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. Author manuscript; available in PMC: 2026 Feb 28.
Published in final edited form as: Dev Biol. 2025 Nov 17;530:38–48. doi: 10.1016/j.ydbio.2025.11.010

Zebrafish pou3f3b controls saccular/auditory development and marks non-neuronal cells that delaminate from the otic vesicle to promote neuroblast maturation

Sydney E Christensen 1,1, Maria Ali 1,1, Jorden N Holland 1, Bruce B Riley 1,*
PMCID: PMC12947660  NIHMSID: NIHMS2145457  PMID: 41260345

Abstract

The zebrafish otic vesicle initially develops with only two sensory maculae, each with distinct functions. The anterior utricular macula is indispensable for vestibular function, while the posterior saccular macula is the primary auditory endorgan in zebrafish. The unique identities of these maculae are specified in the early otic vesicle by differing levels of Fgf vs. Shh signaling, but few downstream effectors have been identified. pou3f3b is the only saccule-specific marker known, but its function has not been established. We generated a knockout allele of pou3f3b and found that it causes a persistent delay in accumulation of saccular hair cells due to a failure to activate saccular expression of fgf3. In addition, saccular hair cells exhibit reduced expression of Otoferlin caused by ectopic expression of neurog1. Defects in saccular hair cell development are fully rescued by misexpressing fgf3 or knocking down neurog1. Misexpression of pou3f3b causes loss of utricular pax5 expression and further truncates neurog1 in the posterior otic vesicle but does not otherwise alter macular development. In addition to regulating saccular development, pou3f3b is also expressed in a previously undescribed population of non-neuronal cells that delaminate from the otic vesicle and migrate together with developing neuroblasts to promote their maturation. Mutant neuroblasts show a marked delay in activation of expression of neurod1, causing a transient delay in accumulation of mature SAG neurons. Thus pou3f3b is required for timely development of SAG neurons and saccular/auditory hair cells.

Keywords: Hair cell, Neuroblast, Statoacoustic ganglion, atoh1a, Neurog1, Fgf3, Otoferlin

1. Introduction

The vertebrate inner ear is a complex sensory system containing a number of discrete sensory epithelia, each specialized for either vestibular or auditory function. In zebrafish, the first sensory epithelia to form in the otic vesicle are the anterior utricular and posterior saccular maculae (Haddon and Lewis, 1996). The utricular macula is critical for vestibular function and begins to function by 3 days post-fertilization (dpf) (Riley and Moorman, 2000; Kwak et al., 2006), whereas the saccular macula is the main auditory epithelium and begins to function robustly by 5 dpf (Zeddies and Fay, 2005; Schuck and Smith, 2009; Smith et al., 2011). Although other sensory epithelia eventually contribute to inner ear function, the utricular and saccular maculae are sufficient to provide all vestibular and auditory function, respectively, for the first several weeks of larval development (Beck et al., 2004; Bever and Fekete, 2002; Lau and Vasconcelos, 2023). This provides an opportunity to explore early development of auditory vs. vestibular function in sensory maculae that otherwise exhibit highly similar molecular profiles in adults (Yao et al., 2020; Shi et al., 2023). Functional specialization arises partly from differential innervation patterns, as auditory and vestibular epithelia are innervated by different classes of neurons that transmit sensory signals to different processing centers in the brain. There are also differences in macular shape and of hair cell distribution that emerge during early development. The first hair cells are already established in pairs at the anterior and posterior poles of the nascent otic vesicle by 18 h post-fertilization (hpf) (Riley et al., 1997). These are specified during placodal development (Millimaki et al., 2007) and are referred to as tether cells because their kinocilia serve to nucleate formation of the first otoliths, crystalline masses that facilitate hair cell activation in response to acceleration or sound (Riley et al., 1997). Subsequent accumulation of the utricular hair cells is initially much faster than in the saccule, with new hair cells forming steadily from 24 hpf through 48 hpf (Kantarci et al., 2020). The saccular macula does not begin to expand until after 30 hpf but then forms new hair cells at an accelerated pace, catching up to the utricular macula by 48 hpf. The characteristic shapes of these maculae are also established during this period. The utricular macula assumes a roughly circular, or C-shaped pattern on the anterior floor of the otic vesicle, while the saccular macula forms a horizontal sensory ribbon along the posterior-medial wall and is tonotopically organized along its length (Haddon and Lewis, 1996; Schuck and Smith, 2009; Breitzler et al., 2020).

