(Danio Rerio) Inner Ear

10438 • The Journal of Neuroscience, October 11, 2006 • 26(41):10438–10451

Development/Plasticity/Repair The Transcription Factor six1 Inhibits Neuronal and Promotes Hair Cell Fate in the Developing Zebrafish (Danio rerio) Inner Ear

Olivier Bricaud and Andres Collazo Leslie and Susan Gonda (Goldschmied) Cell and Molecular Biology Department, House Ear Institute, Los Angeles, California 90057

The developmental processes leading to the differentiation of mechanosensory hair cells and statoacoustic ganglion neurons from the early otic epithelium remain unclear. Possible candidates include members of the Pax–Six–Eya–Dach ( paired box–sine oculis homeobox–eyes absent–dachshund) gene regulatory network. We cloned zebrafish six1 and studied its function in inner ear development. Gain- and loss-of-function experiments show that six1 has opposing roles in hair cell and neuronal lineages. It promotes hair cell fate and, conversely, inhibits neuronal fate by differentially affecting cell proliferation and cell death in these lineages. By independently targeting hair cells with atoh1a (atonal homolog 1a) knockdown or neurons with neurog1 (neurogenin 1) knockdown, we showed that the remain- ing cell population, neurons or hair cells, respectively, is still affected by gain or loss of six1 function. six1 interacts with other members of the Pax–Six–Eya–Dach regulatory network, in particular dacha and dachb in the hair cell but not neuronal lineage. Unlike in mouse, six1 does not appear to be dependent on eya1, although it seems to be important for the regulation of eya1 and pax2b expression in the ventral otic epithelium. Furthermore, six1 expression appears to be regulated by pax2b and also by foxi1 ( forkhead box I1) as expected for an early inducer of the otic placode. Our results are the first to demonstrate a dual role for a member of the Pax–Six–Eya–Dach regulatory network in inner ear development. Key words: six1; zebrafish; inner ear; hair cell; neuron; development

