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)27–32. At E10.5, Sox2 is present in both the nonsensory cochlear floor and roof31. By E12.5, Sox2 is exclusively expressed in OC sensory cells31. In order to confirm complete knockout of Gata3 from HCs, SCs, and SGNs, in situ hybridization was performed using a Gata3 riboprobe. While the control shows high expression of Gata3 in all cell types from base to apex, the homozygous mutant shows no expression in the HCs and SCs and greatly reduced expression in the SGN cell bodies (Fig. 1A-D’), demonstrating that our model is indeed reducing levels of Gata3 in the cell types of interest.
Gata3 is required for sustained formation and organization of HCs
We first analyzed the effect of deletion of Gata3 on HCs, as previous Gata3 CKOs show either no HC development or only patches of HCs5–7, 10. In order to assess the phenotype of the deletion of Gata3, two different controls were used: Gata3 f/f (Fig. 2A-A”) and Sox2-creERT2 (Fig. 2B-B”). Other studies have previously demonstrated that the knock-in Sox2-creERT2 line shows inner hair cell (IHC) duplets, which was confirmed in our study (Fig. 2B-B’’; white circles). It was important to investigate the IHC duplets in the heterozygous mutant compared to the Sox2-creERT2 control to ensure that the phenotype seen is not the result of using this Cre line (Fig. 2C-C”). While the base, middle and apex of the heterozygous mutant all contain IHC duplets similar to the Sox2-creERT2 control, it should be noted that the third row of outer hair cells (OHCs) is lost in the middle region of the OC into the apical region (Fig. 2C-C”). We found that the heterozygous genotype shows a continuous formation of HCs from base to apex, while the homozygous mutant shows some disturbances in the apical region, similar to the previous Gata3 CKO study that showed the presence of patches6. The homozygous mutant also shows a worsening phenotype compared to the heterozygous mutant. While the base contains all three rows of OHCs, progressive rows of OHCs are lost (Fig. 2D-D’). Just two rows of OHCs are present in the middle region and almost no rows of OHCs are present in the apex (Fig. 2E-F”). Additionally, the entire cochlea contains Myosin VIIa positive cells in the GER with the highest number appearing in the apex, which is similar to a postnatal Gata3 CKO from SCs using this same Cre line11. Furthermore, these cells in the GER are associated with SGN endings. Ectopic HCs have been seen in the GER in both CKO and over-expressor models previously33–38. While ectopic HCs generally are 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 this specific cell type.
Gata3 is required for corresponding SC formation and organization
Previous studies that have deleted Gata3 have shown either no SC development or only limited patches of SC formation localized to the HC patches5,6,10. However, we still observe SCs in our model throughout the majority of the length of the cochlea. Similar to the HC phenotype in this model, we found that the homozygous mutant shows almost continuous formation of SCs, except for some patches in the apex (Fig. 3). The apex also shows disorganization of the SC rows. The homozygous mutant shows a worsening phenotype compared to the heterozygous mutant, similar to that seen in the HCs. The base and middle show disorganized SCs and complete loss of some outer SC rows in the middle region. The apex contains the most severe phenotype in which SC appear to cluster together which is very similar to the SC phenotype seen in other Gata3 CKO studies10. Ultimately, the phenotype in the HCs and SCs are consistent in their appearance of progressive loss of OHCs from base to apex, mirroring the loss of SCs from base to apex in the homozygous mutant. While this SC disorganization in our model is also similar to the phenotype seen in other Gata3 CKO studies, it is important to note that the previous study did not also observe ectopic HCs in the GER10. Further studies are needed in order to tease apart the specific requirement for Gata3 within this specific time window to determine if Gata3 deletion in one cell population can influence another cell population.
