Introduction

The mammalian inner ear is comprised of six unique sensory organs that are responsible for our senses of hearing and balance. The cochlea contains the organ of Corti (OC), the hearing organ, which is comprised of mechanosensory hair cells (HCs) and their corresponding supporting cells (SCs). HCs transduce sound energy into electrical impulses via innervation by spiral ganglion neurons (SGNs), which then project into the hindbrain for further auditory processing. The early development of these three cell types has been extensively studied, but there are still gaps in knowledge regarding the transcriptional regulatory networks that control the spatial and temporal aspects of their development at later proneurosensory stages.

The inner ear is derived from the otic placode, which invaginates to form the otic cup before developing into the otocyst around embryonic day 8 (E8)1,2. While several transcription factors are important for neurosensory development in this time frame, the zinc-finger transcription factor Gata3 is particularly interesting due to its dynamic expression throughout inner ear development. While Gata3 is initially expressed as early as E8.5 throughout the otocyst, by E10.5 its expression is restricted to the proneurosensory regions3,4,5,6,7,8,9. Gata3 continues to be expressed in SGNs until postnatal day 14 (P14) and remains highly expressed in SCs, with lower levels of expression in HCs, throughout adulthood10,11,12,13,14. Therefore, it has been postulated that Gata3 plays an important and dynamic role in inner ear development and neurosensory cell formation during this temporal window.

Previous studies have shown that loss of Gata3 in the early proneurosensory region around E8.5 leads to loss of all cochlear neurosensory cells5,15, while loss of Gata3 1 day later around E9.5 leads to a patchy loss of HCs and SCs, and disorganization and patchy loss of SGNs6. Other studies have investigated the role of Gata3 postnatally in the maintenance of HCs and SCs11,12. These studies found that Gata3 is necessary later on to maintain outer hair cells (OHCs) and to functionally develop inner hair cells (IHCs), while loss of Gata3 from postnatal SCs results in an increase in some types of SCs through downregulation of other genes. However, there exists a gap in knowledge about the role of Gata3 later in embryonic development during the period where proneurosensory cells begin differentiating into HCs, SCs, and SGNs. Specifically, it remains to be seen how long Gata3 is required for proper embryonic development of neurosensory cells before switching to its postnatal maintenance role. Additionally, while we know the presence of Gata3 is necessary for proper neurosensory development, it is also critical that expression levels of Gata3 are precise for maintenance and function of neurosensory cells. For example, both Gata3 haploinsufficiency and Gata3 over-expression (as a result of gene duplication) cause human hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome16,17,18,19,20,21. While the triad of symptoms of HDR syndrome range in severity, nearly all patients exhibit deafness16,19,22,23. Uniquely, deafness is the only symptom of HDR syndrome which can present singularly16,19,21,23. This suggests that not only is continued expression of Gata3 required for proper inner ear development, but specific levels of Gata3 are also required. Continued investigation of the dose-dependent requirements of Gata3 will also contribute to the field’s overall understanding of inner ear gene regulatory networks.

In this study, we explored the window of developmental plasticity which is governed by Gata3 as a follow-up to previous studies showing that loss of Gata3 is detrimental to cochlear neurosensory epithelia5,6,7,10,21,24,25. Using the Sox2-creERT2 mouse line26, we conditionally deleted Gata3 from proneurosensory cells via tamoxifen injection at E11.5. Our results show that deletion of Gata3 causes severe loss and disorganization of HCs, SCs, and SGNs in a basal to apical gradient, with a more severe phenotype presenting in the apex. Interestingly, the mutant ears were morphologically normal in that they presented a full-length cochlea structure, unlike previous Gata3 deletion studies5,6. Overall, we show that while Gata3 is not necessary at E11.5 for overall morphological development and elongation of the cochlea structure, Gata3 is required in later proneurosensory development for proper neuronal and cochlear sensory epithelia cell formation.

