorsal part of the future otic placode (Fig. 2A,B). At HH8 (3–5 somites), non- neural ectoderm rostral to the first somite is presumptive otic placode and is marked by Pax2 expression at the four to five somite stage (Groves and Bronner-Fraser, 2000). By HH9 (six to eight somites), the ectoderm has thick- ened and an overt otic placode is dis- cernible (Fig. 2B). Fgf16 expression, thus, is first detectable at the cusp of otic placode formation. Expression was detected exclusively in otic tissue at all stages examined. By HH12, expression was restricted to the dorsomedial lip of the invaginating otic placode (Fig. 2C,D). This expression expanded ventrally at HH13, with a weaker domain extending ventrally from the original dorsomedial expression domain (Fig. 2E,F). By HH16, the lips of the otic cup have fused to form the otocyst and two distinct, equally strong expression zones were observed in dorsal view: an anterior–medial and separate posterolateral domains (Fig. 2G). Transverse sections of the embryo confirm strong staining in both the anterior (Fig. 2H) and posterior zones (not shown). Dy- namic changes within the expression Fig. 2. A–X: Fibroblast growth factor (Fgf) 16 mRNA expression analysis. A,C,E,G: Dorsal views domains occur between stages HH16– of whole-mount embryos from Hamburger and Hamilton stage (HH) 9–16 with anterior to the top 20. From the two initially strong do- of the page. B,D,F,H: Transverse gelatin sections (50 ␮m) through the more anterior part of the mains, the anterior–medial domain expression domain. The black bar in A indicates the level of the transverse section shown in B. weakens at HH17 (compare Fig. Similarly for C, E, and G the black bar indicates the level of the section for D, F, and H, respectively. I,M,Q,U: Lateral views of the developing otocyst from HH/17–20, with anterior to the right. J,N,R,V: 2G,J, dorsal views). The dorsal view Dorsal views of stages HH/17–20. The anterior bar labeled K and more posterior bar labeled L confirms down-regulation of the an- indicate the level of the respective anterior and posterior transverse sections shown in the two terior–medial domain, whereas the panels to the right, K and L. N, R, and V were similarly sectioned. K, O, S, W are transverse sections dorsoposterior domain in the otocyst (50 ␮m) of the left otocyst (lateral to left) through the more anterior expression domain within the otocyst. L, P, T, X are sections from the more posterior domain (50 ␮m) of the same embryos. Scale remains unchanged (Fig. 2J). Dorsal bars ϭ 200 ␮m in A–J,M,N,Q,R,U,V, 100 ␮m in K,L,O,P,S,T,W,X. expression of Fgf16 begins to separate into two separate domains when viewed from the lateral side, with and stronger lateral domain (Fig. posterior spots (Fig. 2M,N) that bear the anterior spot being weaker than 2J,K). The more posterior section re- a striking resemblance to bone mor- the posterior spot (Fig. 2I). The an- capitulates this pattern (Fig. 2J,L). phogenetic protein 4 (Bmp4) and terior transverse section of the oto- Within a few hours, Fgf16 expres- platelet-derived growth factor alpha cyst reveals that the more anterior sion has resolved into two distinct (PDGFA) expression within the puta- spot is composed of a weak medial domains, with separate anterior and tive cristae domains (see below). A dor- FGF16 EXPRESSION 2279 sal view of the expression domain con- timing to Fgf16, Bmp4 expression be- lying within the ventral domain of firms that the spots are now fully comes confined to two spots, an anterior Fgf16 expression (Fig. 3C,F). separated, with a defined posterior and posterior domain, between HH16 domain now visible (Fig. 2N). Trans- and HH20. We show here that, at Double In Situ Hybridization verse sections from the anterior (Fig. HH19, both in the anterior and poste- Analysis: Fgf16 and PDGFA 2O) and posterior otocyst reveal the pos- rior expression domain that Fgf16 (Fig. terior spot to be stronger laterally than 3G) and Bmp4 (Fig. 3H) partially over- Of interest, PDGFA is also expressed medially (Fig. 2P). From HH18, the lap (Fig. 3A–F), with Bmp4 expression at HH19 in the anterior and posterior anterior domain is progressively restricted to the front edge of the otocyst (Fig. 2M,Q,U, lateral view; 2N,R,V, dorsal view), with stronger expression on the lateral side in transverse section (Fig. 2O,S,W). The posterior domain shifts from strong posterolateral expression to more posteromedial expression (Fig. 2P,T,X). The anterior and posterior spots are strongly reminiscent of Bmp4 expression from HH17 onward (see below).