Introduction and hair cells remain unclear (Fekete, 1999; Fekete and Wu, The inner ear of vertebrates is a complex organ for hearing and 2002). Members of the Pax–Six–Eya–Dach ( paired box–sine ocu- maintaining balance consisting of a nonsensory epithelium hous- lis homeobox–eyes absent–dachshund) gene regulatory network, ing the 6–9 sensory organs, containing the mechanosensory hair involved in the development of numerous organs and tissues in cells (Fritzsch et al., 2002). These are innervated by neurons of the both Drosophila (Bui et al., 2000) and vertebrates (Heanue et al., VIIIth cranial or statoacoustic ganglion (SAG) (Rubel and 1999; Wawersik and Maas, 2000; Li et al., 2003; Brodbeck and Fritzsch, 2002). All inner ear cell types, including SAG neurons, Englert, 2004) have been proposed to play an important role in derive from an ectodermal thickening adjacent to the hindbrain, inner ear development (Baker and Bronner-Fraser, 2001; Whit- the otic placode (Hudspeth, 1989; Haddon and Lewis, 1996; field et al., 2002). The vertebrate Six genes, homologous to Dro- Fritzsch et al., 1998). The lineage relationship between these dif- sophila sine oculis and optix (Seo et al., 1999; Kawakami et al., ferent cell types are just beginning to be understood (Fekete and 2000), are of particular interest. Not only can they act on cell Wu, 2002). Single-cell lineage studies have demonstrated that differentiation by activating cell type-specific genes (Kawakami hair cells and neurons share a common progenitor in chickens et al., 2000), recent studies have shown that they directly interact (Satoh and Fekete, 2005), although it has yet to be demonstrated with the cell cycle machinery (Ford et al., 1998; Coletta et al., in zebrafish (Riley and Phillips, 2003). 2004). Two Six genes, Six1 and Six4, are expressed in the devel- Although otic placode induction has been the focus of exten- oping mouse inner ear. Whereas Six4 null mutant mice do not sive research (Noramly and Grainger, 2002), the mechanisms and have any ear phenotype and the adults hear normally (Ozaki et underlying molecules controlling the formation of SAG neurons al., 2001), Six1 null mutants have severe inner ear defects (Li et al., 2003; Zheng et al., 2003; Ozaki et al., 2004). These defects include Received Nov. 9, 2005; revised Aug. 25, 2006; accepted Aug. 27, 2006. an increase in programmed cell death and a decrease in cell pro- This work was supported by a grant (RO1) from the National Institutes of Health and National Institute for liferation, resulting in a lack of development much beyond the Deafness and Other Communication Disorders. This work was also supported by the Oberkotter Foundation. We thankthemembersofAndyK.Groves,NeilSegil,andAndresCollazolaboratoriesfortheirstimulatingdiscussionsof otocyst stage. the data presented here. We studied the function of the recently described zebrafish Correspondence should be addressed to Olivier Bricaud, Section on Inner Ear Development, Gonda Cell and six1 (Bessarab et al., 2004) during inner ear development. In the Molecular Biology Department, House Ear Institute, 2100 West 3rd Street, Los Angeles, CA 90057. E-mail: inner ear, its expression is restricted to the ventral otocyst in [email protected]. DOI:10.1523/JNEUROSCI.1025-06.2006 which the first hair cells differentiate and prospective SAG neu- Copyright © 2006 Society for Neuroscience 0270-6474/06/2610438-14$15.00/0 rons delaminate. Gain- and loss-of-function studies showed that Bricaud and Collazo • Role of Zebrafish six1 in Ear Development J. Neurosci., October 11, 2006 • 26(41):10438–10451 • 10439 six1 promotes formation of hair cells by increasing cell prolifer- ylated UTP nick end labeling (data not shown) and bromodeoxyuridine ation and independently inhibits neuronal development by in- (BrdU) labeling, respectively. ducing apoptosis. Furthermore, epistasis experiments and gene BrdU Incorporation. Two BrdU pulses of 30 min each separated by a 30 expression analyses are consistent with six1 acting before neurog1 min recovery period at 28°C in egg water (Westerfield, 1995) were done (neurogenin 1) in the neuronal lineage. In the hair cell lineage, starting at 28 hpf. For each pulse, dechorionated embryos were incubated in 10 mM BrdU (Invitrogen), made up in egg water with 15% DMSO in a six1 likely acts via two dachshund genes. We also showed that six1 Petri dish resting on top of ice. After the final pulse, the embryos were expression in the inner ear is controlled by pax2b, pax8, and allowed to develop at 28°C in egg water for up to 3 days post-fertilization foxi1 ( forkhead box I1) and that six1 promotes pax2b and inhibits (dpf). After fixation in 4% paraformaldehyde at 4°C overnight, the em- eya1 expression. bryos were subsequently dehydrated in methanol for 30 min at Ϫ20°C and permeabilized with a 10 ␮g/␮l Proteinase K treatment. The embryos Materials and Methods were then processed for cryosectioning as described below. Cloning of zebrafish six1. A 19 hours post-fertilization (hpf) embryonic Detection of SAG neurons after BrdU incorporation. To label SAG neu- cDNA library (gift from B. Appel, Vanderbilt University, Nashville, TN) rons, we used the mouse anti-HuC antibody described above. Because was screened by PCR using a high-fidelity thermostable polymerase, Pwo anti-HuC is an IgG2b and the mouse anti-BrdU is an IgG1 (at a concen- polymerase (Roche Applied Science, Indianapolis, IN). We used a se- tration of 1:20; Invitrogen), we were able to perform the double immu- quence directed against the 3Ј end of the zebrafish expressed sequence tag nostainings as described previously (Marusich et al., 1994). Briefly, we similar to the human Six1 gene (GenBank accession number AI965224) first performed the anti-HuC immunostaining as described above, using as a target. The full-length open reading frame (ORF) (GenBank acces- an anti-mouse IgG2b secondary antibody coupled to Alexa 568 (at a sion number AY254196) was amplified and subcloned into the expres- concentration of 1:200; Invitrogen). After the first immunostaining was sion vector pCS2ϩ (Turner and Weintraub, 1994). completed, we incubated the sections in 2N HCl for 30 min to denature Whole-mount in situ hybridization. Whole-mount in situ hybridiza- the DNA and make the BrdU detectable. BrdU immunostaining was then tion was performed as described previously (Thisse et al., 1993). Exper- performed using the mouse anti-BrdU antibody described above and an imental and control embryos were developed for the same amount of anti-mouse IgG1 secondary antibody coupled to Alexa 488 (at a concen- time in the final colorimetric reaction for every probe used. The neuroD tration of 1:200; Invitrogen). The sections were then mounted in (Korzh et al., 1998), dacha, dachb, dachc (Hammond et al., 2002), pax2a, Fluoromount-G (Southern Biotechnology, Birmingham, AL) and docu- pax2b (Pfeffer et al., 1998), atoh1a (atonal homolog 1) (Kim et al., 1997), mented with our laser scanning confocal microscope as described above. and eya1 (Sahly et al., 1999) probes were described previously. Detection of hair cells after BrdU incorporation. To detect hair cells, we Morpholino-modified oligonucleotides and mRNA injections. The had to use the mouse monoclonal antibody Zn1 (at a concentration of method for injecting RNA and morpholinos (MO) into zebrafish em- 1:50; Developmental Studies Hybridoma Bank, University of Iowa, Iowa bryos has been described previously (Westerfield, 1995; Nasevicius and City, IA) (Kornblum et al., 1990) because its epitope was less vulnerable Ekker, 2000). The morpholinos (Gene Tools, Philomath, OR) used were than HCS-1 to the subsequent acid treatment necessary for BrdU detec- as follows (complementary bases to the predicted start codon are under- tion. Because Zn1 and the anti-BrdU are both mouse IgG1 antibodies, we lined): six1,5Ј-ACCCGAAAGAAGGCAACATTGACAT-3Ј; dacha,5Ј- had to modify the double-immunostaining protocol as follows. Zn1 im- CGGAGGAGTTGCAGATACGGCCATA-3Ј; dachb,5Ј-GCCATAGTG- munostaining was performed as described above using an anti-mouse GTCATTGAACTTAAAG-3Ј; dachc,5Ј-CGACATCGGAGGCGCGT- IgG1 secondary antibody coupled to Alexa 488 (at a concentration of GGGCCATC-3Ј; pax2a,5Ј-GGTCTGCTTTGCAGTGAATATCCAT-3Ј; 1:200; Invitrogen). After completing the first immunostaining, sections pax2b,5Ј-GGTCTGCCTTACAGTGAATATCCAT-3Ј; eya1,5Ј-AGC- were treated with 2N HCl for 30 min. BrdU immunostaining was then TAGATCCTGCATTTCCATAGAC-3Ј; foxi1,5Ј-TAATCCGCTCTC- done using the mouse anti-BrdU antibody, previously described. To de- CCTCCAGAAACAT-3Ј; neurog1,5Ј-TATACGATCTCCATTGTTG- tect the anti-BrdU antibody without interfering with the previous immu- ATAACC-3Ј; atoh1a,5Ј-TCTCTTGTATCCGTGCTCATTCCAT-3Ј; nostaining, we directly coupled the anti-BrdU antibody to Zenon 568 pax8.1, 5Ј-GTTCACAAACATGCCTCCTAGTTGA-3Ј; pax8.2/3, 5Ј- according to the recommendations of the manufacturer (Invitrogen). GACCTCGCCCAGTGCTGTTGGACAT-3Ј; STD, Gene Tools standard Labeled sections were then mounted in Fluoromount-G (Southern control oligo. Biotechnology) and documented with our laser scanning confocal As an additional control, the obtained phenotypes were confirmed by microscope. comparison with those already described in mutants when available. In Cryosections. Cryosectioning was done as described previously (Link et the eya1 experiments, a splice-blocking MO (Kozlowski et al., 2005) gave al., 2000). Briefly, after overnight fixation in 4% paraformaldehyde at the same results as the eya1 morpholino mentioned above. 4°C, the embryos were infiltrated with a graded series of sucrose, up to Whole-mount immunofluorescence. Whole-mount immunofluores- 30%, in PBS. They were then incubated in 30% sucrose in tissue freezing cence was performed as described previously (Bricaud et al., 2001). After media (Triangle Biomedical Sciences, Durham, NC) for several hours staining, the embryos were mounted and documented with a laser scan- until they sank to the bottom of the well. Embryos were then transferred ning confocal microscope (LSM 410; Zeiss, Oberkochen, Germany), by into tissue freezing media overnight. After incubation, they were embed- collecting and projecting confocal z-series. The antibody directed against ded in cryomolds, frozen on dry ice, and sectioned at 20 ␮m. Antibody HCS-1 (hair cell soma 1) (used at a concentration of 1:50; gift from J. T. staining was then performed as described above. Corwin, University of Virginia, Charlottesville, VA) has been shown to Administration of caspase inhibitor. The method for administrating specifically recognize otoferlin (Cyr et al., 2006) and label hair cells in caspase-3 inhibitors has been described previously (Williams and fish, amphibians, birds, and mammals (Finley et al., 1997; Gale et al., Holder, 2000). Briefly, the caspase inhibitor zVADfmk [benzyloxycar- 2000). The antibody directed against HuC (used at a concentration of bonyl Val-Ala-Asp (O-methyl) fluoromethyl ketone] (Sigma, St. Louis, 1:1000; Invitrogen, Carlsbad, CA) has been shown previously to label the MO) was applied to live embryos at a concentration of 300 ␮g/ml. To neurons of cranial ganglia (Marusich et al., 1994; Andermann et al., ensure both penetration and that the inhibition would be effective during 2002). To confirm the phenotype of hair cells and neurons in our exper- the period hair cells and neurons are developing, embryos were incu- iments, rhodamine–phalloidin and Islet-1 were also used as hair cell and bated in the presence of the inhibitor from 15 hpf (before when hair cells neuronal markers, respectively (data not shown). As a cell proliferation and neurons form) to 3 dpf. In other experiments, the inhibitor was marker, we used an antibody directed against human phospho-histone added at correspondingly later time points. The chemically similar ca- H3 (used at a concentration of 1:100; EMD Biosciences, San Diego, CA). thepsin B inhibitor zFAfmk [benzyloxycarbonyl Phe-Ala (O-methyl) flu- Programmed cell death was studied with an antibody against the human oromethyl ketone] (Sigma, St. Louis, MO) was used as a negative control activated caspase-3 (used at a concentration of 1:100;R&DSystems, for zVADfmk at a concentration of 300 ␮g/ml. Minneapolis, MN). The cell death and proliferation findings were also Cell counts. To assess cell numbers accurately, multiple focal planes confirmed with terminal deoxynucleotidyl transferase-mediated biotin- (confocal z-sections) encompassing all labeled cells around the inner ear 10440 • J. Neurosci., October 11, 2006 • 26(41):10438–10451 Bricaud and Collazo • Role of Zebrafish six1 in Ear Development

Figure 1. Expression of six1 mRNA during zebrafish embryogenesis. A–J, Whole-mount in situ hybridization of six1 full-length cDNA probe in zebrafish embryos at 16.5 hpf (A, B), 19 hpf (C, D), 24 hpf (E, F), 32 hpf (G, H), 48 hpf (I), and 72 hpf (J). Position of otic placode or vesicle is noted by a white arrow in A, C, E, and J and surrounded by a dashed line in B, D, and F–I. Dotted lines in B, D, and F indicate the lumen of the otic vesicle. Arrows in F–I point to the six1-expressing cells in the ventral otic epithelium. Note that six1 expression is stronger in the anteriormost (G) and medialmost (H) parts of the ventral otic epithelium. White arrowhead in E points to the developing otic ganglion. Arrowheads in F–I point to cells delaminating from the ventral otic epithelium. Stars in G and I indicate the position of the otic ganglion. The arrowhead in C and the black arrow in E point to the anteroventral edge of the head, possibly the pituitary. The black arrowhead in E pointstosix1expressionintheposteriorlaterallineprimordium.ThearrowheadsinJpointtolaterallineneuromasts.TheinsetinJrepresentsalargermagnificationofaneuromast(surroundedby a dashed line), with the centralmost cells (possibly the hair cells) expressing six1. All panels are lateral views with anterior to the left, except for A, which is an anterodorsal view, and H, which is a dorsal view with medial to the top. a, Anterior lateral line; o, olfactory placodes; p, posterior lateral line; t, trigeminal placode. Scale bars: B, D, F–I,30␮m; A, E, 400 ␮m; C, 350 ␮m; J, 450 ␮m.

