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doi: 10.1242/10.1242/dev.00445

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Fgf3 and Fgf8 dependent and independent transcription factors are required for otic placode specification

Dong Liu1, Hsin Chu*, Lisa Maves1, Yi-Lin Yan1, Paul A. Morcos2, John H. Postlethwait1 and Monte Westerfield1,{dagger}

1 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
2 Gene Tools, LLC, 1 Summerton Way, Philomath, OR 97370, USA



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  		Fig. 1. Cells of the otic placode and vesicle express three transcription factor genes, dlx3b, dlx4b and sox9a required for inner ear development. (A,D,G) Cells of the presumptive otic placode express all three genes at the four-somite stage (4s); other cells along the lateral edge of the neural plate express dlx3b and dlx4b. (B,E,H) By the 10-somite stage (10s) cells throughout the placode express all three genes. (C,F,I) Later, by prim-5 stage (24 h), a subset of cells in the vesicle expresses dlx3b and dlx4b; other cells express sox9a. Throughout these stages, dlx4b expression is similar to but weaker than dlx3b expression. Other sites of expression are not shown. (J,L) Wild-type embryos exhibit an otic vesicle in live embryos (DIC, differential interference contrast optics) at prim-5 (24h) stage (J) and pax2a expression in the presumptive otic placode at the six-somite stage (L). (K,M) Neither the vesicle (K) nor early pax2a expression in the presumptive placode (M) is apparent in Dfb380 mutants (38/38 Dfb380 embryos). (N) The dlx3b, dlx4b and sox9a genes are closely linked on the same chromosome and are removed by the Dfb380 deficiency mutation. (Left) Schematic map of LG12 showing the region of the Dfb380 deficiency in red (not to scale). (Right) We localized dlx3b, dlx4b and sox9a to LG12 by mapping them relative to SSLP markers and other genes (as shown) using the LN54 radiation hybrid panel (Hukriede et al., 1999Go). We estimated the extent of the Dfb380 deficiency based on our ability to amplify flanking SSLP markers, z1176 and z1473, by PCR using Dfb380 homozygous mutant DNA. These two SSLP markers are separated by 21 cM on the HS and 24 cM on the MGH meiotic panels, and by 296 cR on the T51 radiation hybrid panel (http://zfin.org). Missing genes are shown in red. (A,B,D,E,G,H,L,M) Dorsal views, anterior towards the left; (C,F,I,J,K) side views, anterior towards the left, dorsal towards the top. Scale bar in N: 60 µm for A,D,G; 33 µm for B,E,H; 22 µm for C,F,I; 77 µm for J,K; 145 µm for L,M; 5 cM for N.

 


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  		Fig. 2. Knockdown of Dlx3b, Dlx4b and Sox9a functions mimics the Dfb380 mutant ear phenotype. (A,D,G) Otic vesicles of wild-type embryos express pax2a, fn1 and cldna. (B,E,H) A few cells in Dfb380 mutants form a small epithelial ball and express the same markers (83/196 Dfb380 mutants expressed pax2a, 11/17 expressed fn1 and 10/20 expressed cldna). (C,F,I) Injection of a mixture of dlx3b-MO, dlx4b-MO and sox9a-MO produces a similar, small ball of cells that express the same markers (24/56 expressed pax2a, 8/20 expressed fn1 and 2/7 expressed cldna). Developmental stage: Prim-5 (24 h). Side views, anterior towards the left, dorsal towards the top. Scale bar: 25 µm.

 