Results

Gata3 is deleted from HCs, SCs, and SGNs at E11.5

Previous studies have characterized Sox2-creERT2 expression at the placode stage (E8.5), otocyst stage (E10.5), and the late otocyst stage (E12.5)27,28,29,30,31,32. At E10.5, Sox2 is present in both the nonsensory cochlear floor and roof31. At E11.5, Sox2 is expressed in the anteroventral region of the otocyst30. By E12.5, Sox2 is expressed in OC sensory cells and greater epithelial ridge (GER) cells29,31. In order to confirm knockout of Gata3 from HCs, SCs, and SGNs, in situ hybridization was performed using a Gata3 riboprobe. While the control showed high expression of Gata3 in all cell types from base to apex, the homozygous mutant displayed minimal expression in the HCs and SCs and greatly reduced expression in the SGN cell bodies (Fig. 1A–D′), demonstrating that our model reduces levels of Gata3 in the cell types of interest.

Figure 1
figure 1

Gata3 is conditionally deleted from HCs, SCs, and SGNs at E11.5 (A–D′) Whole mount in situ hybridization was performed with a Gata3 riboprobe on a Gata3 f/f control and a Sox2-creERT2: Gata3 f/f mutant and imaged at the cochlear base and apex. Gata3 expression appears in the HCs, SCs, and SGNs of the control and is absent in the HCs and SCs and decreased in the SGNs of the homozygote mutant. Scale bar: 100 µm.

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Gata3 is required for sustained formation and organization of HCs

Previous Gata3 conditional knockout (CKO) models show either no HC development or only patches of HCs5,6,7,10. We therefore wanted to assess the effect of deleting Gata3 at E11.5 on HC development. For this assessment, two different controls were used: Gata3 f/f (Fig. 2A–A″) and Sox2-creERT2 (Fig. 2B–B″). Other studies have demonstrated that the knock-in Sox2-creERT2 line shows IHC duplets, which was confirmed in our study (Fig. 2B–B″; white circles). It was important to investigate the IHC duplets in the Sox2-creERT2: Gata3 f/+ (heterozygous) mutant compared to the Sox2-creERT2 control to ensure that any resultant phenotype in the heterozygous mutant was not attributed to using this Cre line (Fig. 2C–C″). While the base, middle, and apex of the heterozygous mutant all contained IHC duplets similar to the Sox2-creERT2 control, it should be noted that the third row of OHCs was lost in the middle and into the apical region of the OC (Fig. 2C–C″). The heterozygous phenotype showed continuous formation of HCs from base to apex; however, conditional deletion of both alleles of Gata3 (homozygous mutants) resulted in disruptions in HC formation in the apical region, similar to the previous Gata3 CKO study that observed HC patches in the absence of Gata36. Sox2-creERT2: Gata3 f/f mutants also showed a worsening phenotype compared to the heterozygous mutants. In homozygous mutants, the base contained all three rows of OHCs, but a progressive loss of OHC rows occurs spatially along the OC (Fig. 2D–D′). Only two rows of OHCs were present in the middle region and almost no rows of OHCs were present in the apex (Fig. 2E–F″). Quantification of the total number of HCs in the basal, middle, and apical regions was performed between Gata3 f/f controls (base: 51.67 ± 2.31; middle: 55.67 ± 1.53; apex: 59.33 ± 4.73), Sox2-creERT2: Gata3 f/+, and Sox2-creERT2: Gata3 f/f ears, as indicated by mean ± standard deviation of three ears, in 100 µm sections, with significance set at P = 0.05 using a one-way ANOVA (Fig. 2G). In heterozygous mutants, loss of HCs was only statistically significant in the apex (35.00 ± 8.89, P = 0.0063); HC loss in the base and middle were not significantly different from control (47.33 ± 5.51, P = 0.4956; 42.00 ± 10.15, P = 0.0735, respectively). However, the homozygous mutant revealed a statistically significant loss of HCs from all three regions of the cochlea (base: 31. 67 ± 6.11, P = 0.0046; middle: 32.33 ± 4.73; P = 0.0083, apex: 18.67 ± 4.73, P = 0.0005, respectively).