were collected with a Zeiss LSM 410 laser scanning confocal microscope. arrow). Six1 expression is also detectable in the prospective SAG By careful comparison with the previous and following sections, we made neurons (Fig. 1G,I, stars) and in cells that appear to be migrating sure to only count each cell once. Sample sizes listed for the cell counts out of the otic epithelium (Fig. 1F–I, arrowheads), possibly cor- represent individual embryos because, for each embryo, only the cells on responding to delaminating SAG neuroblasts. At 48 hpf, six1 is one side have been scored. Statistical analyses were done using Student’s t test and the Smith’s Statistical Package (Pomona College, Claremont, still expressed in the ventral epithelium of the otic vesicle and in CA) with p Ͻ 0.05 considered as statistically significant. the developing otic ganglion (Fig. 1I). After 48 hpf, six1 expression is no longer detectable in the inner ear but remains visible in Results lateral line neuromasts (Fig. 1J). Cloning and expression of the zebrafish six1 gene Although our main focus has been to study six1 expression in We isolated a new zebrafish member of the Six gene family related the developing inner ear, we confirmed that most of the other to vertebrate six1 by PCR screening a zebrafish 15–19 hpf library cranial placode-derived sensory systems expressed various levels (Appel and Eisen, 1998). The longest clone we obtained is 1088 of six1 mRNA, including the olfactory, trigeminal, and lateral line bp long and contains a full-length ORF of 284 amino acids. The placodes (Fig. 1E). Weak expression in the anteroventral edge of sequence of the ORF contained in this clone is identical to one the head (Fig. 1C,E) could represent six1 expression in the pitu- recently published (Bessarab et al., 2004). itary as described recently (Bessarab et al., 2004). However, we We subsequently studied its expression with a focus on the were unable to detect any six1 expression in the muscle and pec- developing inner ear by in situ hybridization. At 16.5 hpf, six1 toral fin as described previously (Bessarab et al., 2004). This dis- mRNA is expressed in three distinctive domains at the periphery of the neural plate (Fig. 1A) that correspond to the sensory pla- crepancy could be attributable to differences in the probes used to codes (Sahly et al., 1999). It is initially expressed throughout the detect six1 mRNA. We used the full-length cDNA, which includes Ј otic placode (Fig. 1B), but, by 19 hpf, six1 expression becomes a large fragment of 3 untranslated region (UTR), whereas Besar- restricted to the ventral half of the otic vesicle (Fig. 1C–F). This is rab and colleagues used a 598 bp fragment corresponding to the the region from which the first sensory hair cells and neurons will evolutionarily conserved six domain and homeodomain, gener- arise between 24 and 48 hpf and 22–42 hpf, respectively (Haddon ated by reverse transcription-PCR from embryonic total RNA. By and Lewis, 1996). An anteroposterior expression gradient begins using a smaller probe that only included the 3Ј UTR, we observed to emerge with stronger six1 expression anteriorly (Fig. 1F). Be- an even more restricted pattern, mainly to just the inner ear (data tween 24 hpf (Fig. 1F) and 32 hpf (Fig. 1G,H), six1 expression not shown). This leads us to think that six1 could have several becomes more restricted to the anterior ventral part of the otic splice variants, as described for Six3 in the mouse retina epithelium (Fig. 1G, arrow). This expression appears to be stron- (Kawakami et al., 1996) or a closely related paralog as reported for ger in the medial wall of the ventral otic epithelium (Fig. 1H, six4 (Kobayashi et al., 2000). Bricaud and Collazo • Role of Zebrafish six1 in Ear Development J. Neurosci., October 11, 2006 • 26(41):10438–10451 • 10441

morpholino and a morpholino with four mismatches. Both result in a wild-type phenotype (data not shown). A second, overlapping morpholino targeting the ATG region but centered on it also had the same effect as our experimental morpholino (data not shown).

Six1 gain-of-function results in more hair cells and fewer SAG neurons Overexpression of six1 during inner development was achieved by injecting a synthetic six1 mRNA at early stages. Such gain-of- function experiments gave the opposite phenotype to that seen after six1 loss-of-function. At 3 dpf, more hair cells (Figs. 2, 3A,K) are present. This overproduction of hair cells is detectable as early as 28 hpf, with an average of four hair cells observed (Fig. 3L) instead of the two in wild-type embryos (Fig. 3B). We assayed for the presence of differentiated neurons at 3 dpf (Fig. 3C,D,M,N) and neural precursors at 32 hpf (Fig. 3E,O) with the neuronal markers HuC and neuroD, respectively. At 32 hpf in the six1 overexpressing embryos, fewer neuroD-positive cells are detectable in the otic ganglion (Fig. 3O) than in control embryos (Fig. 3E), suggesting that fewer neural progenitors are formed when six1 is overexpressed. At 3 dpf, the decrease in number of SAG neurons versus controls (Figs. 2, 3C,D) is even more dramatic. In extreme cases, SAG neurons are completely eliminated (Fig. 3M,N). These results indicate that, when six1 is overexpressed, not only are fewer neural progenitors formed but many of these progenitors do not go on to differentiate into neurons. In conclusion, these, together with the six1 loss-of-function results, suggest that six1 is necessary and sufficient for the normal formation of hair cells in the anterior macula, although it inhibits neu- Figure 2. Average number of hair cells and neurons in 3 dpf anterior maculae or SAG. Hair ronal fate in the developing inner ear. cells and neurons have been detected by HCS-1 and HuC, respectively, using confocal microscopy.ValuesrepresentmeanϮSDcellcountswithasamplesizeof20anteriormaculaeforthe Six1 loss- and gain-of-function appears to only affect hair hair cell counts and 15 SAG for the neuron counts. Statistical analysis was performed with cells of the anterior macula and SAG neurons Student’s t test; all comparisons were made with embryos injected with standard MO control To determine whether the role of six1 in hair cell development (STD). **p Ͻ 0.005. was restricted only to the anterior macula, we counted hair cells stained by HCS-1 in the posterior macula in either STD-MO-, Six1 loss-of-function results in fewer hair cells and more six1-MO-, or six1 mRNA-injected embryos. At 36 hpf, we scored, SAG neurons respectively, 6.1 Ϯ 1.29, 6.3 Ϯ 1.49, or 6.0 Ϯ 1.15 hair cells for To study the role of six1 in inner ear development, we scored for each of the three conditions (10 embryos were counted for each changes in number of hair cells and neurons after six1-MO injec- condition). Statistical analysis with Student’s t test shows that the tion (Figs. 2, 3). The timing and number of hair cells forming in number of hair cells in the posterior macula under these three the first two macula, as well as the timing of neuron delamination conditions were not significantly different ( p ϭ 0.7), suggesting from the otocyst, have been well studied in developing zebrafish that the role of six1 in sensory organ formation is restricted to the and are relatively stereotyped (Haddon and Lewis, 1996). At 3 anterior macula. dpf, the anterior (or utricular) macula of a wild-type zebrafish Six1 is also expressed in most of the cranial ganglia. To deter- larva contains ϳ30 hair cells (Figs. 2, 3A). When six1-MO is mine whether neurogenesis in these ganglia could be inhibited or injected, the number of hair cells is reduced by half to ϳ14 (Figs. induced by six1 perturbation, we counted the number of HuC- 2, 3F). Reduction in the number of hair cells is detectable as early positive cells in all of the major cranial ganglia (Fig. 4) at 3 dpf (20 as when the first hair cells start differentiating. At 28 hpf, in embryos for each condition) in embryos injected with STD-MO, six1-MO-injected embryos, either one or no hair cell is detected six1-MO, or six1 mRNA. No significant differences ( p values (Fig. 3G) where normally two would be present (Fig. 3B). ranged from 0.1 to 0.92) in the number of HuC-positive cells We also scored for number of neurons in the otic ganglion at have been found in any of the cranial ganglia except for the otic two different time points. At 3 dpf in six1-MO-injected embryos, ganglion (Fig. 4). Therefore, it seems that six1 is only involved in there are more HuC-positive cells (Fig. 3C,H). At this stage, the the formation of neurons in the SAG. Nonetheless, we cannot otic ganglion in a wild-type larva contains ϳ20 neurons (Fig. 2), rule out that six1 plays a role in other aspects of cranial ganglia whereas, when six1 is knocked down, the number of SAG neu- formation, such as neuronal identity. Also, because we could not rons doubles to ϳ40 (Fig. 2). Furthermore, the expression do- always individually distinguish the overlapping trigeminal, facial, main of neuroD, an early marker of neuroblasts, at 32 hpf is anterodorsal, or anteroventral lateral line ganglia, lumping all of expanded when six1 is knocked down (Fig. 3E,J). their neurons into one category, it is possible that the number of To test for the specificity of our morpholino, we rescued the neurons in one or more of these ganglia is being reduced or six1-MO phenotype by separately injecting six1 mRNA (Fig. 2), increased. However, this would have to be balanced by corre- titrating the morpholino and demonstrating that the six1 mRNA sponding increases or reductions in other ganglia to keep the total itself was not toxic. As negative controls, we used a sense six1 number the same. 10442 • J. Neurosci., October 11, 2006 • 26(41):10438–10451 Bricaud and Collazo • Role of Zebrafish six1 in Ear Development