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  		Fig. 3. Dlx3b function is required for generation of the full complement of otic sensory hair cells and semi-circular canals. (A,C,I) In normal (control) embryos, pax2a expression marks the presumptive otic placode at the three-somite (3s; A) and eight-somite (8s; C) stages. By high-pec stage (43h; I), otic sensory hair cells express Pax2a as indicated by antibody ({alpha}-Pax2; I) labeling. (B,D,J) In embryos injected with dlx3b-MO, pax2a expression is absent early (B; three-somite stage; 16/60 embryos) and reduced later (D; eight-somite stage; 5/17 embryos). By high-pec stage (J; 43 h) about half the normal number of Pax2-expressing otic sensory hair cells are apparent after knock down of dlx3b function by morpholino injection. Hair cell numbers as indicated by {alpha}-Pax2 labeling: wild-type 11.9±0.8 (n=34), dlx3b-MO 6.9±1.5 (n=49), dlx4b-MO 9.4±1.2 (n=7), dlx3b + dlx4b MOs 0.2±0.4 (n=18). (E,F) A Dlx3b antibody ({alpha}-Dlx3b) labels Dlx3b-expressing cells at the six-somite stage in normal embryos (E), but not after injection of dlx3b-MO (F). (G,H,K,L) Knock down of dlx3b function blocks formation of the otoliths (G,H; prim-5 stage, 24 h) and subsequent formation of the semi-circular canals (K,L; pec-fin stage). Twenty-eight percent of 356 injected embryos lacked both otoliths and semi-circular canals; all embryos injected with dlx3b-MO and dlx4b-MO lacked otoliths (data not shown). (A-F) Dorsal views, anterior towards the left; (G-L) side views, anterior towards the left, dorsal towards the top. Scale bar: 107 µm for A-D; 110 µm for E,F; 73 µm for G,H,K,L; 25 µm for I,J.

 


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  		Fig. 4. Restoring Dlx3b, Dlx4b and Sox9a functions together rescues the Dfb380 mutant ear phenotype at the 10-somite stage. (A,E) Wild-type embryos express pax2a in the presumptive otic placodes. (B,F) Dfb380 mutant embryos lack early otic expression of pax2a (50/51 embryos). (C,G) Injection of wild-type dlx3b mRNA into Dfb380 mutants partially restores otic pax2a expression (13/30 embryos). (D,H) Injection of wild-type dlx3b, dlx4b and sox9a mRNAs into Dfb380 mutants restores otic pax2a expression to nearly normal levels (22/65 embryos). In other experiments, 8/30 Dfb380 embryos injected with dlx3b and dlx4b and 11/33 injected with dlx3b and sox9a showed rescued pax2a expression. (A-D) Dorsal views, anterior towards the left; (E-H) side views, anterior towards the left, dorsal towards the top. Scale bar: 100 µm in A-D; 31 µm in E-H.

 


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  		Fig. 5. sox9b functions in otic specification. (A,B) The splice-blocking morpholino against sox9b (sox9bMO) significantly reduces the size of the otic vesicle (B, n=40 embryos), compared with that of wild-type control embryos (A). (C,D) In jellyfish (jef) mutants that harbor a retroviral insertion in the sox9a gene (jefhil134) (Amsterdam et al., 1999Go; Yan et al., 2002Go), injecting sox9b-MOs results in the loss of a morphologically recognizable otic vesicle (n=9 embryos), although a few dispersed pax2a-positive cells persist (D). In uninjected jef (sox9a) mutants (C, n=6 embryos), otic vesicles are slightly smaller than in wild-type control embryos (A). (E,F) Injecting sox9b-MO blocks formation of the residual otic cells of Dfb380 (4/6 Dfb380 embryos had residual pax2a-expressing cells, only 2/12 Dfb380 embryos had residual otic cells after injection with sox9b-MO). (A,B,E,F) prim-12 (28 h); (D,F) prim 25 (36 h). Side views, anterior towards the left, dorsal towards the top. Scale bar: 25 µm.

 


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  		Fig. 6. Fgf3 and Fgf8 signaling together is required for otic expression of sox9a but not sox9b or dlx3b. (A-D) Cells of the presumptive otic placodes express sox9a in wild-type embryos (A), at somewhat reduced levels after fgf3-MO injection (B), at more reduced levels in fgf8– mutant embryos (C) and at undetectable levels in fgf8– embryos injected with fgf3-MO (D; n=8). (E-H) Cells of the presumptive otic area and cranial neural crest (Li et al., 2002Go) express sox9b in wild-type embryos (E) and fgf3-MO injected embryos (F). Expression of sox9b in the otic region is slightly reduced in fgf8– embryos (G) and more significantly reduced, although not completely eliminated, in fgf8– embryos injected with fgf3-MO (H; n=10). sox9b expression in cranial crest is apparently unaffected by reduced fgf3 and fgf8 expression. (I-L) Otic expression of dlx3b is much less affected by reduction of Fgf signaling (n=16). Similar results were obtained for dlx4b (not shown). Expression of egr2 (krox20) in red indicates the locations of rhombomeres 3 and 5. Four-somite stage. Dorsal views, anterior towards the left. Scale bar: 110 µm.