Differential growth and morphogenesis of the utricular and saccular maculae depend on different levels of Fgf and Hh signaling (Hammond and Whitfield, 2011). Fgf is required for sensory specification in both the utricular and saccular maculae, with early Fgf signals emanating from the hindbrain during placodal development, and later from the maculae themselves after formation of the otic vesicle (Millimaki et al., 2007). The utricular macula requires a high level of Fgf signaling, which is necessary and sufficient to induce local expression of pax5 (Kwak et al., 2002, 2006), as well as increased nuclear accumulation of Pax2a protein (Sweet et al., 2011). Together, Pax2a and Pax5 serve to upregulate cdh1/E-cadherin to stabilize hair cells within the utricular epithelium (Roberson et al., 2025). This compensates for the destabilizing effects of elevated Fgf, which tends to cause disassembly of junctional complexes (Lilien and Balsamo, 2005). Disruption of pax2a or pax5 prevents local upregulation of cdh1 and causes sporadic and progressive ejection of intact hair cells from the utricular macula and severe impairment of vestibular function (Kwak et al., 2006; Roberson et al., 2025). In contrast, the saccular macula requires low Fgf plus high Hh signaling, which in combination are required to activate expression of pou3f3b in the saccular domain (Hammond and Whitfield, 2011; Tan et al., 2023). pou3f3b is expressed in an elongate band extending from the nascent saccular tether cells into an anterior region that presages future growth of the saccular macula. Expression of pou3f3b is eliminated by blocking Hh signaling (Tan et al., 2023). Expression is also reduced or lost by eliminating early fgf8 signaling. Interestingly, pou3f3b expression expands into the anterior utricular domain in fgf3−/− mutants, consistent with a requirement for a discrete low level of Fgf signaling (Tan et al., 2023). In mouse, too, the orthologous gene Pou3f3 is expressed in an Fgf-dependent manner in the region of the otic vesicle that later gives rise to the cochlear duct and organ of Corti, the auditory epithelium in mammals (Urness et al., 2018)). Knockout of mouse Pou3f3 has no clear impact on development of the organ of Corti in neonatal pups, but adults homozygous for a hypomorphic allele of Pou3f3 exhibit strongly impaired hearing at all frequencies (Mutai et al., 2009; Kumar et al., 2016). Whether disruption of pou3f3b in zebrafish alters development of the saccular macula has not been previously investigated but could provide insight into potential conservation of function.

In addition to activating sensory development, Fgf signaling simultaneously initiates development of neuroblasts in the floor of the otic vesicle by inducing a broad zone of neurog1 expression (Vemaraju et al., 2012). Neuroblasts eventually differentiate into neurons of the statoacoustic ganglion (SAG), but to do so they must first delaminate from the otic vesicle. Delamination occurs in a progressive fashion and requires Fgf-dependent expression of goosecoid (gsc) within the lateral-most neuroblasts (Kantarci et al., 2016). Gsc acts as a transcriptional repressor that downregulates cdh1/E-cadherin, thereby promoting epithelial-mesenchymal transition. Interestingly, pou3f3b is also expressed in cells that delaminate from the otic vesicle and accumulate around the anterior outer wall of the otic vesicle, previously interpreted as delaminating neuroblasts (Tan et al., 2022). Whether this domain of pou3f3b expression affects development of neuroblasts is unknown.

Here we have analyzed the function of pou3f3b in development of the saccular macula and SAG neuroblasts. We generated a knockout allele and compared it with a previously reported allele. Both alleles cause comparable defects in saccular development. Specifically, mutants exhibit abnormal differentiation and delayed accumulation of saccular hair cells, defects caused by ectopic expression of neurog1 in hair cell progenitors and loss of activation of fgf3 in the saccular domain. These defects are rescued by knocking down neurog1 or misexpressing fgf3. Mutants also exhibit delayed differentiation of newly delaminated neuroblasts, characterized by prolonged expression of neurog1 and delayed activation of neurod1, resulting in delayed accumulation of mature SAG neurons. Surprisingly, pou3f3b + delaminating cells are not eliminated by disrupting neurog1, indicating that they are non-neuronal cells that are required to promote early neuroblast differentiation. Thus, pou3f3b is required for timely differentiation of saccular/auditory hair cells, and acts non-autonomously to promote maturation of SAG neurons.

2. Materials & methods

Fish strains and developmental conditions. Wild-type zebrafish embryos were derived from the AB line and incubated under standard conditions at 28.5 °C and treated with PTU (1-phenyl 2-thiourea, Sigma P-7629) for time points after 24hpf to prevent melanin formation (Kimmel et al., 1995). Previously published mutant lines used herein include fgf3t21142 (Herzog et al., 2004), gscx59 (Kantarci et al., 2016), and pou3f3be1502 (Barske et al., 2020). We also generated a new mutant line, pou3f3bx77, using CRSPR/Cas9 mutagenesis. Transgenic lines used for misexpression include Tg(hsp70:fgf3)x27 (Sweet et al., 2011), Tg(hsp70: neurog1)x28, Tg(hsp70:gsc)x58 (Kantarci et al., 2016), Tg(hsp70:gal4) (Sheer et al., 2002), and Tg(uas:pou3f3b)e1578 (Barske et al., 2020).

2.1. In situ hybridization, whole-mount antibody staining, and acridine orange staining

Whole-mount in situ hybridization was performed as previously described (Phillips et al., 2001). Immunohistochemistry was performed using primary antibodies anti-GFP (Invitrogen A11122, 1:250), anti-Otoferlin to visualize hair cells (Developmental Studies Hybridoma Bank HCS-1, 1:100 for hair cell counts, 1:300 for Otoferlin photo-bleaching time) and anti-Islet1/2 for SAG neurons (Developmental Studies Hybridoma Bank 39.4D5, 1:75). Secondary antibodies used were either AlexaFluor 488 goat anti-mouse (Invitrogen A11001, 1:50) or AlexaFluor 546 goat anti-mouse (Invitrogen A11003, 1:50). To label dying/apoptotic cells, live dechorionated embryos were incubated for 1 h in aqueous solution containing 1 μg/ml of Acridine Orange (Invitrogen A1301) and briefly washed in phosphate buffered saline (PBS) before imaging via fluorescence microscopy.

2.2. Heat shock misexpression and morpholino injections

Heat shock inducible transgenes were activated by incubating heterozygous embryos in a water at 35 °C for 6 h or 39 °C for 1 h, as described in the text. Following heat shock treatment, embryos were incubated at 33 °C until time of fixation. To knock down expression of neurog1, embryos were injected at the one cell stage with 5 ng of morpholino oligomer using a previously published MO sequence (Andermann et al., 2002).