Figure 3. Effects of six1 loss- and gain-of-function in the developing inner ear are complementary, differentially affecting hair cells and neurons. HCS-1 immunostaining of hair cells in 3 dpf embryos injected with standard MO control (A), six1-MO (F), and six1 mRNA (K) and 28 hpf embryos with standard MO control (B), six1-MO (G), and six1 mRNA (L) is shown. HuC immunostaining ofneuronsin3dpfembryonicoticgangliainjectedwithstandardMOcontrol(C),six1-MO(H),andsix1mRNA(M)isshown.ThewhiteboxesinC,H,andMoutlineoticganglionneuronsmagnified in D, I, and N, respectively. Whole-mount in situ hybridization to neuroD in 32 hpf embryos injected with standard MO control (E), six1-MO (J), and six1 mRNA (O) is shown. Whole-mount in situ hybridization to neurog1 in 26 hpf embryos injected with standard MO control (P), six1-MO (Q), and six1 mRNA (R) is shown. All panels are lateral views with anterior to the right, except A, F, and K, which are dorsal views. Arrows point to hair cells in A, B, F, G, K, L and to SAG neurons in C, D, H, I, M, N. White dashed lines in B, C, E, G, H, J, L, M, O, P, Q, and R outline the otic vesicle. The area surrounded by a black line in E, J, and O is the same in all three panels and shows the difference in the size of neuroD expression. The changes in expression levels have been confirmed in at least 30 embryos for each marker and time point. ad, Anterodorsal lateral line ganglion; av, anteroventral lateral line ganglion; f, facial epibranchial ganglion; g, glossopharyngeal epibranchial ganglion; o, octaval/statoacoustic ganglion precursors; p, posterior lateral line placode/ganglion; t, trigeminal ganglion; v, vagal epibranchial placode/ganglion. Scale bar, 10 ␮m. t/ad/av/f, Trigeminal/ anterodorsal lateral line/anteroventral lateral line/facial epibranchial ganglion complex

and inhibiting programmed cell death (Ford et al., 1998; Zheng et al., 2003; Ozaki et al., 2004). We, therefore, determined the number of cells undergoing cell proliferation and death in the otocyst, at 24, 32, and 48 hpf of six1-MO-injected and six1-overexpressing embryos, using phospho-histone H3 and activated caspase-3, respectively, as markers (Fig. 5). Interestingly, under both conditions, we observed significant increases in both cell proliferation and death compared with control embryos. These extra cells seem to be mainly localized to the ventral otic epithelium at these time points. This is where six1 is normally expressed and is a hot spot for cell division (Lang et al., 2000) and cell death (Bever and Fekete, 1999) during inner ear development. However, some of the extra dividing and dying cells are observed in more dorsal positions and suggest that six1 could have a non-cell autonomous function, although we cannot rule out the possibility of cell mix- Figure4. Averagenumberofneuronsin3dpfcranialgangliaaftersix1loss-of-function(six1 ing, as has been shown in the developing Xenopus inner ear (Kil MO) or gain-of-function (six1 mRNA). Neurons labeled with HuC were scored using confocal and Collazo, 2001). Another caveat is, because we only count cells microscopy. Values represent mean Ϯ SD cell counts with a sample size of 20 embryos, except within the otocyst itself, we are missing cell proliferation and for the values for the otic ganglion, which have been taken from Figure 2. All comparisons were death occurring in delaminated neuroblasts and differentiating made with embryos injected with standard (STD) MO control. o, Otic ganglion; t/ad/av/f, tri- otic ganglion neurons, especially at 36 and 48 hpf. geminal/anterodorsal lateral line/anteroventral lateral line/facial epibranchial ganglion com- To determine the identity of the supernumerary-dividing plex; g, glossopharyngeal epibranchial ganglion; v, vagal epibranchial ganglion; m, middle lateral line ganglion; p, posterior lateral line ganglion. **p Ͻ 0.005. cells, we performed two BrdU pulses at 28 hpf. Normally, at this time, most of the cells in the otic epithelium are not dividing (Fig. 5). Consistent with this observation, our control embryos in- Six1 affects hair cell and neuronal number through jected with STD-MO had very few BrdU-positive hair cells (Fig. differential cell proliferation and death in these lineages 6A,J). Although more neurons were labeled, it was still far from Although as a transcription factor six1 is known to play a role in all (Fig. 7A). When six1 is knocked down, most of the SAG neu- cell type-specific differentiation (Spitz et al., 1998; Heanue et al., rons (86% in nine embryos scored) were BrdU positive (Fig. 7B), 1999), better characterized is its role in promoting proliferation showing that they were derived from cells dividing at the time of Bricaud and Collazo • Role of Zebrafish six1 in Ear Development J. Neurosci., October 11, 2006 • 26(41):10438–10451 • 10443

2000) (25 embryos each). These results suggest that different cell populations undergo cell death after six1 loss-of-function versus six1 gain-of-function; the former population consists of cells fated to form hair cells, whereas the latter consists of cells fated to form SAG neurons.