Figure 2
figure 2

Deletion of Gata3 results in loss of HCs in a basal to apical gradient (A–F′) Representative images from the basal, middle, and apical regions of the cochlea for HCs indicated by MYOSIN7A+ staining. Two different controls were used, Gata3 f/f and Sox2-creERT2, in order to account for the haploinsufficent phenotype of the Cre line used. Both the heterozygous and homozygous mutant show IHC duplets (white circles) and missing rows of OHCs (white brackets), while the homozygous mutant also shows ectopic HCs in the GER. (G) Total hair cell quantification was performed in the base, middle, and apex in 100 µm sections using a One-way ANOVA with post hoc Dunnett’s test (P** ≤ 0.01; P*** ≤ 0.001) Scale bar: 50 µm.

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Homozygous mutants contained MYOSIN7A+ cells in the GER along the length of the cochlea, with the highest number appearing in the apex, similar to a postnatal Gata3 CKO from SCs using this same Cre line11. Ectopic HCs have previously been seen in the GER in both CKO and over-expressor models33,34,35,36,37,38. While ectopic HCs are generally not seen in combination with missing rows of OHCs, previous studies have shown that loss of Gata3 results in missing OHCs postnatally12,21. The phenotype of both ectopic HCs and missing rows of OHCs as a result of embryonic loss of Gata3 is unique and further supports a role for Gata3 in this specific temporal window in HCs.

Gata3 is required for corresponding SC formation and organization

Previous studies that have examined the effects of deleting Gata3 on inner ear development have shown either no SC development or disorganization and limited formation of SCs in variable phenotypic severity5,6,10. However, SCs were present in our model throughout the majority of the cochlear length, as indicated by SOX2+ SCs (Fig. 3). Similar to the HC phenotype in this model, heterozygous mutants showed continuous formation of SCs with some loss and disorganization of SCs in the apex (Fig. 3B–B″). As with HCs, homozygous mutants showed a worsening phenotype compared to heterozygous mutants (Fig. 3C–C″). The basal and middle regions contained disorganized SCs and complete loss of some outer SC rows in the middle region. The apex contained the most severe phenotype in which SCs appear to cluster together, similar to the SC phenotype seen in other Gata3 CKO studies10. Ultimately, the phenotype of HCs and SCs in homozygous mutants are consistent in their progressive loss from base to apex. While SC disorganization in our model is similar to the phenotype seen in another Gata3 CKO study, it is important to note that this previous study did not observe ectopic HCs in the GER10. Therefore, further experiments are needed in order to tease apart the specific requirement for Gata3 within proneurosensory development and differentiation to determine if Gata3 deletion in one cell population can influence another cell population.

Figure 3
figure 3

Deletion of Gata3 results in loss of SCs in a basal to apical gradient (A–C″) Representative images from the basal, middle, and apical regions of the cochlea showing SCs indicated by SOX2+ staining. The homozygous mutant shows a worsening disorganization of SCs from base to apex, with entire rows of SCs missing in the middle and apex. Scale bar: 25 µm.

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Gata3 is required for organization of SGN peripheral projections

Previous studies examining the effect of Gata3 deletion from the proneurosensory region of the developing otocyst noted a severe reduction in the number of SGNs present in CKO mutant samples and SGNs that did form had aberrant projection patterns towards the developing OC6,10. A study in which Gata3 deletion was restricted to SGNs resulted in present SGNs; however, peripheral projections were disorganized14,24. As the Sox2-creERT2 model results in Gata3 deletion from multiple cell types, including SGNs, we hypothesized that deletion of Gata3 from multiple cell types would result in an aberrant neuronal phenotype. We first examined peripheral projections in a Sox2-creERT2 mutant sample to determine whether the Cre knock-in displays a SGN phenotype. When compared with control samples (Fig. 4D–D″), there was no obvious difference in SGN number or organization (data not shown), suggesting that any potential phenotype in mutant samples would be attributed to deletion of Gata3. We then examined the peripheral projections in a heterozygous mutant. The gross organization of SGNs in the heterozygous mutant is disrupted when compared to control samples in the base and middle regions (Fig. 4E–E′). However, the radial bundles in the apex of the heterozygous mutant appear to have an increased area separating them relative to the control (4F”). The homozygous mutants had an even more striking phenotype, including a further increase in the distances between radial bundles relative to the control and progressively increased disorganization along the length of the cochlea (Fig. 4F–F″). The mutant base, middle, and apex (Fig. 4F–F″) reveal irregular distances between radial bundles, which were statistically significant (P < 0.0001; Fig. 4J), in addition to extra branches from radial bundles towards the OC. The method for radial bundle area quantification can be found as Supplementary Fig. S1 online. This phenotype was most profound in the apex (Fig. 4F″). Additionally, the areas between radial bundles in mutant samples were highly variable, further supporting that loss of Gata3 results in disorganization of peripheral projections of SGNs (Fig. 4J).

