Contributions of mirror-image hair cell orientation to mouse otolith organ and zebrafish neuromast function: Part 1/2, Zebrafish data

Otolith organs in the inner ear and neuromasts in the fish lateral-line harbor two populations of hair cells oriented to detect stimuli in opposing directions. The underlying mechanism is highly conserved: the transcription factor EMX2 is regionally expressed in just one hair cell population and acts through the receptor GPR156 to reverse cell orientation relative to the other population. In mouse and zebrafish, loss of Emx2 results in sensory organs that harbor only one hair cell orientation and are not innervated properly. In zebrafish, Emx2 also confers hair cells with reduced mechanosensory properties. Here, we leverage mouse and zebrafish models lacking GPR156 to determine how detecting stimuli of opposing directions serves vestibular function, and whether GPR156 has other roles besides orienting hair cells. We find that otolith organs in Gpr156 mouse mutants have normal zonal organization and normal type I-II hair cell distribution and mechano-electrical transduction properties. In contrast, gpr156 zebrafish mutants lack the smaller mechanically-evoked signals that characterize Emx2-positive hair cells. Loss of GPR156 does not affect orientation-selectivity of afferents in mouse utricle or zebrafish neuromasts. Consistent with normal otolith organ anatomy and afferent selectivity, Gpr156 mutant mice do not show overt vestibular dysfunction. Instead, performance on two tests that engage otolith organs is significantly altered – swimming and off-vertical-axis rotation. We conclude that GPR156 relays hair cell orientation and transduction information downstream of EMX2, but not selectivity for direction-specific afferents. These results clarify how molecular mechanisms that confer bi-directionality to sensory organs contribute to function, from single hair cell physiology to animal behavior.

Methods
Zebrafish immunofluorescence and imaging (Figures 5, Figures 9-10)

Immunohistochemistry was performed on whole larvae at 5 dpf. Whole larvae were fixed with 4% paraformaldehyde in PBS at 4°C for 3.5 hr. For pre- and post-synaptic labeling all wash, block and antibody solutions were prepared with 0.1% Tween in PBS (PBST). For Emx2 labeling performed on sparse afferent labeling (see below) all wash, block and antibody solutions were prepared with PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). After fixation, larvae were washed 4 × 5 min in PBST or PBDTT. For synaptic labeling, larvae were permeabilized with Acetone. For this permeabilization larvae were washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at −20°C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 4 × 5 min in PBST. For all immunostains larvae were blocked overnight at 4°C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST or PBDTT). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST or PBDTT) overnight, nutating at 4°C. The next day, the larvae were washed for 4 × 5 min in PBST or PBDTT to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature, with minimal exposure to light. Secondary antibodies were washed out with PBST or PBDTT for 4 × 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. Primary antibodies used were:

Rabbit anti-Myo7a (Proteus 25-6790; 1:1000)

Mouse anti-Ribeye b (IgG2a) (Sheets et al., 2011)

Mouse anti-pan-MAGUK (IgG1) (Millipore MABN7; 1:500)

Mouse anti-Myo7a (DSHB 138-1; 1:500)

Rabbit anti-Emx2 (Trans Genic KO609; 1:250).

The following secondaries were used at 1:1000 for synaptic labeling: goat anti-rabbit Alexa 488, goat anti-mouse IgG2a Alexa 546, goat anti-mouse IgG1 Alexa 647, along with Alexa 488 Phalloidin (Thermofischer; #A-11008, #A-21133, #A-21240, #A12379). For Emx2 co-labeling the following secondaries were used at 1:1000: goat anti-mouse Alexa 488, and goat ant-rabbit Alexa 647, along with Alexa 488 Phalloidin (Thermofischer; #A12379, #A28175, #A27040).

Fixed samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with an Airyscan attachment (Carl Zeiss AG, Oberkochen, Germany) using an 63 × 1.4 NA oil objective lens. Airyscan z-stacks were acquired every 0.18 µm. The Airyscan Z-stacks were processed with Zen Black software v2.1 using 2D filter setting of 6.0. Experiments were imaged with the same acquisition settings to maintain consistency between comparisons. Processed imaged were further processed using Fiji.