Six1 acts early in both hair cell and neuronal lineages The developmental events (hair cell differentiation, neuroblast delamination, and ganglion formation) leading to the formation of the anterior macula and the SAG all happen concomitantly in the ventral otic epithelium (Fig. 8I). The lack of suitable markers for hair cell or SAG neuronal precursors means that assaying the identity of the dividing cells before they actually differentiate is currently not possible. Therefore, to begin determining when six1 perturbation may be inducing programmed cell death, we used the ability of the caspase-3 inhibitor zVADfmk to rescue six1 loss- and gain-of-function phenotypes. We assayed what was the latest time point we could begin applying zVADfmk and still see rescue when scored at 3 dpf (Fig. 8G,H). No rescue is observed in hair cell or neuronal lineages when zVADfmk is added at 48 hpf (Fig. 8G,H). In contrast, zVADfmk application beginning at 24 hpf produced results similar to application beginning at 15 hpf, al- most complete rescue. When the caspase-3 inhibitor is added at an intermediate time point (36 hpf), the rescue is only partial. Figure 5. A–F, Analysis of cell proliferation (A–C) and cell death (D–F) in the otocyst of 32 hpf embryos injected with standard-MO control (A, D), six1-MO (B, E), and six1 mRNA (C, F). The timing for this rescue coincides with the initial wave of hair Lateral views of otocyst immunostained for the mitosis marker phospho-histone H3 (A–C) and cell and neuronal differentiation between 24 and 48 hpf observed the cell death marker activated caspase-3 (D–F). Otic vesicle is outlined by a dashed line. Scale during inner ear development (Haddon and Lewis, 1996). These bar, 30 ␮m. G, Quantification of cell proliferation and death in injected and non-injected oto- results suggest that the promotion by six1 of programmed cell cysts at 24, 32, and 48 hpf. Values are mean Ϯ SD cell counts of cells labeled with either death is required early in both sensory and neuronal lineages, phospho-histone H3 or activated caspase-3, sample size of 10 embryos for each experiment. possibly in their common precursor (Satoh and Fekete, 2005). Statistical analysis was with Student’s t test; all comparisons were made with embryos injected To begin to understand the relationship between six1 and with standard MO control. For all three time points, the number of cells counted in both six1- early proneural genes, we studied the expression of neurog1 in the Ͻ MO- and six1 mRNA-injected embryos are significantly higher than controls ( p 0.05). Only otic epithelium in embryos in which six1 has been either knocked labeled cells within the otocyst were counted. down or overexpressed at 26 hpf, near the onset of neurogenesis (Fig. 3P–R). When six1 is knocked down, neurog1 expression is the pulses, whereas the few hair cells were not (Fig. 6B,K). When increased within the ventral wall of the otic epithelium (Fig. 3Q). six1 is overexpressed, most of the hair cells (84% in nine embryos Conversely, neurog1 expression is dramatically reduced when scored) were BrdU positive (Fig. 6C,L), whereas the few SAG six1 is overexpressed (Fig. 3R). These results are consistent with neurons formed were BrdU negative (Fig. 7C). We conclude that six1 being necessary for neuroblast formation before the onset of the extra cells observed undergoing proliferation when six1 func- neurog1 expression in the ventral otic epithelium. tion is perturbed mainly represent cells fated to form SAG To further determine whether six1 acts before or after neuro- neurons in the case of six1 loss-of-function and hair cells of genesis, we studied the epistatic relationship between six1 and the anterior macula or their progenitors in the case of six1 neurog1. The transcription factor neurog1 has been shown to be gain-of-function. crucial for the formation of SAG neurons in the otic epithelium, Because we also observed an increase in the number of cells before the neuroblasts delaminate (Andermann et al., 2002). undergoing apoptosis in the otic epithelium when six1 is knocked When neurog1 is knocked down, fewer neurons are formed (An- down or overexpressed, we assayed whether inhibiting apoptosis dermann et al., 2002), a phenotype that is the opposite of that could rescue the phenotype of six1 loss- or gain-of-function in observed after six1 knockdown. We therefore knocked down sensory and neuronal lineages. We allowed the embryos to de- both six1 and neurog1 together and scored for number of SAG velop in the presence of a caspase-3 inhibitor, zVADfmk, and neurons (Fig. 2). The number of neurons (7.27 Ϯ 1.8; n ϭ 15) in subsequently assayed for number of hair cells (Fig. 8). When six1 the double knockdown is statistically the same as that observed is knocked down in the presence of zVADfmk, the number of hair after neurog1 knockdown (6.53 Ϯ 2.2; n ϭ 15; p ϭ 0.3) but cells observed at 3 dpf (Fig. 8C) is comparable with the number of opposite to that seen after six1 loss-of-function (39.67 Ϯ 2.0; n ϭ hair cells observed in wild-type larvae (Fig. 8A). A similar rescue 15; p Ͻ 0.005); therefore, neurog1 is likely to be epistatic to six1. of neurons is observed when six1 is overexpressed, and caspase- This means that six1 is not downstream of neurog1 and is consis- 3-dependent apoptosis is inhibited (Fig. 8F). The increased num- tent with six1 acting upstream of neurog1, early in the neuronal bers of hair cells or neurons observed when six1 is overexpressed lineage. Because neither of these loss-of-function phenotypes has or knocked down, respectively, are not affected by the presence of been shown to be null, the epistatic relationship between six1 and zVADfmk (Fig. 8G,H and data not shown). The phenotypes of neurog1 proposed here has to be taken as tentative. However, either six1 loss-of-function (Fig. 8B) or gain-of-function (Fig. these results, together with the dependency of neurog1 expression 8E) remain unaffected in controls treated with the chemically on six1 function (Fig. 3P–R), lead us to think that six1 acts before similar cathepsin B inhibitor zFAfmk (Williams and Holder, neurog1, early in the neuronal lineage. 10444 • J. Neurosci., October 11, 2006 • 26(41):10438–10451 Bricaud and Collazo • Role of Zebrafish six1 in Ear Development

Six1 likely acts in both hair cell and neuronal lineages Because it seems that six1 function is required very early in otic development, we cannot rule out that six1 is acting on a common precursor cell whose asymmetri- cal division or survival results in the increase of one cell population at the expense of the other. Another possibility, which is not mutually exclusive, is that six1 is acting after hair cell and neuronal precursors separate but its effects are restricted to one lineage whose absence or promotion then results in increases or decreases, respectively, in the other cell lineage. If so, one prediction would be that six1 loss- or gain- of-function would not have any effect when the cell type in which six1 is sup- posed to be acting is missing. We therefore decided to study the effects of six1 loss- and gain-of-function on one lineage when the other is missing or decreased. We impaired the formation of hair cells by knocking down atoh1a (Itoh and Chit- nis, 2001; Whitfield et al., 2002), the zebrafish homolog of MATH-1 (mouse atonal homolog), which has been shown to be necessary for hair cell formation in mice (Bermingham et al., 1999). In the developing zebrafish inner ear, loss-of-function of atoh1a leads to fewer hair cells (21.6 Ϯ 2.1; n ϭ 20; p Ͻ 0.005), but the number of neurons remains unaffected (19.8 Ϯ 1.9; n ϭ 15; p ϭ 0.9) (Fig. 2). The number of SAG neurons is still decreased by six1 overexpression, even when hair cell formation is inhibited by atoh1a loss-of-function (10.3 Ϯ 2.2 neurons; n ϭ 15; p Ͻ 0.005) (Fig. 2). Conversely, the number of SAG neurons is still increased by six1 loss-of- function when hair cell formation is prevented (38.73 Ϯ 2.0 neurons; n ϭ 15; p Ͻ 0.005) (Fig. 2). These results suggest that six1 function in the neuronal lineage is independent of the presence of hair cells. Interestingly, in embryos in which atoh1a has been knocked down and six1 overexpressed, the number of hair cells in the anterior macula is reduced to a level comparable with that observed in the atoh1a knockdown alone (22.3 Ϯ 2.8 hair cells; n ϭ 20; p ϭ 0.4) (Fig. 2). Conversely, the number of hair cells in the anterior macula of embryos in which both six1 and atoh1a have been knocked down (13.8 Ϯ 2.7; n ϭ 20) (Fig. 2) is reduced to a level statistically closer to that observed in six1 knockdown alone (14.1 Ϯ 2.5; n ϭ 20; p ϭ Figure 6. Effects of six1 perturbation on hair cell proliferation. Zn1 (hair cell marker in green) and BrdU (in red) staining in the 0.7) (Fig. 2) than to that observed in anterior macula of 3 dpf zebrafish injected with standard-MO control (A, D, G, J, M, P), six1-MO (B, E, H, K, N, Q), and six-1 mRNA atoh1a knockdown alone (21.6 Ϯ 2.1; n ϭ (C, F, I, L, O, R). Arrows point to Zn1-positive cells that have incorporated BrdU. Arrowheads point to Zn1-positive cells that have 20; p Ͻ 0.005) (Fig. 2). notincorporatedanyBrdU.A–I,Transversecryosections.J–R,Frontalcryosectionsthroughsixdifferentembryos.Theorientation To decrease the neuronal population, and the planes of section are shown on the schematic of a 3 dpf embryo in S. The dashed lines in A–I outline the lumen of the otic we knocked down neurog1 as described vesicle (o). am, Anterior macula; Ant., anterior; Dors., dorsal; Lat., lateral; Med., median; Post., posterior. Scale bar, 5 ␮m. Bricaud and Collazo • Role of Zebrafish six1 in Ear Development J. Neurosci., October 11, 2006 • 26(41):10438–10451 • 10445

gest that, just as neurons and hair cells are differentiating, loss of one population does not necessarily affect the formation of the other.