2.3. Quantitation, reproducibility and statistics

Statistical analysis was performed using student’s t-test for pairwise comparisons. One-way ANOVA with Tukey post-hoc HSD tests were performed for experiments with three or more groups. Cell counts were obtained by counting relevant cell populations under a Zeiss compound microscope using brightfield or fluorescence imaging. At least 10 specimens were used for each measurement, except where indicated in the text. Hair cell numbers were quantified by counting cells marked by anti-Otoferlin staining in whole-mount embryos. To quantify Otoferlin staining intensity, the anti-Otoferlin antibody was diluted 1:300 and saccular hair cells were viewed with a 40× objective under fluorescence using an X-Cite series 120 UV source. Time to complete photobleaching was measured in seconds. SAG neurons were quantified by counting cells marked by anti-Islet1/2 staining in whole mount embryos. Numbers of cells expressing gsc in the otic vesicle or pou3f3b in ONND cells that have delaminated from the otic vesicle were counted from whole-mount specimens stained by whole-mount in situ hybridization. Neuroblasts marked by neurod1 expression or neurog1 expression were quantified by counting cells in 10 μm serial sections of embryos stained by whole-mount in situ hybridization.

3. Results

3.1. pou3f3b knockouts impair saccular development

To investigate the function of pou3f3b, we generated a CRISPR/Cas9 knockout (allele x77) featuring an 8 bp deletion just downstream of the translation start site, causing a frameshift and premature stop (Fig. 1A). Homozygous pou3f3bx77/x77 mutants are viable and fertile, similar to a previously described knockout allele, pou3f3be1502 (Barske et al., 2020). Comparison of pou3f3b expression in wild-type and pou3f3bx77/x77 mutants showed no change in saccular expression at 24–36 hpf (Fig. 1B–E). Development of hair cells was examined by staining embryos with anti-Otoferlin antibody, an early marker of hair cell differentiation that regulates synaptic transmission and is required for auditory and vestibular function (Goodyear et al., 2010; Pangrsic et al., 2010; Chaterjee et al., 2015; Spaiardi et al., 2022). Anti-Otoferlin staining revealed several specific defects in saccular hair cells in both x77/x77 and e1502/e1502 mutants: First, the number of saccular hair cells was reduced by roughly 25 % at 36, 48, and 72 hpf (Fig. 1F–L). Second, the intensity of anti-Otoferlin staining was specifically reduced in saccular hair cells in mutant embryos through at least 72 hpf (Fig. 1F–K), suggesting a defect in hair cell differentiation. To confirm the latter change, we measured the amount of time required to photo-bleach staining in saccular hair cells under fluorescence imaging using a 40× objective. At 36 hpf, saccular hair cells in wild-type embryos photo-bleached in 70 ± 2.8 s. In contrast, saccular hair cells photo-bleached in 34 ± 5.3 s in x77/x77 mutants and 34 ± 7.0 s in e1502/e1502 mutants (Fig. 1M). Development of utricular hair cells appeared normal in pou3f3b mutant embryos (Fig. 1F–L), showing that disruption of pou3f3b specifically impairs early saccular development.

Fig. 1. Disruption of pou3f3b impairs development of the saccular macula.

Fig. 1.

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(A) Schematic for a new CRISPR-mediated knockout allele of pou3f3b, x77. (B–E) Expression of pou3f3b in wild-type embryos (B, D) and pou3f3bx77/x77 mutants (C, E) at 24 and 36 hpf. Expression is evident in the saccular domain (sac), delaminating cells (dc) in the neurogenic region of the otic vesicle, and pharyngeal arches (pa). Oval marques delimit the otic vesicle and pharyngeal arch domains. (F–K) Developing hair cells in the utricle (ut) and saccule (sac) stained with anti-Otoferlin antibody in wild-type embryos (F, I), pou3f3bx77/x77 mutants (G,J), and pou3f3be1502/e1502 mutants (H, K) at 36 and 72 hpf. (L) Means and standard deviations of the number of utricular (utr) and saccular (sac) hair cells in wild-type and mutant embryos at the indicated times. Significant differences are indicated with asterisks and p-values. (M) Means and standard deviations of the number of seconds required to photo-bleach anti-Otoferlin staining in saccular hair cells in wild-type and mutant embryos (see Materials & Methods). Embryos were viewed with a 40× objective under UV fluorescence for the indicated times. Significant differences are indicated with asterisks and p-values.