 Zebrafish immunostain quantification (Figure 5, Figures 9-10)

Images were processed in ImageJ. Researcher was not blinded to genotype. Hair bundle orientation was scored relative to the midline of the muscle somites. HC number per neuromast were quantified based on Myo7a labeling and presence of a paired/complete synapse. For quantification of Emx2 labeling, HCs were scored as Emx2 positive if they labeled with both Emx2 and Myo7a. To quality as a ribbon or presynapse, the following minimum size filters were applied to images: Ribeye b: 0.025 μm2, MAGUK: 0.04 μm2. A complete synapse was comprised of both a Ribeye b and MAGUK puntca. An unpaired presynapse consisted of only a Ribeye b puntca, while an unpaired postsynapse consisted of only a MAGUK puncta. In each neuromast all HCs (~15 HC per neuromast) were examined for our quantifications.

Zebrafish functional calcium imaging in hair bundles (Figure 5)

GCaMP6s-based calcium imaging in zebrafish hair bundles has been previously described (Lukasz and Kindt, 2018). Briefly, individual 5-6 dpf larvae were first anesthetized with tricaine (0.03% Ethyl 3-aminobenzoate methanesulfonate salt, SigmaAldrich). To restrain larvae, they were then pinned to a Sylgard-filled recording chamber. To suppress the movement, alpha-bungarotoxin (125 μM, Tocris) was injected into the heart. Larvae were then rinsed and immersed in extracellular imaging solution (in mM: 140 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2 and 10 HEPES, pH 7.3, OSM 310 +/- 10) without tricaine. A fluid jet was used to mechanically stimulate the apical bundles of HCs of the A-P neuromasts of the primary posterior lateral-line. To stimulate the two orientations of HCs (A>P and P>A) a 500 ms ‘push’ was delivered. Larvae were rotated 180° to deliver a comparable ‘push’ stimulus to both the A>P and P>A HCs.

To image calcium-dependent mechanosensitive responses in apical hair bundles, a Bruker Swept-field confocal system was used. The Bruker Swept-field confocal system was equipped with a Rolera EM-C2 CCD camera (QImaging) and a Nikon CFI Fluor 60✕ 1.0 NA water immersion objective. Images were acquired in 5 planes along the Z-axis at 0.5 mm intervals (hair bundles) at a 50 Hz frame rate (10 Hz volume rate). The 5 plane Z-stacks were projected into one plane for image processing and quantification. The method to create spatial ∆F heatmaps has been described (Lukasz and Kindt, 2018). For GCaMP6s measurements, a circular ROI with a ~1.5 mm (hair bundles) diameter was placed on the center of each individual bundle. The mean intensity (∆F/F0) within each ROI was quantified. F0 represents the GCaMP6s intensity prior to stimulation. We examined the GCaMP6s signal in each hair bundle to determine its orientation. The GCaMP6s responses for each neuromast were averaged to quantify the magnitude of the A>P and P>A responses.

Zebrafish sparse labeling of single afferents in the lateral-line (Figure 9)

To visualize the innervation pattern of single afferent neurons, a neuroD1:tdTomato plasmid was injected into zebrafish embryos at the 1-cell stage. This plasmid consists of a 5kb minimal promoter, neurod1, that drives tdTomato expression in lateral-line afferents (Ji et al., 2018). This plasmid was pressure at a concentration of 30 ng/µl. At 3 dpf larvae were anesthetized with 0.03% ethyl 3-aminobenzoate methanesulfonate (Tricaine), to screen for tdTomato expression. Larvae were screened for mosaic expression of tdTomato expression in the lateral-line afferents using a Zeiss SteREO Discovery V20 microscope (Carl Zeiss) with an X-Cite 120 external fluorescent light source (EXFO Photonic Solutions Inc). After selecting larvae with tdTomato expression, larvae were prepared for immunostaining at 5 dpf, and imaged as outlined above.