Figure 4
figure 4

Deletion of Gata3 results in fewer SGN processes in a worsening gradient from base to apex (A–C) Overview of SGN processes in the control, heterozygous mutant, and homozygous mutant apex labeled by NF200 staining. (D,D′,D″) Radial bundles of a control sample. (E,E′,E″) Radial bundles of a heterozygous mutant. Slightly increased space between the radial bundles is observed in all three regions. (F–F″) Radial bundles of a homozygous mutant. The distances between radial bundles are significantly increased relative to those of the control sample. The dotted white outline and white arrowheads in F’ indicate increased branching in the middle. The radial bundles in (F″) exhibit an even greater degree of branching as well as an increase and irregular distance between the fibers. (F″) (G,G′,G″) Peripheral projections of the control where they reach the OC are well organized in the control. (H) In the heterozygous mutant, peripheral neurites are present and relatively organized. (H′) Peripheral neurites in the middle of the heterozygous mutant have some peripheral projections that misturn towards the apex instead of the base. Additionally, there appear to be fewer neurites present than in the control. (H″) In the apex of the heterozygous mutant, there are fewer peripheral projections and those that are present show disorganization relative to the control sample. (I) In the base of the homozygous mutant, peripheral projections are present but are fewer in number and show an increased misturning of neurites towards the apex. (I′) The middle of the homozygous mutant has drastically fewer neurites reaching the OC relative to the control, particularly those neurites that should project to the OHC region. (I″) The apex of the homozygous mutant has some peripheral neurites approaching the IHC region of the OC but no peripheral neurites extending to the OHC region. The dotted white lines indicate where the OC should be. (J) Quantification of the distances between radial bundles in all three regions of control, heterozygous mutant, and homozygous mutant samples. The distance between radial bundles is greater in homozygote mutant samples than in controls and homozygous samples show greater variability in the distance between radial bundles as indicated by a TTEST (P**** ≤ 0.0001). Scale bar: 100 µm.

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We then examined the peripheral projections where the neurites reach the OC (Fig. 4G–I″). The basal region of the heterozygous mutant was comparable to the control (Fig. 4G,H), but peripheral projections were progressively fewer and became disorganized, with increasing severity from the middle to apical regions (Fig. 4G′–G″,H′–H″). Upon examination of homozygous mutants, peripheral projections in the base appeared slightly disorganized upon reaching the OC. Additionally, the density of neurites in the homozygous mutant appeared to be less when compared to the base of the control (Fig. 4I). The disorganization of the neurites and decreased density was even more pronounced in the middle and apical regions of the homozygous mutant (Fig. 4I′–I″). Fewer neurites projected into the OHC region of the OC in the middle and few-to-no neurites projected to the OHC region in the apex. In these regions, not all neurites that were present within the OHC region properly turned towards the base, but rather, turned towards the apex.

Based upon our results, Gata3 expression is important for the formation of radial bundles with regards to appropriate density and distance between bundles, as well as for proper branching patterns and overall organization. Additionally, Gata3 is needed for peripheral neurites to reach the OC, particularly into the OHC region. Importantly, the loss of Gata3 has a phenotype that progressively worsens along the length of the cochlea, with the most severe phenotype observed in the apex.