3.2. pou3f3b is required for saccular fgf3 expression

Although saccular tether cells become morphologically mature by 21–22 hpf (Riley et al., 1997), additional hair cells do not begin to accumulate in the saccule until around 30 hpf, marked by elevated expression of atoh1a in sensory precursors (Fig. 2A). This corresponds to the time when fgf3, a known inducer of sensory development (Kwak et al., 2002; Millimaki et al., 2007), normally begins to be expressed in the saccular domain (Fig. 2D). In pou3f3b−/− mutants, saccular expression of atoh1a is weaker and marks fewer cells than normal at 30 hpf (Fig. 2B and C). At the same time, pou3f3bx77/x77 and pou3f3be1502/e1502 mutants exhibit little or no expression of fgf3 in the saccular domain, whereas expression in the utricular macula and pharyngeal arches appear normal at 30 hpf (Fig. 2E and F). Likewise, expression of fgf8a and fgf10a appear normal in pou3f3b−/− mutants (Fig. 2G–J). These data suggest that reduced expression of fgf3 contributes to the deficiency of saccular hair cells in pou3f3b−/− mutants. To test this idea, we examined hair cell development in fgf3−/− mutants. Staining with anti-Otoferlin showed that accumulation of saccular hair cells was reduced by 30–40 % in fgf3−/− mutants from 36 to 72 hpf, whereas the number of utricular hair cells was normal during this interval (Fig. 2K, L, Q). However, the intensity of anti-Otoferlin staining appeared normal in all hair cells in fgf3−/− mutants (Fig. 2L). We next tested whether misexpression of a heat shock-inducible transgene, hs:fgf3, could rescue hair cell production in pou3f3bx77/x77 mutants. Low level activation of hs:fgf3 for 6 h (35 °C from 24 to 30 hpf) restored the proper number of saccular hair cells in pou3f3bx77/x77 mutants at 48 hpf, whereas anti-Otoferlin staining intensity was not rescued (Fig. 2M–P, R). The deficiency of saccular hair cells in mutant embryos does not reflect elevated cell death, as we observed no increase in acridine orange staining at 30–48 hpf (Fig. 2S and T). Together, these data support the conclusion that impaired saccular expression of fgf3 contributes to a reduced rate of saccular hair cell production in pou3f3b−/− mutants but does not affect Otoferlin expression levels.

Fig. 2. Loss of saccular fgf3 expression delays hair cell accumulation.

Fig. 2.

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(A–C) Dorsolateral views showing utricular (utr) and saccular (sac) expression of atoh1a in wild-type and pou3f3b−/− mutant embryos at 30 hpf. The mean and standard deviation of the number of atoh1a + cells in the saccular domain is indicated (n = 27 wt, 21 x77, 12 e1502). Significant differences are indicated with p-values. Oval marques delimit the otic vesicle. (D–J) Dorsolateral views showing expression of fgf3 (D–F), fgf8a (G, H), and fgf10a (I, J) at 30 hpf in embryos with the indicated genotypes. Utricular (utr), saccular (sac), and pharyngeal arch (pa) domains are marked, and oval marques encircle the otic vesicle. Mutant embryos fail to activate saccular expression of fgf3 (n = 10 each). (K–P) Dorsolateral views showing utricular (utr) and saccular (sac) hair cells marked by anti-Otoferlin staining in the indicated genotypes at 48 hpf. Embryos in (M–P) were heat shocked at 35 °C from 24 to 30 hpf. (Q, R) Means and standard deviations of the number of utricular (utr) and saccular (sac) hair cells in embryos at the indicated times. Keys indicate genotypes. Embryos in (R) were heat shocked at 35 °C from 24 to 30 hpf. Significant differences are indicated (asterisks, p < 0.0001). (S, T) Lateral views of the otic vesicle in a wild-type (S) and pou3f3bx77/x77 mutant embryo (T) stained with acridine orange (AO) at 48 hpf, n = 5 each.

3.3. pou3f3b represses neurog1 in the saccular domain

Expression of neurog1 marks early stages of neuroblast development while also restricting sensory development by transcriptionally repressing atoh1a/b (Raft et al., 2007; Gou et al., 2018). In wild-type embryos at 24 hpf, neurog1 is expressed broadly in the floor of the otic vesicle between the utricular and saccular maculae, with a posterior limit ending roughly 20 μm from the posterior pole (Fig. 3A–L). In pou3f3b−/− mutants, the posterior end of the neurog1 domain extends to within 8–12 μm of the posterior pole, encroaching on the saccular domain (Fig. 3B, C, L). Posterior expansion of neurog1 was more pronounced in pou3f3bx77/x77 than in pou3f3be1502/e1502 mutants (Fig. 3L), possibly because in these experiments the former lack both maternal and zygotic function. To test whether expanded posterior expression of neurog1 contributes to defects in saccular development, we injected morpholino oligomer (MO) to knock down neurog1 in pou3f3bx77/x77 mutants. As previously reported (Sapède et al., 2012), injection of neurog1-MO into wild-type embryos caused precocious expansion of the saccular macula at 36 hpf and 48 hpf (Fig. 3F–M). Knockdown of neurog1 in pou3f3bx77/x77 mutants restored both the proper number of saccular hair cells and Otoferlin staining levels at 48 hpf (Fig. 3G–M). To test whether weak misexpression of neurog1 is sufficient to impair saccular development in an otherwise wild-type background, we activated transgenic hs:neurog1 at a low level for 6 h (35 °C at 24–30 hpf, or at 30–36 hpf). With either heat shock regimen, utricular hair cells developed normally but the number of saccular hair cells was reduced by ~30 % at 48 hpf (Fig. 3H–K, N). Moreover, Otoferlin staining levels were reduced in a manner similar to pou3f3b−/− mutants (compare Fig. 3E–I, K). These findings support the conclusion that pou3f3b normally restricts neurog1 expression from the saccular region, and encroachment of neurog1 into the saccular domain in pou3f3b−/− mutants contributes to both reduced accumulation of saccular hair cells and reduced Otoferlin staining intensity.

Fig. 3. Expanded expression of neurog1 impairs saccular hair cell differentiation.

Fig. 3.