 


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  		Fig. 7. Removal of both Fgf3 and Fgf8 signaling reduces the number, but does not eliminate Dlx3b-expressing otic cells. (A,B) Early expression of dlx3b in cells of the presumptive otic placode (A) is reduced in fgf8– mutants injected with fgf3-MO (B; n=18). (C-F) Later, at prim-6 stage (25h), dlx3b-expressing cells persist in fgf8– embryos injected with fgf3-MO (D) and the {alpha}-Dlx3b antibody shows that the residual Dlx3b-expressing cells are scattered (F), rather than being organized into an otic epithelium (E). Similar results were obtained in 3/15 injected mutants. (A,B) Dorsal view, anterior towards the left; (C-F) side view, anterior towards the left and dorsal towards the top. Scale bar: 100 µm for A-D; 25 µm for E,F.

 


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  		Fig. 8. Dlx3b and Sox9a crossregulate each other's expression in the otic placode. (A,B) dlx3b (blue) and sox9a (red) colocalize in the presumptive otic placode at the four-somite stage as shown with brightfield (A) and fluorescence (B) optics. sox9a is also expressed in presumptive rhombomere 4. (C,D) dlx3b expression is reduced in the otic placode after injection of sox9a-MO (30/37 embryos). (E,F) sox9a expression (blue) is reduced in the otic placode after injection of dlx3b-MO (44/50 embryos). pax2a expression is shown in red. (G,H) sox9b-MO produces a very slight reduction in sox9a expression in the otic region (15/18 embryos). (I,J) sox9b expression is significantly reduced by dlx3b-MO plus dlx4b-MO injection (16/18 embryos). We observe a similar reduction in sox9b expression in Dfb380 mutants (not shown). Expression of egr2 (krox20) in red indicates the locations of rhombomeres 5 and 6. Four-somite stage (11 h). Dorsal views, anterior towards the left. Scale bar: 100 µm in A-F.

 


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  		Fig. 9. The residual otic cells that form in Dfb380 mutants require Fgf3 and Fgf8 signaling. (A,B) Loss of dlx3b, dlx4b and sox9a function in Dfb380 mutants results in formation of only a few residual otic cells, as indicated by pax2a expression, that form a small epithelial ball (19/44 Dfb380 embryos). (C) Injection of fgf3-MO into Dfb380 mutants further reduces the number of otic cells, but the residual cells still form an epithelial-like structure (9/21 embryos). (D) Dfb380;fgf8– double mutants lack all detectable otic cells (11/11 embryos). (E,F) fgf8– mutants form a somewhat variably smaller but otherwise fairly normal otic vesicle. (G) Injection of fgf3-MO into wild-type embryos results in a small and somewhat disorganized otic vesicle. (H) Injection of fgf3-MO into fgf8– mutants results in a very reduced number of disorganized otic cells (25/26 embryos). More than half of the injected embryos form a relatively large placode or vesicle-like otic mass (not shown), suggesting a less severe otic phenotype compared with fgf8– mutants injected with fgf3-MO. Prim-5 (24h). Side views, anterior towards the left, dorsal towards the top. Scale bar: 25 µm.

 


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  		Fig. 10. Fgf3- and Fgf8-dependent and -independent pathways for otic development. Model to summarize potential interactions between hindbrain Fgf signals and otic placode transcription factors. (Top) Fgf3 and Fgf8 (FGF) are expressed at high levels by hindbrain rhombomere 4 (Maves et al., 2002Go; Walshe et al., 2002Go). Adjacent rhombomeres are labeled R5 and R6. (Middle) Cells of the presumptive otic placode express sox9b and the three transcription factors deleted by the Dfb380 mutation: dlx3b, dlx4b and sox9a (Fig. 1). Both sox9a (Fig. 6A-D) and to some extent sox9b (Fig. 6E-H) require Fgf signaling, whereas dlx3b (Fig. 6I-L) and dlx4b do not. dlx3b and sox9a crossregulate each other's expression in the placode (Fig. 8C-F) and dlx3b and dlx4b regulate sox9b (Fig. 8I,J). Thus, interactions between the Fgf3- and Fgf8-dependent factor, sox9a, and the relatively Fgf3- and Fgf8-independent factors (sox9b, dlx3b and dlx4b) are required for formation of the epithelium of the otic vesicle (bottom) and subsequent differentiation of the inner ear.

 

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