内耳的耳石器官（otolith organs）与鱼类侧线的神经丘（neuromasts）均拥有两类朝向相反方向以检测刺激的毛细胞（hair cells）。其潜在分子机制高度保守：转录因子EMX2仅在一类毛细胞群体中区域特异性表达，并通过受体GPR156发挥作用，逆转该类毛细胞相对于另一类毛细胞的朝向。在小鼠与斑马鱼中，Emx2的缺失会导致感觉器官仅存在单一朝向的毛细胞，且无法完成正常的神经支配。在斑马鱼中，Emx2还会赋予毛细胞降低的机械感受特性。本研究利用缺失GPR156的小鼠和斑马鱼模型，探究朝向相反的刺激检测如何介导前庭功能，以及GPR156是否除调控毛细胞朝向外还具备其他功能。研究发现，Gpr156小鼠突变体的耳石器官具备正常的分区结构、I型-II型毛细胞分布与机械电转导特性。与之相反，gpr156斑马鱼突变体缺乏表征Emx2阳性毛细胞的小型机械诱发信号。GPR156的缺失不会影响小鼠椭圆囊（utricle）或斑马鱼神经丘中传入纤维的朝向选择性。与正常的耳石器官解剖结构及传入纤维选择性一致，Gpr156突变小鼠未表现出明显的前庭功能障碍。相反，两项涉及耳石器官的行为测试结果出现显著改变：游泳运动与垂直轴外旋转实验。综上，我们认为GPR156在EMX2下游传递毛细胞朝向与转导信息，但不参与对方向特异性传入纤维的选择性调控。本研究阐明了赋予感觉器官双向性的分子机制如何从单个毛细胞的生理特性层面延伸至动物行为层面发挥功能。

## 方法
### 斑马鱼免疫荧光染色与成像（图5、图9-10）
本研究对5日龄（5 dpf）的完整幼鱼开展免疫组织化学染色。将完整幼鱼置于4℃下用4%多聚甲醛的PBS溶液固定3.5小时。对于突触前与突触后标记，所有洗涤、封闭及抗体孵育液均采用含0.1%吐温的PBS（PBST）配制。而针对稀疏传入纤维标记的Emx2染色（详见下文），所有洗涤、封闭及抗体孵育液均采用含1% DMSO、0.5% Triton-X100及0.1% Tween-20的PBS（PBDTT）配制。固定完成后，幼鱼用PBST或PBDTT洗涤4次，每次5分钟。对于突触标记，幼鱼需经丙酮透化处理：先用去离子水洗涤5分钟，弃去后加入预冷的丙酮，置于-20℃孵育5分钟，随后再用去离子水洗涤5分钟，最后用PBST洗涤4次，每次5分钟。所有免疫染色实验中，幼鱼均在封闭液（含2%山羊血清、1%牛血清白蛋白、2%鱼皮明胶的PBST或PBDTT）中于4℃封闭过夜。之后将幼鱼置于抗体液（含1%牛血清白蛋白的PBST或PBDTT）配制的一抗中，于4℃摇床孵育过夜。次日，用PBST或PBDTT洗涤幼鱼4次，每次5分钟以去除未结合的一抗。加入抗体液配制的二抗后，将幼鱼置于室温避光孵育2小时。随后用PBST或PBDTT洗涤4次，每次5分钟以去除未结合的二抗。最终将幼鱼用ProLong Gold封片剂（赛默飞世尔科技）置于载玻片上，使用1.5号盖玻片封片。
本研究使用的一抗如下：
兔抗-Myo7a（Proteus 25-6790；1:1000）
小鼠抗-Ribeye b（IgG2a）（Sheets等，2011）
小鼠抗泛-MAGUK（IgG1）（默克MABN7；1:500）
小鼠抗-Myo7a（发育杂交研究银行DSHB 138-1；1:500）
兔抗-Emx2（Trans Genic KO609；1:250）。
突触标记实验中使用的二抗稀释比例均为1:1000：山羊抗兔Alexa 488、山羊抗小鼠IgG2a Alexa 546、山羊抗小鼠IgG1 Alexa 647，同时搭配Alexa 488鬼笔环肽（赛默飞；#A-11008、#A-21133、#A-21240、#A12379）。针对Emx2共标记实验，使用的二抗稀释比例同样为1:1000：山羊抗小鼠Alexa 488、山羊抗兔Alexa 647，同时搭配Alexa 488鬼笔环肽（赛默飞；#A12379、#A28175、#A27040）。
固定样本采用配备Airyscan模块的蔡司LSM 780倒置激光扫描共聚焦显微镜（卡尔蔡司集团，德国奥伯科亨）进行成像，使用63×1.4 NA油浸物镜。以0.18 μm的步距采集Airyscan Z轴堆叠图像序列。Airyscan Z轴堆叠图像使用Zen Black软件v2.1进行处理，采用6.0的二维滤波参数。所有实验采用统一的采集参数以保证组间比较的一致性。处理后的图像再通过Fiji软件进行后续加工。