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(A–C). Dorsolateral views showing expression of neurog1 in wild-type and mutant embryos. The dashed line marks the posterior limit of the otic vesicle lumen, and black arrows mark the posterior limit of neurog1 expression in the otic vesicle floor. (D–K) Dorsolateral views showing utricular (utr) and saccular (sac) hair cells stained with anti-Otoferlin antibody at 48 hpf. Genotypes are indicated across the top. Embryos in (H–K) were heat shocked at 35 °C from 24 to 30 hpf (H, I) or 30–36 hpf (J, K). (L) Scatter plots showing the mean and standard deviation of the posterior proximity of neurog1 expression, or the distance in micrometers between the posterior limit of neurog1 expression and the inner posterior wall of the otic vesicle, as marked in (A–C). Significant differences are indicated with asterisks and p-values. (M, N) Mean and standard deviation of the number of utricular (utr) and saccular (sac) hair cells for the indicated times and genotypes. Embryos in (N) were heat shocked as indicated. Significant differences are indicated with asterisks and p-values.

3.4. Misexpression of pou3f3b

To test the effects of pou3f3b misexpression, we used hs:gal4 transgene to globally activate a uas:pou3f3b transgene (Barske et al., 2020). We chose to activate hs:gal4; uas:pou3f3b expression beginning at 22 hpf when only the first two tether cells are present in the utricular and saccular maculae. Full activation of hs:gal4 (39 °C for 1 h) leads to a strong pulse of Gal4 accumulation that can continue activating uas target transcription for 17 h or more (Sheer et al., 2002). Global misexpression of pou3f3b beginning at 22 hpf was sufficient to repress utricular expression of pax5 at 24 hpf (Fig. 4A and B). Expression of fgf3, which is required for pax5 expression in the utricular domain (Kwak et al., 2006), was not altered under these conditions (Fig. 4E and F), suggesting that forced expression of pou3f3b might alter utricular identity. However, examination of later sensory development showed that both the morphology and number of hair cells were normal in utricular and saccular maculae at 36 and 48 hpf (Fig. 3C, D, I). Thus, misexpression of pou3f3b repressed pax5 but was not sufficient to fully alter utricular morphology or to accelerate saccular development. Interestingly, misexpression of pou3f3b led to dramatic truncation of neurog1 expression in the posterior half of the otic vesicle (Fig. 4G, H, J), in sharp contrast to posterior expansion of neurog1 in pou3f3b−/− mutants. Thus, pou3f3b represses neurog1 expression to varying degrees, depending on the level of pou3f3b expression.

Fig. 4. Misexpression of pou3f3b represses pax5 and neurog1.

Fig. 4.

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(A–J) Wild-type and uas:pou3f3b transgenic embryos carrying hs:gal4 were heat shocked at 39 °C from 22 to 23 hpf and fixed and stained for the indicated gene products at the indicated times. (A, B) Dorsolateral view showing pax5 expression in the utricular domain at 24 hpf. (C, D) Utricular (utr) and saccular (sac) hair cells stained with anti-Otoferlin at 48 hpf. (E, F) Dorsolateral views showing fgf3 expression in the utricular domain (utr) and pharyngeal arches (pa) at 24 hpf. (G–H) Dorsolateral views showing neurog1 expression at 24 hpf. Black arrows mark the posterior limit of neurog1 expression and the dashed line marks the posterior limit of the otic vesicle lumen. (I) Means and standard deviations of the number of utricular (utr) and saccular (sac) hair cells at 36 and 48 hpf. (J) Scatter plots showing means and standard deviations of posterior proximity of neurog1 (distance in micrometers between the posterior limit of expression and the posterior limit of the lumen). Significant differences are indicated with asterisks and p-values.

3.5. pou3f3b promotes maturation of early neuroblasts

In addition to robust expression in the saccular domain, pou3f3b is also expressed in a group of cells that appear to delaminate from the neurogenic region of the otic vesicle, which we initially interpreted as neuroblasts (Fig. 1B–E, Fig. 5A). Delaminating pou3f3b + cells were markedly reduced or missing in pou3f3bx77/x77 mutants at 24 hpf (Figs. 1C and 5B) but appear normal by 36 hpf (Fig. 1E). Despite the early deficiency of delaminating pou3f3b + cells at 24 hpf, mutant embryos did not appear to show a corresponding deficiency of delaminating neurog1+ neuroblasts (Fig. 3A–C). This raised the possibility that delaminating pou3f3b + cells are not neuroblasts, but are instead a non-neuronal cell type. To investigate this possibility, we examined the spatial relationship between expression of pou3f3b and neurod1:Gfp in transverse sections around the front of the otic vesicle (Fig. 5C–E’). Expression of pou3f3b marks cells in the anterior ventrolateral edge of the otic vesicle (Fig. 5D and E) and delaminated cells that abut the lateral and dorsal edges of the mass of neurod1:Gfp + transit-amplifying (TA) neuroblasts (Fig. 5C’–E′). We observed no cells co-expressing both genes, consistent with the idea that they represent different cell types. Using a more definitive approach, we observed that morpholino knockdown of neurog1, which blocks neuroblast formation (Andermann et al., 2002), did not alter development of delaminating pou3f3b + cells (Fig. 5F–I). Together, these data show that delaminating pou3f3b + cells represent a previously undescribed non-neuronal cell type, which we have termed otic non-neuronal delaminating (ONND) cells. Delamination of neuroblasts requires the transcriptional repressor Goosecoid to promote epithelial-mesenchymal transition (Kantarci et al., 2016), so we tested whether altering gsc function also affects ONND cells. Activation of heat shock inducible transgene hs:gsc at 22 hpf caused a 50 % increase in the number of delaminated ONND cells by 24 hpf (Fig. 5G). In gsc−/− mutants, the number of delaminated ONND cells was reduced by 66 % at 24 hpf (Fig. 5H). This shows that gsc is required for proper delamination of both neuroblasts and ONND cells. To examine upstream signaling requirements, we treated embryos with Fgf antagonist SU5402 or the Wnt agonist BIO, both of which block neurogenesis (Tan et al., 2022). Similarly, these treatments fully blocked formation of ONND cells (Fig. 5J and K). Thus, ONND cells and neuroblasts share key upstream requirements for specification and delamination, but then exhibit distinct, complementary migration patterns around the front of the otic vesicle.