Gata3 is required for proper central pathfinding of SGNs

Given that homozygous mutants display aberrant peripheral projections of SGNs, with the phenotype progressively increasing in severity from base to apex (Fig. 4), we next investigated whether central projections of SGNs to the cochlear nucleus (CN) were also affected. Previous studies examining the role of Gata3 in SGN central pathfinding have shown varied results depending on the location and timing of Gata3 deletion6,24. Early deletion of Gata3 throughout the entire inner ear at E9.5 results in central SGN fibers bifurcating at several branch points with terminal fibers projecting non-specifically throughout the CN6. However, deletion of Gata3 exclusively from delaminated SGNs at E9.5 results in normal projection of SGNs to the CN with tonotopy maintained24. Taken together these two studies suggest that Gata3 may affect SGN central pathfinding in a cell non-autonomous and time-dependent manner. In order to investigate this further, lipophilic dyes were applied to the base (red) and apex (green) of Sox2-creERT2 control, heterozygous mutant, and homozygous mutant cochleas (Fig. 5A) to visualize the projections of SGNs into the CN. Sox2-creERT2 control SGNs entered the hindbrain and bifurcated, sending ascending and descending process towards the anteroventral cochlear nucleus (AVCN) and dorsal cochlear nucleus (DCN)/posteroventral cochlear nucleus (PVCN), respectively (Fig. 5B). Sox2-creERT2 control SGNs remained segregated, with basal fibers extending more dorsally and apical fibers more ventrally (Fig. 5B). This stereotyped central wiring was also maintained in heterozygous samples (Fig. 5C). In contrast, SGNs in homozygous mice displayed less segregation between apical and basal fibers. Apical fibers often projected more dorsally into spaces occupied by basal fibers. Additionally, some apical fibers, upon reaching the hindbrain, projected outside of cranial nerve VIII into areas outside of the CN (Fig. 5D). These results provide further evidence that Gata3 plays an important role in the development and wiring of SGNs centrally. Our data, along with previous studies6,24, suggest that Gata3 is acting in a cell non-autonomous manner at or before E11.5 to promote proper central wiring of SGNs. Further investigations are needed to elucidate what cell populations require early Gata3 expression in order to promote proper central pathfinding of SGNs.

Figure 5
figure 5

SGN central pathfinding is altered by Gata3 deletion (A) Schematic view of lipophilic dye placement and visualization of SGNs in the CN. (B–D) Lipophilic dye was applied to the base (red) and apex (green) of control and mutant cochleas at E18.5 and their central projections were analyzed. (B) In the Sox2-creERT2 control, SGNs bifurcate and send processes towards the AVCN and DCN/PVCN. Basal and apical SGN fibers also remain segregated throughout the CN (C) Heterozygous SGNs bifurcate and maintain tonotopic segregation similar to controls. (D) Homozygous mutants have aberrant SGN central projections with apical fibers projecting more dorsally and sometimes projecting outside the CN. Additionally, some SGN neurites project outside cranial nerve VIII before reaching the CN.

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Gata3 deletion at E11.5 results in full morphologic development of the cochlear duct and vestibular system, but shows progressive neurosensory epithelial loss and disorganization

Previous Gata3 deletion studies have shown a variety of phenotypes that include cochlear structure and cochlear neurosensory epithelia cell defects5,6,7,10,21,24,25. Gata3 null mice display a severely truncated cochlea and vestibular system devoid of sensory epithelia except for a small patch of HCs and SGNs in a portion of the saccule5,7. Gata3 deletion at E8.5 using the Foxg1-cre mouse line resulted in a truncated cochlea, which contained no HCs and abnormal morphologic development of the vestibular system6. Gata3 deletion at E9.5 using the Pax2-cre mouse line resulted in similar morphologic defects, including a truncated cochlea and abnormal vestibular system. However, unlike deletion at E8.5, deletion at E9.5 resulted in patchy sensory cell development and a limited population of SGNs6,10. In studies where Gata3 has been conditionally deleted from only SGNs, HCs and SCs form properly24,25. We contribute results for Gata3 deletion at E11.5, a time in development in which proneurosensory cell differentiation is occurring. Our findings show that Gata3 deletion at E11.5 results in a morphologically sound structure with a full-length cochlea and well-developed vestibular system (data not shown). Within the homozygous mutant, the sensory cells in the OC are mostly present and have a varying phenotype depending on the cochlear region. In the homozygous mutant cochlear base, HCs and SCs are present with only mild disorganization (Fig. 6), while the homozygous mutant basal radial bundles have larger spacing than normal but the neurons are relatively organized. This contrasts the phenotype seen in the apex where the peripheral projection density of the mutant apex is decreased and those projections which are present appear disorganized (Fig. 6). Additionally, the tonotopy of SGN central projections is largely maintained within the CN in both heterozygous and homozygous mutants (Fig. 5). In comparison, the mutant apical HCs are severely reduced to patchy clusters with some ectopic HCs that appear in the GER, while the apical SCs are not organized in rows and instead cluster together (Fig. 6). Our data demonstrates a role for Gata3 in all neurosensory cells after their initial specification.