Gata3 Is Required For Organization Of Sgn Peripheral Projections
Our observation of Myosin VIIa positive cells associated with SGN endings in the homozygous mutant led us to investigate the peripheral projections of SGNs to confirm that they were developing properly. Previous studies examining the effect of Gata3 deletion from the proneurosensory region of the developing otocyst observed a severe reduction in the number of SGNs present in mutant samples, while the SGNs that did form displayed aberrant projection patterns towards the developing OC6,10. Another study in which Gata3 deletion was restricted to SGNs saw proper formation of SGNs with disorganized peripheral projections14,24. We first examined peripheral projections in a Sox2-creERT2 mutant sample to establish whether the Cre knock-in displays a SGN phenotype. When compared with control samples (Fig. 4D-D”), we saw no obvious difference in SGN number or organization (data not shown). We then looked at the peripheral projections in a heterozygous mutant. The number and overall organization of SGNs in the heterozygous mutant largely resemble control samples in the base and middle (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 (F”). The homozygous mutant has a striking phenotype that displays an increase in the distance between radial bundles as well aberrant projections of the radial bundles which becomes progressively more disorganized along the length of the cochlea (Fig. 4F-F”). The mutant base and middle (Fig. 4F-F’) reveal irregular distances between radial bundles in addition to extra branches from radial bundles. There are also inconsistencies in their organization, as some gaps are large and others are reduced (Fig. 4C). This phenotype is even more profound in the apex (Fig. 4F”).
The area between the radial bundles in the apex of control and homozygous mutant samples was measured and quantified (Fig. 4J). The method for radial bundle distance quantification can be found as Supplementary Fig. S1 online. Analysis of the data showed a statistically significant increase in the distance between radial bundles in the homozygous mutant relative to the control (p < 0.0001). Additionally, the values for the area in mutant samples was highly variable, further supporting that loss of Gata3 results in disorganization of peripheral projections of SGNs.
We also examined the peripheral projections where the neurites reach the OC (Fig. 4G-I”). The basal region of the heterozygous mutant is comparable to the control (Fig. 4G-H), but peripheral projections are progressively fewer and become disorganized in the heterozygous mutant with progression to the middle and apex (Fig. 4G’-G”, H’-H”). Peripheral projections in the base of the homozygous mutant appear slightly disorganized upon reaching the OC. Additionally, the density of neurites in the homozygous mutant appears to be less when compared to the base of the control (Fig. 4I). The disorganization of the neurites and decreased density is even more pronounced in the middle and apex of the homozygous mutant (Fig. 4I’-I”). Fewer neurites project into the OHC region of the OC in the middle and few-to-no neurites project to the OHC region in the apex. In these regions, not all neurites that are present within the OHC region properly turn towards the base but rather, turn 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 the OHC region, and to form proper connections with HCs. Importantly, the loss of Gata3 has a phenotype that progressively worsens along the length of the cochlea, with the greatest 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 getting more severe in a basal to apical manner (Fig. 4), we next investigated whether central projection of SGNs to the cochlear nucleus (CN) was also affected. Previous studies examining the role of Gata3 in spiral ganglion neuron 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 within delaminated SGNs at E9.5 results in normal projection of SGNs within the CN with tonotopy maintained24. Taken together these two studies suggest that Gata3 may be affecting SGN neuron 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, as well as heterozygous and homozygous mutant cochlea (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 (Fig. 5C) mice. In contrast SGNs in homozygous mice display less segregation between apical and basal fibers. Apical fibers often project more dorsally into spaces occupied by basal fibers. Additionally, some apical fibers upon reaching the hindbrain project outside of cranial nerve VIII into areas outside of the CN (Fig. 5D). These results provide further evidence for the idea that Gata3 expression 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.
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 morphologic and cochlear neurosensory epithelia defects5–7, 10,21,24,25. Gata3 null mice display a severely truncated cochlear and vestibular system which were 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, deletion at E9.5 resulted in patchy sensory cell development of HCs, SCs, and SGNs6,10. In studies that have conditionally deleted Gata3 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 cochlea, 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 well organized. This contrasts the phenotype seen in the apex since 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 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.