DEVELOPMENTAL DYNAMICS 235:2276–2281, 2006 PATTERNS & PHENOTYPES Restricted Expression of Fgf16 Within the Developing Chick Inner Ear 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). © 2006 Wiley-Liss, Inc. FGF16 EXPRESSION 2277 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), us- ing embryos from prestreak stages, HH1 to HH20 (Hamburger and Hamil- ton, 1951). The earliest stage with de- tectable expression was HH8/9 in an anterior-to-posterior stripe of dorsally expressing non-neural ectoderm adja- cent 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 ven- trally 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 postero- lateral 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.