Double In Situ Hybridization Analysis: Fgf16 and Bmp4 To determine the relationship between Fgf16 and Bmp4 expression patterns, we used double ISH. The anterior and posterior Bmp4 expression domains mark the areas that give rise to the respective anterior and posterior crista ampullaris of the semicircular canals (Wu and Oh, 1996). In an example of remarkably similar developmental

Fig. 3. Double in situ hybridization with Fgf16/ Bmp4 and Fgf16/PDGFA at Hamburger and Hamilton stage (HH) 19. A–F: Transverse sections (50 ␮m) from the same embryo double labeled with Fgf16/Bmp4. A–C: Anterior sections through the otocyst. D–F: Posterior sections through the otocyst. G: Insert G is a close-up of a lateral view of a whole-mount embryo showing two spots of Fgf16 expression in the otocyst at HH19; anterior is to the right. H: Similarly, insert H shows two spots of Bmp4 expression in an HH19 lateral view of a whole- mount embryo. Note in the merged images, that Bmp4 expression overlaps with the ventral domain of expression of Fgf16 in both the anterior (C) and posterior (F) otocyst. I–N: Transverse sections (50 ␮m) from the same embryo double labeled with Fgf16/PDGFA. I–K: Anterior sections through the otocyst. L–N: Posterior sections through the otocyst. O: Insert O is a close-up of a lateral view of a whole-mount embryo showing PDGFA in the otocyst at HH19; anterior is to the right. Note in the merged images that PDGFA overlaps the expression domain of Fgf16 in both the anterior (K) and posterior (N) otocyst. FGF, fibroblast growth factor; Bmp, bone morphogenetic protein; PDGFA, platelet-derived growth factor alpha. Scale bar ϭ 200 ␮m in A–F,I–N; 300 ␮min lateral views G,H,O. 2280 CHAPMAN ET AL. domains (Fig. 3O). PDGFs are impor- terest, experiments that disrupt or en- Total RNA was extracted using tant during embryonic development, hance FGF signaling provide evidence RNeasy Mini Kit (Qiagen). Using the as demonstrated by genetic analysis that FGFs (Fgf2, 3, 10) are upstream manufacturer’s GeneRacer protocol in mouse involving both knockout of of BMPs (Bmp2 but not Bmp7) in the yielded a polymerase chain reaction ligands and receptors and transgenic induction of cristae and associated fragment of 160 bp that was ligated overexpression of ligands (Betsholtz, semicircular canals (Chang et al., into pCRII-TOPO Vector (Invitrogen) 2004). PDGFs are involved in prolifer- 2004). Functional experiments are un- and sequenced. Based on this 5Ј se- ation, migration, differentiation, and der way to determine the role of Fgf16 quence a Forward Primer ATG GCC cell survival in vertebrate develop- in inner ear patterning. GAG GTG GGC GGC TT and Reverse ment. Previously, the PDGFA ligand Primer TCA CCT GTA GTG GAA was identified in later stages as a po- EXPERIMENTAL GAG GTC were designed to clone out tentially important mitogen in the the full-length gene, which was li- growth and proliferation of hair cells PROCEDURES gated into pCRII-TOPO Vector (In- in the developing rat cochlea. These Incubation, harvesting, staging, in vitrogen) and confirmed by sequenc- data, however, are not conclusive, situ hybridization and Vibratome sec- ing (NCBI full-length sequence lacking an analysis of the spatial and tioning were performed according to accession number DQ640740). The temporal expression pattern within our standard protocols as described primers used to make the in situ hy- otic tissue. Moreover, in the study by previously (Chapman et al., 2002). bridization probe were as follows: For- Lee and coworkers, both sense and an- Fgf16 digoxigenin (DIG) -labeled ward primer GTA CGC CTC AAC tisense oligonucleotides inhibited probe is visualized using anti-DIG an- GCT CTA C, reverse primer AAC AGC DNA synthesis in otic tissue, suggest- tibody and colored with nitroblue tet- AGT TGT CTG GAT TC. ing that toxic effects from the oligonu- razolium/5-bromo-4-chloro-3-indolyl cleotides lead to a decrease in cell pro- phosphate (NBT/BCIP) staining as ACKNOWLEDGMENT liferation rather than loss of PDGFA previously described (Fig. 3, left col- activity (Lee et al., 2004). Our results umn). Bmp4 and PDGFA fluorescein S.B. is supported by a grant from the demonstrate specific expression of isothiocyanate (FITC) -labeled probes American Heart Association and by PDGFA in the chick otocyst at HH19. are visualized by using anti-FITC an- the NIH. 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