Figure 6
figure 6

Loss of Gata3 is depicted by the timeline with TMX injection at E11.5 and collection of tissue at E18.5. A Gata3 mutant ear at E18.5, indicates the cochlea (red), vestibular system (green), and endolymphatic duct (purple). Images are representative of the phenotype observed along the length of the cochlea and are taken from Figs. 2, 3, and 4. Our study deleted Gata3 at E11.5, later than previous studies, and found that the overall morphology of the inner ear is intact, unlike in the previous studies. In addition, there was a loss of sensory cells with increasing severity from base to apex, indicating that Gata3 is still required for their formation and organization.

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Discussion

Gata3 was previously shown to be necessary for both proper cochlear structure and cochlear neurosensory epithelia cell formation early in inner ear development when its expression is high throughout the entire otocyst5,6,7,10,21,39. However, the role of Gata3 in HC, SC, and SGN formation after its restriction to the proneurosensory region was unknown. Our study reveals novel findings that Gata3 plays both a necessary and dose-dependent role in the formation and organization of neurosensory cell types but does not have an impact on the overall morphology of the inner ear at this specific developmental time point.

This project contributes new knowledge about the role of Gata3 in proneurosensory epithelia cell formation in a temporal window that fills a gap between previous studies investigating Gata3 deletion. Our results demonstrate that deletion of Gata3 from the proneurosensory domain at E11.5 results in a fully-formed cochlear duct (data not shown), regardless of the single or dual loss of Gata3 alleles. Therefore, Gata3 is not required for morphologic development at or after E11.5. Given that previous Gata3 deletion studies did not see normal morphology of the cochlea5,6, it is intriguing that deletion of Gata3 approximately 2 days later than previous studies results in a morphologically sound inner ear with a fully formed cochlea structure. We hypothesize that the resulting phenotype from this deletion of Gata3 prior to neurosensory cell differentiation may be the result of abnormal differentiation that occurs as a result of the loss of a key transcription factor, rather than cell cycle exit.

Previous lineage tracing analysis using the Sox2-creERT2 line has shown that tamoxifen administration at E11.5 results in nearly complete labeling of neurosensory cells in the apex, compared to sparser labeling in the base29. Consistent with this, our model appears to show increased recombination efficiency of Gata3 in the apex when compared to the base (Fig. 1). Despite this, homozygous mutant cochleas still displays near-complete deletion of Gata3 in the base when compared to controls (Fig. 1). Deletion of Gata3 at E11.5 using the Sox2-creERT2 line results in formation of HCs, SCs and SGNs. However, they are highly disorganized and this phenotype increases in severity from base to apex. In Gata3 heterozygous null mice, OHC loss occurs in the absence of Gata312,21,40. This phenotype is mirrored in our study, despite the difference in timing at which Gata3 is deleted. It is also noteworthy that the heterozygous mutant had a subtler phenotype compared to the homozygous mutant, suggesting that precise levels of Gata3 are needed for proper formation and organization of the proneurosensory epithelia. If precise levels of Gata3 are truly necessary, then increased levels of Gata3 should also have a phenotype in our model. Several other over-expressor studies have been published that demonstrate ectopic HCs in the GER33,34,35,36,38. Previous studies have even used the Gata3 over-expressor model in combination with upregulation of other sensory genes in order to increase the efficiency of ectopic HC formation35,36. Investigating the over-expression of Gata3 using this Sox2-creERT2 mouse line would be useful in determining any detrimental effects of elevated levels of Gata3 in the cochlea. While this would further elucidate the specific role of Gata3 in the cochlea, the investigation of Gata3 over-expression is especially pertinent since extra alleles of Gata3 have also been known to cause HDR syndrome20.