Six1 likely acts via dacha and dachb genes in the sensory lineage In the developing compound eye of the fly, dachshund expression is directly dependent on sine oculis (Halder et al., 1995; Chen et al., 1997; Shen and Mardon, 1997). Three dachshund genes have been shown to be expressed in the developing zebrafish inner ear (Hammond et al., 2002). In an effort to begin to understand which genes six1 is acting through, we assayed the expression of dacha, dachb, and dachc after six1 loss-of-function (Fig. 9A– F). When six1 is knocked down, the expression of both dacha and dachb is reduced in the ventral otic epithelium (Fig. 9A,D and B,E), whereas the expression of dachc remains unaffected (Fig. 9C,F). Moreover, in the dorsal otic epithelium, in which six1 is not expressed, dacha expression is still present after six1-MO injection (Fig. 9A,D, arrows). Consistent with the expression data, only dacha or dachb, but not dachc, loss- of-function lead to a reduction in the number of hair cells in the anterior macula at 3 dpf, as observed with six1 loss-of- function (Fig. 9G–J,M). Dach loss-of- function has no effect on the number of SAG neurons (Fig. 2). We also scored hair cell numbers at two earlier time points (32 hpf and 2 dpf) after knocking down dacha or dachb (data not shown). Already by 32 hpf, we were able to observe fewer hair cells. Reduced numbers of hair cells are also observed at 2 dpf. Based on these re- Figure 7. Effects of six1 perturbation on neuronal proliferation. HuC (neural marker in red) and BrdU (in green) coimmunos- sults, it is likely that both dacha and dachb taining in the SAG of 3 dpf zebrafish injected with standard-MO control (A, D, G), six1-MO (B, E, H), and six1 mRNA (C, F, I)on act early in the hair cell lineage, possibly in transverse cryosections. Both are nuclear markers. Arrows and arrowheads in A, B, D, E, G, and H point to BrdU-labeled SAG their progenitors, as proposed for six1. neurons and to SAG neurons that have not incorporated BrdU, respectively. Arrows and arrowheads in C, F, and I point to SAG Given that their expression is reduced neurons that have not incorporated BrdU and to a BrdU-positive cell that is not an SAG neuron, respectively. The orientation and after six1 knockdown and that their plane of section are shown on the schematic of a 3 dpf embryo in J. The dashed lines outline the outer part of the otic vesicle (ov). knockdown results in a similar reduction Ant., Anterior; Dors., dorsal; Med., median; sag, statoacoustic ganglion. Scale bar, 10 ␮m. in hair cell number to that seen after six1 loss-of-function, it is likely that dacha and above (Fig. 2), which results in fewer neurons. Even so, six1 gain- dachb act downstream of six1 in the hair cell lineage. That six1 of-function still results in an increase in hair cell numbers even expression is not affected by either dacha or dachb loss-of- when neurog1 function is reduced (37.5 Ϯ 2.6, n ϭ 18 vs 29.9 Ϯ function (Fig. 9N–Q) further supports this conclusion. Another 2.7, n ϭ 20 in the STD-MO-injected embryos; p Ͻ 0.005) (Fig. 2). possibility that is not mutually exclusive is that the two dach genes Conversely, the six1 loss-of-function phenotype of fewer hair act together with six1. All three genes appear to act early in the cells is still seen, even in the absence of SAG neurons (16.2 Ϯ 2.7; developing hair cell lineage. In mouse, it has been shown that Six1 n ϭ 20; p Ͻ 0.005) (Fig. 2). These results suggest that the role of and Dach form a transcriptional complex important for organo- six1 in the hair cell lineage is independent of the presence of SAG genesis (Li et al., 2003). neurons. To test for synergistic interactions between dacha/dachb and Because atoh1a and neurog1 are likely acting later in hair cell six1 in the hair cell lineage, we scored for numbers of hair cells in and neuronal lineages, respectively, than six1, these results do not the anterior macula at 3 dpf after dacha and/or dachb knock- rule out the possibility that six1 could be acting only in one lin- down(s) in either a six1 loss- or gain-of-function background eage after they separate but before atoh1a or neurog1 acts, with (Figs. 2, 9K,L). SAG neuron counts and dachc knockdowns in consequences on the other lineage. However, these data do sug- similar backgrounds served as negative controls (Fig. 2). When 10446 • J. Neurosci., October 11, 2006 • 26(41):10438–10451 Bricaud and Collazo • Role of Zebrafish six1 in Ear Development dacha, dachb, or both were knocked down in conjunction with six1 loss-of-function, the loss of hair cells seen was indistinguish- able from that observed in six1, dacha,or dachb morphants alone (Figs. 2, 9K,L). When either dacha or dachb is knocked down in a six1 gain-of-function background, the number of hair cells in the anterior macula is similar to that observed in wild-type embryos (Fig. 2). However, when both dacha and dachb are knocked down and six1 overexpressed, the number of hair cells seen at 3 dpf in the anterior macula is similar to that observed in individual six1, dacha,ordachb morphants (Fig. 2). Although the dacha and dachb knockdowns in an embryo overexpressing six1 are consistent with these two dach genes acting downstream of six1 in the hair cell lineage, these experiments do not rule out the reverse relationship. The difference in phenotypes seen after knocking down one versus both dach genes suggests that there are interactions between dacha and dachb that remain to be studied. We propose that dacha and dachb both act downstream of or possibly with six1 to promote hair cell fate in the anterior macula (summarized in supplemental Fig. S1, available at www.jneurosci.org as supplemental material).