Fig. 5. pou3f3b marks otic non-neural delaminating (ONND) cells.

Fig. 5.

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(A, B) Transverse sections through the anterior pole of the otic vesicle showing pou3f3b in ONND cells (arrows) in a wild-type embryo (A) and pou3f3bx77/x77 mutant (B) at 24 hpf. Mutant embryos exhibit a marked deficiency of pou3f3b + cells. The outer edges of the otic vesicle are marked with a dashed line. (C-E′) Serial transverse sections through a transgenic embryo showing pou3f3b (dark blue) and neurod1:Gfp (green, prime letters) at 24 hpf. The most anterior section (C, C′) passes though the group of neurod1:Gfp + transit-amplifying (TA) neuroblasts that accumulate just anterior to the otic vesicle; the next section (D, D′) passes through the anterior pole of the otic vesicle; and the third section (E, E′) passes through the otic vesicle just anterior to the utricular macula. pou3f3b + ONND cells (arrows) form a contiguous band wrapping around the dorsal and lateral edges of anterior mass of TA neuroblasts. Edges of the otic vesicle are marked with a dashed line. (F–K) Dorsolateral views showing expression of pou3f3b at 24 hpf in a wild-type embryo (F), a hs: gsc transgenic embryo (G), a gsc−/− mutant (H), a neurog1 morphant (I), a wild-type embryo treated with SU5402 from 12 hpf (J), and a wild-type embryo treated with BIO from 12 hpf (K). Transgenic hs:gsc embryos were heat shocked at 39C for 1 h beginning at 22 hpf. Expression of pou3f3b marks ONND cells as they delaminate and accumulate around that anterior surface of the otic vesicle (o), pharyngeal arches (p), and trigeminal ganglion (g). Treatment with SU5402 or BIO from 12 hpf prevents formation of neuroblasts and pou3f3b + ONND cells, whereas saccular expression of pou3f3b continues. The mean number (±S.D.) of delaminated ONND cells in front of the otic vesicle is indicated (n = 8 each). Significance relative to wild-type control embryos is indicated by p-values. Note: development with 5 %DMSO or heat shocking wild-type embryos did not alter accumulation of delaminated ONND cells (24.9 ± 2.6 cells and 25.2 ± 1.6 cells, respectively, images not shown).

We next investigated whether pou3f3b function is required non-autonomously for neuronal development. Following delamination, neuroblasts normally undergo a rapid developmental progression in which they activate expression of neurod1 and lose expression of neurog1. At the same time, neuroblasts persist in a state of transit-amplification (TA) in which they continue to proliferate as they migrate towards the hindbrain. TA cells eventually differentiate as post-mitotic neurons of the statoacoustic ganglion (SAG), marked by expression of islet1 and islet2 (Andermann et al., 2002; Vemaraju et al., 2012). We compared developmental progression of TA cells in wild-type and pou3f3bx77/x77 embryos by counting the number of neurog1+ and neurod1+ cells in serial sections. As expected from whole-mount staining, pou3f3bx77/x77 mutants exhibited a nearly 50 % increase in the number of neurog1+ cells in the floor of the otic vesicle (Fig. 6A, B, E), reflecting posterior expansion of the neurogenic domain into the saccular region (Fig. 3A–C). Mutant embryos also exhibited a nearly 70 % increase in neurog1+ cells outside the otic vesicle compared to wild-type embryos (Fig. 6A, B, E). The increase in neurog1+ TA cells outside the otic vesicle likely does not arise from excess delamination, as the number and distribution of gsc-expressing cells in the otic vesicle was normal in mutant embryos (Fig. 6G and H). Moreover, gsc expression occupies a lateral domain in the floor that is well separated from the saccular region where ectopic neurog1+ cells appear in mutant embryos (Kantarci et al., 2016). In contrast to the excess of neurog1+ TA cells in pou3f3bx77/x77 mutants, the number of neurod1+ TA cells was reduced by ~15 % compared to wild-type embryos (Fig. 6C, D, F). Although this difference was not statistically significant (p = 0.195), the ratio of neurod1+ cells to neurog1+ cells was much smaller in mutants (0.73) compared to wild-type embryos (1.44). Thus, the transition from neurog1 expression to neurod1 expression was considerably slower in pou3f3bx77/x77 mutants, suggesting a delay in early neuroblast differentiation. To test this idea further, we counted the number of mature SAG neurons at various time points by staining embryos with anti-Islet1/2 antibody. Consistent with a delay in neuroblast differentiation, we observed a significant 20–25 % decrease in accumulation of Isl1/2+ neurons in pou3f3bx77/x77 mutants at 24 and 30 hpf (Fig. 6I–L, O). However, SAG accumulation appeared normal in mutant embryos at 36–48 hpf (Fig. 6J, K, M–O). Interestingly, although the number of SAG neurons returns to normal in pou3f3b−/− mutants, the pattern of Isl1/2+ neurons showed irregular gaps (Fig. 6L–N), similar to other mutants that delay maturation of SAG neurons (Kantarci et al., 2015; Ali et al., 2025). Together, these data show that pou3f3b is required non-autonomously for timely differentiation of nascent neuroblasts through 30 hpf but is no longer required at later stages, allowing SAG development to recover in mutant embryos. How pou3f3b expression in ONND cells regulates early neuroblast development remains to be established (see Discussion).