Finally, it should also be noted that Gata3 was deleted from three different cell types: HCs, SCs, and SGNs. While these cell types work together, it is unclear if loss of Gata3 in just one of the cell types is enhancing the overall phenotype we see in our model. Despite that HCs in our model appear to be innervated by SGNs throughout the entire OC, we are unable to determine if the HC disorganization is causing the improper SGN peripheral projections when using the Sox2-creERT2 model, or vice versa. Likewise, since the SCs and HCs are connected via tight junctions in the OC, our model is unable to determine if a phenotype in one of these cell types is exacerbating the overall phenotype. Therefore, in order to tease apart the role of Gata3 during proneurosensory development, future studies could use cell-specific Cre lines to delete Gata3. Comparison of the phenotype in our model to Gata3 CKO in HC-specific, SC-specific, or SGN-specific lines could help elucidate the exact role of Gata3 in this stage of proneurosensory development.

In conclusion, our work demonstrates that Gata3 is essential for proper cochlear neurosensory epithelia cell development and organization in the late proneurosensory stage at E11.5. Because our study demonstrates a phenotype in the heterozygous mutant in addition to a more severe phenotype in the homozygous mutant, we propose that correct levels of Gata3 are also required for proper development. Furthermore, our study performs the latest embryonic Gata3 deletion from the inner ear yet. We contribute to the understanding of neurosensory development that Gata3 is required for proper formation and organization of cochlear afferent neurons and sensory epithelia at E11.5, but not for overall cochlear morphology.

Methods

Mouse model and genotyping

All animal care and procedures were approved by Western Michigan University Institutional Animal Care and Use Committee (IACUC) following the guidelines for use of laboratory animals (IACUC #20-11-01). All experiments were carried out in accordance with the ARRIVE guidelines, and all methods were carried out in compliance with all relevant regulations. The Sox2-creERT2 (Jackson Labs)26 and Gata3 Flox (provided by Dr. Maxime Bouchard41) mouse strains were used in this study. Sox2-creERT2 males were bred with Gata3 f/f females to produce males that were Sox2-creERT2: Gata3 f/+, who were viable. Sox2-creERT2: Gata3 f/f mice were produced by breeding Sox2-creERT2: Gata3 f/+ or Sox2-creERT2: Gata3 f/f males with Gata3 f/f or Gata3 f/+ females. Genotyping was performed using the following primers: Cre 5′ CCT GTT TTG CAC GTT CAC CG 3′ and 5′ ATG CTT CTG TCC GTT TGC CG 3′ yield a 280 base pair (bp) mutant, IL2 5′ CTA GGC CAC AGA ATT GAA AGA TCT 3′ and 5′ GTA GGT GGA AAT TCT AGC ATC ATC C 3′ yield a 324 bp control band, and Gata3 5′ GAT TCA GTC TCC CTC CTT CTT C 3′ yield a 430 bp mutant band and 5′ GTT CAC ACA CTC CCT GCC TTC TG 3′ yield a 400 bp control band. Breedings were performed with E0.5 specified as noon on the day of vaginal plug. Pregnant females received an intraperitoneal injection of 3 mg/40 g tamoxifen and 2 mg/40 g progesterone at E11.5 between 9 and 11 a.m.30. On the day of collection, the pregnant female was given a lethal intraperitoneal injection of Avertin (500 mg/kg 2.2.2-tribromoethanol). Embryos were dissected from the uterus, perfused with 4% paraformaldehyde (PFA) and stored at 4 °C. All images are representative of at least three biological replicates.