The formation of hair cells and neurons in the inner ear requires a complex network of transcriptional interactions We studied the interactions between six1 and other members of the Pax–Six–Eya– Dach gene regulatory network, specifically pax2a, pax2b, and eya1 (Fig. 10). The loss- of-function of six1 does not affect pax2a expression at early stages (24 hpf; data not shown), but later (32 hpf), when pax2a is expressed in the developing hair cells, its expression in the developing otocyst is reduced, notably in the utricular hair cells (Fig. 10A,B). At 28 hpf, six1 loss-of- function significantly reduces pax2b ex- Figure8. Caspase-3inhibitorcanrescuethephenotypeofsix1loss-andgain-of-function.A–C,HCS-1immunostainingofhair pression (Fig. 10C,D) and expands the ex- cells in 3 dpf embryos, untreated standard-MO control (A), treated with zFAfmk and six1-MO (B), and treated with zVADfmk and pression domain of eya1 (Fig. 10E,F). six1-MO(C).D–F,HuCimmunostainingofSAGneuronsin3dpfembryos,untreatedstandard-MOcontrol(D),treatedwithzFAfmk These results suggest that six1 activates and overexpressing six1 (E), treated with zVADfmk and overexpressing six1 (F). The zVADfmk compound is a caspase-3 inhibitor, whereas zFAfmk, a structurally similar cathepsin B inhibitor, was used as a negative control. The otocyst is outlined by a dashed pax2b expression but inhibits eya1 expres- lineinA–C.ArrowsarepointingtoanteriormaculahaircellsinA–C(confocalopticalsectionthroughmacula)andtoSAGneurons, sion and are consistent with the functional which are also surrounded by a white dashed line in D–F (confocal projection). Scale bars: A–C,40␮m; D–F,30␮m. G, H, Mean data on these two genes: pax2b loss-of- numberofHCS-1-positivehaircellsin3dpfanteriormacula(G)orHuC-positiveneuronsin3dpfSAG(H)ofSTD,six1MO,andsix1 function leads to a loss of hair cells (Whit- mRNA-injected embryos treated with zVADfmk until 72 hpf beginning at 15, 24, 36, or 48 hpf with a sample size of 15 for each field et al., 2002), whereas the eya1 muta- condition.I,Summarytimelineofganglionformation,neurondelamination,andhaircellformationinthezebrafishinnerear.The tion leads to a loss of neurons in the otic arrows mark the time points at which zVADfmk application was begun in our experiments. ganglion (Kozlowski et al., 2005). Less consistent is the loss of hair cells observed in the eya1 mutant (Kozlowski et al., 2005). Although the loss of posed that eya1 could act as a suppressor of apoptosis (Kozlowski hair cells is more dramatic in the cristae that develop later, half of et al., 2005). Therefore, both eya1 and six1 could be required to the hair cells in the anterior macula are lost in the eya1 mutant prevent apoptosis in the hair cell lineage, whereas they could have [dog (dog-eared)]. Because of the large number of apoptotic cells opposite actions in the neuronal lineage. observed within the otic vesicle of dog mutants, it has been pro- Because MATH-1 has been shown to be necessary for hair cell Bricaud and Collazo • Role of Zebrafish six1 in Ear Development J. Neurosci., October 11, 2006 • 26(41):10438–10451 • 10447

to be expressed in zebrafish because of al- ternative splicing and two transcription initiation sites (Mackereth et al., 2005). We knocked down all three variants by injecting a mixture of two MOs, each targeting one of the two transcription initiation sites as performed previously (Mackereth et al., 2005). This results in a dramatic effect on inner ear morphology (Hans et al., 2004; Mackereth et al., 2005). Although six1 expression is still detectable in the ventral otic epithelium, it is greatly reduced (Fig. 11E), although not to the level seen after pax2b knockdown. The forkhead family member, foxi1 is an important player not only in the induction of the otic placode (Solomon et al., 2003) but also in the proper activation of differentiation pathways in the inner ear (Hans et al., 2004). We therefore studied whether six1 was dependent on foxi1 function in the zebrafish inner ear. When foxi1 is knocked down, the ear anlagen is either entirely missing or greatly reduced (Solomon et al., 2003) and no expression of six1 is detectable (Fig. 11F). Because, at 28 hpf, the lack of six1 expression could be secondary to the overall absence of the otic placode at- Figure 9. six1 acts via two of the three dach genes to promote the proliferation of hair cell progenitors but not SAG neurons. tributable to foxi1 loss-of-function, we A–F,Whole-mountinsituhybridizationtodacha(A,D),dachb(B,E),anddachc(C,F)inotocystsofSTD(A–C)andsix1-MO(D–F) studied six1 expression at either 28 hpf in injectedembryos.Allstainingshavebeenperformedat28hpfexceptBandE,whichwereperformedat32hpf.Arrowheadspoint embryos with a less severe phenotype (data to ventral expression of dacha (A, D), dachb (B, E), and dachc (C, F). The changed expression was confirmed in at least 21 of 25 not shown) or at 16.5 hpf when the pla- embryos scored. Arrows in A and D point to dorsal expression of dacha. G–M, HCS-1 immunostaining of hair cells in STD (G), code just arises (supplemental Fig. S2, six1-MO (H), dacha-MO (I), dachb-MO (J), six1-MO/dacha-MO (K), six1-MO/dachb-MO (L), and dachc-MO (M) injected 3 dpf available at www.jneurosci.org as supple- embryos. N–Q, Whole-mount in situ hybridization to six1 in otocysts of STD control (N), dacha-MO (O), dachb-MO (P), and mental material). In both cases, no expres- dachc-MO (Q) injected 28 hpf embryos. Arrowheads point to six1 expression in SAG; arrows point to six1 expression in the ventral otic epithelium. At least 30 embryos for each condition have been scored for six1 expression. All panels are lateral views with sion of six1 is detectable (data not shown). anterior to left and dorsal up, except for G–M, which are dorsal views with medial to the top. The otocyst is outlined by a dashed In conclusion, six1 may be activated by line in A–F and N–Q. Scale bars: A–F, N–Q,15␮m; G–M,5␮m. foxi1 and subsequently may activate pax2b, dacha, and dachb, leading to the formation of hair cells; in contrast, six1 may inhibit neuronal fate in part by repressing the expression of eya1 in this lin- formation in mouse (Bermingham et al., 1999), we assayed eage. The genetic relationships between all of these genes and six1 whether six1 could regulate the expression of its homolog, as well as their proposed roles in the formation of hair cells and atoh1a, in the zebrafish inner ear (Fig. 10G,H). When six1 is neurons are summarized in supplemental Figure S1 (available at knocked down, atoh1a expression is dramatically reduced in the www.jneurosci.org as supplemental material). anterior ventral otic epithelium, in which the utricular macula hair cells are going to arise (Fig. 10H). Discussion The Pax–Six–Eya–Dach network has been shown to rely on a Six1 may have a more specific role in zebrafish than in mouse complex series of feedback regulatory loops (Pignoni et al., 1997). to balance hair cell and neuron numbers Therefore, we studied whether any of the genes belonging to this Our results demonstrate that six1 plays a pivotal role in control- pathway had an effect on the expression of six1 (Fig. 11). It ap- ling the balance between utricular hair cells and SAG neurons pears that neither pax2a nor eya1 loss-of-function has an effect during zebrafish inner ear development. Its mode of action in- on the normal expression of six1 (Fig. 11B,D). These results have volves controlling both cell proliferation and programmed cell been confirmed by studying six1 expression pattern in noi (no death in the sensory and neuronal lineages. In zebrafish, genes isthmus) and dog (kindly provided by D. Kozlowski, Medical belonging to the Pax–Six–Eya–Dach network have either the School of Georgia, Augusta, GA) mutants (Brand et al., 1996; Lun same or similar roles in both lineages, such as eya1 (Kozlowski et and Brand, 1998). Conversely, pax2b loss-of-function reduces al., 2005), or a unique role in only one lineage, such as dacha and the level of six1 expression (Fig. 11C), demonstrating that pax2b dachb (this study). Our study is the first report of a gene within can regulate, directly or indirectly, six1 transcription. Pax8,a the developing inner ear that acts in opposite ways in these two third member of the Pax gene family, has been shown to be im- lineages. portant for inner ear development in zebrafish (Hans et al., 2004; Although the expression of Six1 in the ventral otic epithelium Mackereth et al., 2005). Three different pax8 variants are thought appears to be highly conserved across vertebrates (Pandur and 10448 • J. Neurosci., October 11, 2006 • 26(41):10438–10451 Bricaud and Collazo • Role of Zebrafish six1 in Ear Development

Figure 10. Effect of six1 loss-of-function on the expression of pax2a, pax2b, eya1, and atoh1a in the developing inner ear. A–H, Whole-mount in situ hybridization to pax2a (A, B), pax2b (C, D), eya1 (E, F), and atoh1a (G, H) in otocysts of embryos injected with standard-MO control (A, C, E, G) and six1-MO (B, D, F, H). All stainings have been performed on 28 hpf embryos except for A and B,whichare36hpfembryos,andGandH,whichare32hpfembryos.Oticvesicleisoutlinedbyadashedline.Arrowspointtopax2a(A,B)andpax2b(C,D)expressionsitesintheventralepithelium. ThearrowheadsinAandBpointtopax2aexpressioninthedorsalepithelium.ThearrowinEpointstosamepartofotocystasinF,indicatingtheposteriorlimitofeya1expressioninawild-typeotic vesicle. The arrows in G and H indicate the normal site of atoh1a expression in the developing anterior macula. The changed expression was confirmed in at least 28 of 32 embryos scored. All panels are lateral views with anterior to the left. Scale bar, 10 ␮m.