### 斑马鱼免疫染色定量分析（图5、图9-10）
图像在ImageJ中进行处理。实验操作者未对基因型设盲。毛束朝向以肌节中线为参照进行评分。根据Myo7a标记信号及突触是否成对/完整，统计每个神经丘中的毛细胞数量。针对Emx2标记的定量分析，若毛细胞同时被Emx2和Myo7a标记，则判定为Emx2阳性。对于突触结构的判定，需满足以下最小尺寸阈值：Ribeye b：0.025 μm²，MAGUK：0.04 μm²。完整突触需同时包含Ribeye b和MAGUK阳性斑点；未成对的突触前结构仅包含Ribeye b阳性斑点，未成对的突触后结构仅包含MAGUK阳性斑点。本定量分析对每个神经丘内的所有毛细胞（每个神经丘约含15个毛细胞）进行统计。

### 斑马鱼毛束的功能性钙成像（图5）
基于GCaMP6s的斑马鱼毛束钙成像方法已有文献报道（Lukasz和Kindt，2018）。简要流程如下：首先将5~6日龄的幼鱼用三卡因（0.03% 乙基3-氨基苯甲酸甲酯磺酸盐，西格玛奥德里奇）麻醉。随后将幼鱼固定于填充Sylgard的记录槽中以限制其活动。为进一步抑制运动，向心脏注射α-银环蛇毒素（125 μM，Tocris）。之后用无三卡因的细胞外成像液（成分单位mM：140 NaCl、2 KCl、2 CaCl₂、1 MgCl₂、10 HEPES，pH 7.3，渗透压310 ±10）冲洗并浸泡幼鱼。使用流体喷射装置对初级后侧线神经丘的前后（A-P）方向毛细胞的顶端毛束施加机械刺激。为刺激两类朝向的毛细胞（A>P和P>A），施加500 ms的“推送”刺激。将幼鱼旋转180°，即可对A>P和P>A朝向的毛细胞施加等效的“推送”刺激。
为成像毛束顶端的钙依赖性机械敏感响应，本研究使用Bruker扫频共聚焦系统，该系统配备Rolera EM-C2 CCD相机（QImaging）及尼康CFI Fluor 60×1.0 NA水浸物镜。以0.5 μm的步距沿Z轴采集5个平面的图像，采集帧率为50 Hz单平面（体积采集帧率为10 Hz）。将5个平面的Z轴堆叠图像序列投影为单平面图像以用于后续处理和定量分析。生成空间ΔF热图的方法已有文献报道（Lukasz和Kindt，2018）。针对GCaMP6s信号定量，在每个毛束中心放置直径约1.5 μm的圆形感兴趣区域（ROI），量化该ROI内的平均荧光强度变化（ΔF/F₀），其中F₀代表刺激前的GCaMP6s荧光强度。本研究对每个毛束的GCaMP6s信号进行检测以确定其朝向，对每个神经丘的GCaMP6s响应取平均值以量化A>P和P>A方向响应的幅度。

### 斑马鱼侧线单传入纤维的稀疏标记（图9）
为可视化单个传入神经元的支配模式，将neuroD1:tdTomato质粒注射至1细胞期的斑马鱼胚胎中。该质粒包含5 kb的最小启动子neurod1，可在侧线传入纤维中驱动tdTomato的表达（Ji等，2018）。注射浓度为30 ng/µl。在3日龄时，用0.03%乙基3-氨基苯甲酸甲酯（三卡因）麻醉幼鱼，筛选tdTomato的表达情况。使用配备X-Cite 120外置荧光光源（EXFO Photonic Solutions Inc）的蔡司SteREO Discovery V20显微镜（卡尔蔡司），筛选侧线传入纤维中存在马赛克式tdTomato表达的幼鱼。筛选到带有tdTomato表达的幼鱼后，于5日龄时按照前述流程进行免疫染色并成像。