Whole-mount immunohistochemistry

Whole mount immunohistochemistry was performed on previously fixed tissue42. Ears were washed in phosphate buffered saline (PBS), then washed five times five minutes in PBS/0.05% Tween20 followed by blocking for one hour in 5% normal donkey serum, 1% bovine serum albumin, and 0.5% TritonX-100 in PBS. The tissue was incubated in primary antibodies, diluted in blocking buffer, at 4 °C for three nights. The following primary antibodies were used: MYO6 Rabbit (Sigma; 1:1000), MYOSIN7A Mouse (DSHB; 1:200), MYSOIN7A Rabbit (Proteus Biosciences, Inc.; 1:500), Neurofilament 200 (NF200) HC Chicken (Aves; 1:200), and SOX2 Rabbit (Sigma; 1:500). Next, the tissue was washed four times thirty minutes, followed by overnight incubation at 4 °C in secondary antibody in blocking buffer. Secondary antibodies were conjugated to Alexa flour anti-Mouse 488, anti-Rabbit 488, anti-Chicken 555, anti-Goat 647, or anti-Rabbit 647 (Life Tech; 1:1000). Nuclei were labeled using Hoescht Dye (1:2000), received as a gift from Bernd Fritzsch. Images were taken on either a Nikon C2 confocal microscope or a Leica Stellaris 5 confocal microscope and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017).

Total hair cell quantification

For total hair cell quantification, shown in Fig. 2, whole-mount immunohistochemistry samples that were stained with MYO6/MYOSIN7A/MYOSINVIIA were quantified in the base, middle, and apical regions in 100 µm sections using FIJI imaging software (Version 1.8.0_172). Hair cell counts were recorded in GraphPad Prism (Version 8.0.0) and a One-Way ANOVA and post hoc Dunnett’s test were performed with significance set at P < 0.05. All counts are representative of three biological replicates.

Spiral ganglion neuron peripheral process quantification

For radial bundle quantification, shown in Fig. 4 and Supplementary Fig. S1 online, cochleas were imaged at the same magnification in the base, middle, and apex for control and homozygote samples. Using FIJI imaging software (Version 1.8.0_66), eight spaces between radial bundles in each region were outlined and measured. All area results were recorded in GraphPad Prism (Version 9.1.2) and a TTEST analysis was performed. Data point plot graphs were constructed, and significance was set at P < 0.05.

In situ hybridization

Gata3 mRNA labeling was achieved using a previously described in situ hybridization protocol42. Mice were fixed in 4% PFA and inner ears were dissected in 0.4% PFA. Control ears and experimental ears were run together throughout the experiment to ensure both ears received the same experimental conditions. Ears were dehydrated overnight in 100% methanol and rehydrated through a graded methanol series. Ears were digested with Proteinase K in PBS (Ambion, Austin, TX, USA). Samples were hybridized overnight at 60 °C to the Gata3 riboprobe in hybridization solution consisting of 50% (v/v) formamide, 50% 2X saline sodium citrate (SSC), and 6% (w/v) dextran sulphate. Unbound probe was removed by performing washes with 2X SSC. Samples were then incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany) overnight at room temperature. Ears were extensively washed with 1X washing buffer throughout the day, then left overnight in 1X washing buffer at room temperature. Samples were then incubated at room temperature in detection buffer (Roche) before being thoroughly saturated with nitroblue phosphate/5-bromo, 4-chloro, 3-indolil phosphate (BM purple substrate, Roche). Control and mutant samples were developed in BM purple for the same length of time. Ears were mounted in glycerol on a slide and imaged with a Nikon Eclipse E600 microscope and Canon EOS Rebel T7i camera. Images were edited in CorelDraw (version 19.0; 2017).

Lipophilic dye tracing

Neuronal tracing of spiral ganglion neurons was conducted as previously described42. Briefly, the lateral half of the inner ear was exposed, and pieces of lipophilic dye-soaked paper was inserted into the base (NeuroVue® Red) and apex (NeuroVue® Maroon) of the cochlea. Heads were then placed into glass vials filled with 4% PFA and incubated at 37 °C for 3 days to allow for proper dye diffusion. Following incubation, the brains were removed, and the brain stem was flat mounted with the lateral side facing up in glycerol on a slide and imaged within 1 h of dissection. All imaging was performed using a Leica Stellaris 5 confocal microscope with LAS X software and images were compiled in ImageJ and edited in CorelPhoto Paint (Version 19.0; 2017).