Fig. 6. pou3f3b non-autonomously promotes maturation of nascent neuroblasts.

Fig. 6.

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(A–D) Transverse sections (dorsal up, medial to the left) showing neurog1 (A, B) and neurod1 expression (C, D) in wild-type and pou3f3bx77/x77 mutant embryos. Transit-amplifying cells are outlined in red and labeled (ta). (E, F) Scatter plots showing the means and standard deviations of the number of neurog1+ cells in the otic vesicle or in TA cells (E) and the number of neruod1+ TA cells (F) in wild-type and pou3f3b mutant embryos. Numbers were counted from serial sections of n = 3 embryos (E) or n = 4 embryos (F). p-values are indicated. (G, H) Dorsolateral view showing otic expression of gsc in a wild-type embryo (E) and a pou3f3bx77/x77 mutant (F) at 24 hpf. The mean number (±S.D.) of gsc-expressing cells, and p-value in mutant relative to wild-type, are indicated (n = 10 each). (I–N) Dorsolateral views showing anti-Isl1/2 antibody staining in wild-type (I–K) and pou3f3bx77/x77 mutant (L–N) embryos at the indicated times. White arrows in (L–N) indicate variable gaps in the otherwise regular pattern of mature SAG neurons. (O) Means and standard deviations of the number of Isl1+ SAG neurons in wild-type and pou3f3bx77/x77 mutant embryos at the indicated times. Significant differences are indicated with asterisks and p-values. ns, not significant.

4. Discussion

Here we have shown that pou3f3b regulates two fundamental aspects of inner ear development in zebrafish: pou3f3b promotes proper development of the saccular/auditory macula (Fig. 7A), and timely maturation of nascent neuroblasts during early stages of neurogenesis (Fig. 7B). These are discussed in more detail below.

Fig. 7. Summary and model.

Fig. 7.

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(A) Utricular and saccular domains are established in the nascent otic vesicle by the distribution of upstream inductive signals: Strong Fgf signaling from rhombomere 4 (r4) of the hindbrain induces pax5 in a broad circular domain corresponding to the utricular macula, and Shh from the midline combined with weak Fgf (reflecting diffusion from anterior sources) induce pou3f3b in an elongated domain corresponding to the future saccular macula. Expression of pou3f3b promotes saccular expression of atoh1a through fgf3 while repressing posterior expression of neurog1 as needed for proper expression of Otoferlin. (B) expression of pou3f3b marks otic non-neuronal delaminating (ONND) cells that delaminate and accumulate around the front of the otic vesicle to form a band wrapping around the dorsal and lateral edges of emergent migrating TA neuroblasts. pou3f3b function in ONND cells non-autonomously promotes maturation of delaminated neuroblasts by facilitating the transition from neurog1 expression to neurod1 expression. The stimulatory signal from ONND cells remains to be identified.

Saccular development. Activation of pou3f3b in the saccular domain requires signaling interactions that begin during placodal stages: Shh released from the floorplate plus weak Fgf signaling from the hindbrain (Hammond and Whitfield, 2011; Tan et al., 2023). Additionally, pou3f3b is restricted from the utricular domain by strong Fgf signaling from rhombomere 4 and the anterior otic vesicle, as well as transcriptional repression by pax5 (Tan et al., 2023, Fig. 6A). By 30 hpf, pou3f3b directly or indirectly activates expression of fgf3 in the anterior part of the saccular domain to induce progressive formation of new hair cells. Saccular pou3f3b also directly or indirectly represses neurog1 in sensory progenitors, as required for proper differentiation of hair cells. In pou3f3b−/− mutants, neurog1 is expressed ectopically in the saccular region and saccular hair cells accumulate at a reduced rate and show reduced expression of Otoferlin. The Otoferlin deficiency is attributable solely to ectopic neurog1 expression, as knockdown of neurog1 restores Otoferlin levels in pou3f3b−/− mutants, and misexpression of hs: neurog1 in wild-type embryos is sufficient to reduce Otoferlin expression. The effects of neurog1 on hair cell development likely reflect its ability to repress atoh1a (Raft et al., 2007; Gou et al., 2018), although it could also repress downstream genes since activation of hs:neurog1 at 30–36 hpf (after hair cells have already differentiated) is sufficient to reduce Otoferlin expression (Fig. 3K). We note that knockdown of neurog1 also increases the number of saccular hair cells in wild-type embryos, as has been reported previously (Sapède et al., 2012). This likely reflects the existence of sensory/neural bipotential progenitors unique to the saccular region (Sapède et al., 2012). Disrupting neurog1 presumably alters early cell fate choice to strictly favor sensory development, thereby expanding the pool of sensory progenitors. Thus, repression of neurog1 by pou3f3b both increases the number of saccular hair cells and blocks the inhibitory effects of neurog1 on hair cell differentiation. In contrast to neurog1, saccular expression of fgf3 serves to accelerate hair cell development but is not required for proper regulation of Otoferlin. Indeed, fgf3−/− mutants show reduced accumulation of saccular hair cells while Otoferlin expression remains normal; and misexpression of hs:fgf3 in pou3f3b−/− mutants restores proper accumulation of saccular hair cells but not Otoferlin expression. Otoferlin facilitates synaptic transmission from hair cells and is required for both hearing and vestibular function (Pangrsic et al., 2010; Chaterjee et al., 2015; Spaiardi et al., 2022). Future studies will be required to determine whether reduced saccular expression of Otoferlin impairs auditory function in pou3f3b−/− mutants. Similarly, it would also be instructive to determine whether Pou3f3 mutant mice exhibit reduced Otoferlin expression, which might account for their functional deficits (Kumar et al., 2016).