the less severe phenotype could be attributable to the MO knockdown not being a complete null, this does not account for the opposite phenotype seen in regards to SAG neurons. Such differences between phenotypes in zebrafish and mouse have been documented for other genes, such as eya1 (Kozlowski et al., 2005). One explanation for this less severe phenotype is a dupli- cation of either the whole genome or gene families in the evolu- tionary lineage leading to zebrafish (Postlethwait et al., 2000; Robinson-Rechavi et al., 2001) that would have resulted in yet another unknown six1 gene. Interactions between Six1 and other members of the Pax–Six– Eya–Dach gene network, such as Eya1, also seem to differ between mouse and zebrafish. Zebrafish six1 inhibits eya1 expression, although its own expression is independent of the function of eya1. In mouse, Eya1 positively regulates Six1 expression (Xu et al., 1999), although its own expression is Six1 independent (Li et al., 2003; Zheng et al., 2003). Not only may interactions be- Figure11. Effectofpax2a,pax2b,eya1,pax8,andfoxi1loss-of-functionontheexpressionof tween six1 and eya1 differ in zebrafish relative to mouse but so six1.A–F,Whole-mountinsituhybridizationofsix1inotocystsof28hpfembryosinjectedwith might the interactions between six1 and the pax2 genes. In STD (A), pax2a-MO (B), pax2b-MO (C), eya1-MO (D), pax8-MO (E), and foxi1-MO (F). Arrows mouse, Pax2 expression is dependent on Six1 in the kidneys but point to six1 expression in the ventral epithelium of the otic vesicle. The star in F indicates the not the developing ear (Xu et al., 2003; Zheng et al., 2003), position where the ear should form. The changed expression was confirmed in at least 25 of 30 whereas in zebrafish, pax2b is directly or indirectly under the embryosscoredforeachcondition.Allpanelsarelateralviewswithanteriortotheleft.Otocysts control of six1 in the inner ear. Six1 expression in the developing are outlined by a dashed line. Scale bar, 10 ␮m. mouse inner ear is not dependent on Pax2 (Xu et al., 2003; Zheng et al., 2003), whereas in zebrafish, it appears that six1 expression is dependent on pax2b but not pax2a. Moody, 2000; Xu et al., 2003; Bessarab et al., 2004), there are In the inner ear, relatively few genes have been found that some striking differences between the phenotype of the Six1 affect the development of one sensory organ but not another. knockouts in mouse (Li et al., 2003; Zheng et al., 2003; Ozaki et Although six1 is expressed throughout most of the ventral otic al., 2004) and its loss-of-function in zebrafish. Six1-null mutant epithelium at early stages of otic development, our data suggest mice inner ears never develop much beyond the otocyst stage (Li that its function is only required for the formation of the hair cells et al., 2003; Zheng et al., 2003; Ozaki et al., 2004). The develop- of the anterior but not posterior macula. Hair cells of the anterior mental arrest is thought to be caused by increased apoptosis and macula may require different genes for their development from decreased cell proliferation. Sensory organs and the SAG fail to those of other sensory organs because they arise closer to the form or, if they do, undergo cell death. Although none of these main neurogenic region of the otocyst (Haddon and Lewis, 1996; studies scored for presence or absence of differentiated hair cells, Whitfield et al., 2002) and are the only hair cells that appear to two reported a dramatic reduction in the expression of prospec- share a common progenitor with SAG neurons (Satoh and tive sensory organ markers such as Bmp4 (bone morphogenic pro- Fekete, 2005). In contrast to six1 perturbations, vgo (van gogh) tein 4) and Lunatic fringe (Zheng et al., 2003; Ozaki et al., 2004), mutants, defective for the tbx1 (T-box 1) gene, are missing all consistent with a loss of hair cell precursors. sensory organs but the anterior macula (Piotrowski et al., 2003). We show here that, in zebrafish, six1 loss-of-function has a less six1 function seems restricted to the otic ganglia even though severe but more specific phenotype. The number of hair cells is it is expressed in other ganglia. However, we cannot rule out more reduced but the number of SAG neurons is increased. Although subtle effects of six1 in other cranial ganglia, such as controlling Bricaud and Collazo • Role of Zebrafish six1 in Ear Development J. Neurosci., October 11, 2006 • 26(41):10438–10451 • 10449 the type of receptors or neurotransmitters expressed by these fects of six1. It has been shown in Xenopus ectoderm that six1 neurons. The neural crest contribution to other placodes (Col- can promote the formation of the placodal field by repressing lazo et al., 1994; Baker and Bronner-Fraser, 2001) could also the transcription of ectodermal and neural crest genes and make six1 function less obvious than in the SAG. For instance, it concomitantly by activating the transcription of placode- has been shown that, in the nodose ganglion, loss of neurons specific genes (Brugmann et al., 2004). Our results suggest that arising from the placode could be rescued by neural crest-derived six1, by playing different roles, regulates the formation of sen- neurons and vice versa (Kirby, 1988a,b; Harrison et al., 1995). sory versus neuronal cell types, balancing their numbers in the developing inner ear. How can six1 act differently in two cell lineages? Six genes have been shown to be involved in three different pro- References cesses during development: (1) cell differentiation (Kawakami et Andermann P, Ungos J, Raible DW (2002) Neurogenin1 defines zebrafish al., 2000); (2) cell proliferation (Ford et al., 1998; Coletta et al., cranial sensory ganglia precursors. Dev Biol 251:45–58. 2004); and (3) cell death (Li et al., 2003; Xu et al., 2003; Zheng et Appel B, Eisen JS (1998) Regulation of neuronal specification in the ze- al., 2003). These three roles are not mutually exclusive, as has brafish spinal cord by Delta function. 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In eye devel- Bricaud O, Chaar V, Dambly-Chaudiere C, Ghysen A (2001) Early efferent innervation of the zebrafish lateral line. J Comp Neurol 434:253–261. opment, it has been shown that Six3 can prevent the formation of Brodbeck S, Englert C (2004) Genetic determination of nephrogenesis: the a Geminin–Cdt1 (cdc10-dependent transcript 1) complex (by Pax/Eya/Six gene network. Pediatr Nephrol 19:249–255. binding to Geminin), leaving free Cdt1 to play its role in the Brugmann SA, Pandur PD, Kenyon KL, Pignoni F, Moody SA (2004) Six1 pre-replication complex, allowing proliferation (Del Bene et al., promotes a placodal fate within the lateral neurogenic ectoderm by func- 2004). tioning as both a transcriptional activator and repressor. Development Six proteins can either activate or repress the same genes de- 131:5871–5881. Bui QT, Zimmerman JE, Liu H, Bonini NM (2000) Molecular analysis of pending on the presence of certain cofactors. This would suggest Drosophila eyes absent mutants reveals features of the conserved Eya do- that cofactors are differentially localized in the developing inner main. Genetics 155:709–720. ear to restrict six1 action to the anterior macula and SAG neu- Chen R, Amoui M, Zhang Z, Mardon G (1997) Dachshund and eyes absent rons, even when six1 is ectopically overexpressed as in our gain- proteins form a complex and function synergistically to induce ectopic of-function experiments. Gro (Groucho), a transcriptional re- eye development in Drosophila. Cell 91:893–903. pressor, can bind Six genes through their two eh1 (engrailed Coletta RD, Christensen K, Reichenberger KJ, Lamb J, Micomonaco D, homolog) domains (Kobayashi et al., 2001; Zhu et al., 2002; Huang L, Wolf DM, Muller-Tidow C, Golub TR, Kawakami K, Ford HL (2004) The Six1 homeoprotein stimulates tumorigenesis by reactivation Lopez-Rios et al., 2003; Silver et al., 2003). The model proposed of cyclin A1. 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