It is interesting to compare the fgf3 regulatory functions in the utricle vs. the saccule. Utricular expression of fgf3 appears to be induced by Fgf3 from the hindbrain (Kantarci et al., 2020) but does not require atoh1a/b (Millimaki et al., 2007). On the other hand, misexpression of atoh1a is sufficient to induce ectopic expression of fgf3 throughout the medial wall within a few hours (Sweet et al., 2011). This suggests that positive feedback between atoh1a and fgf3 gene expression helps maintain continuing expansion of the utricular macula. Loss of fgf3 does not detectably impair production of utricular hair cells, likely because overlapping expression of fgf8a and fgf10a provide extensive redundancy. In contrast, only fgf10a is expressed broadly in the saccular domain, fgf8a is expressed only weakly at the posterior end of the saccule, and fgf3 is not expressed until 30 hpf. The relatively low level of Fgf signaling likely accounts for the initially slow expansion of the saccular macula, and the pou3f3b−/− mutant phenotype further indicates that fgf3 is critical for boosting overall Fgf signaling level to increase the rate of saccular hair cell production. Because activation of fgf3 begins roughly 10 h after pou3f3b, it is likely that pou3f3b acts indirectly, or combinatorially with other genes, to regulate fgf3.

Neuroblast development. pou3f3b is also expressed in a previously undescribed population of otic non-neuronal delaminating (ONND) cells that delaminate from the neurogenic region and migrate in close association with nascent SAG neuroblasts (Fig. 7B). In pou3f3b−/− mutants, the number of ONND cells is strongly reduced at 24 hpf, and maturation of delaminated neuroblasts is also delayed. During this period, neuroblasts continue to delaminate but their developmental progression from neurog1 to neurod1 expression is much slower than normal, with a corresponding delay in accumulation of mature Isl1/2+ SAG neurons. This suggests that ONND cells normally provide a stimulatory signal that promotes early maturation of neuroblasts. Subsequently, the rate of neural development in mutant embryos returns to normal by 36 hpf. This is probably because the process of neuroblast delamination is nearly completed by 36 hpf, hence most neuroblasts have already made the transition to neurod1 expression. Despite restoration of the proper number of neurons, however, the distribution of mature neurons shows persistent gaps and irregularities (Fig. 5P–R). This is consistent with our previous studies showing that mutations that reduce the rate of neuroblast differentiation disturb the normal arrangement of mature neurons (Kantarci et al., 2015; Ali et al., 2025).

The role of ONND cells likely reflects the observation that multiple steps in neuronal development often require assistance from non-neuronal cell types. For example, Schwann cells and satellite glial cells (SGCs), derived from neural crest, are important for regulating late aspects of development and function of many diverse peripheral neurons. SGCs promote terminal differentiation, regeneration, and homeostasis in various peripheral ganglia, and they produce myelin for spiral ganglion neurons in the mammalian cochlea (Huang et al., 2013; Avraham et al., 2020; Tasdemir-Yilmaz et al., 2021; Mapps et al., 2022; LeBlang et al., 2025). Schwann cells also produce myelin and promote regeneration, in addition to providing axon guidance cues, promotion of axonal excitability, and neurotrophic support of spiral ganglion neurons (Hansen et al., 2001; He et al., 2013; Druckenbrod et al., 2020; Kantarci et al., 2024). By comparison, pou3f3b-expressing ONND cells are unique in that they are derived from the otic vesicle and promote the first steps in development of nascent neurog1-expressing SAG neuroblasts. This initial stage may be critical because neurog1 not only initiates neuronal development but also activates expression of Delta genes, which through Notch activity transcriptionally represses neurog1 (Ma et al., 1998). Thus, interactions with ONND cells may serve to enhance neurog1 activity to avoid prolonged delays in neuronal differentiation. A similar non-neuronal delaminating cell type reported in zebrafish expresses hmx3a, but hmx3a-expressing cells are distinct in that they migrate away from the otic vesicle to join the anterior lateral line ganglion and are required for normal proliferation and migration of the lateral line primordium (Adamska et al., 2000; Feng and Xu, 2010; England et al., 2020). It is not known whether hmx3a-expressing cells also promote transition from neurog1 to neurod1 expression in the lateral line ganglion. Whether ONDD cells exist in mammals is still unknown, but expression of Pou3f3 has also been reported in the developing cochleovestibular ganglion in embryonic mice (Huang et al., 1999), although it is not known whether this marks neurons or associated non-neuronal cells. Characterizing the properties of ONND cells and the nature of their interactions with SAG neuroblasts remain important goals of future research.

Funding

This work was supported by the National Institutes of Health - NIDCD grant R01-DC03806.

Footnotes

CRediT authorship contribution statement

Sydney E. Christensen: Writing – review & editing, Methodology, Investigation, Formal analysis. Maria Ali: Writing – review & editing, Methodology, Investigation, Formal analysis. Jorden N. Holland: Writing – review & editing, Methodology, Investigation, Formal analysis. Bruce B. Riley: Writing – original draft, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Conflict of interest

None.

Data availability

Data will be made available on request.

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