Single-cell RNA-sequencing reveals different subpopulations of hair cells in zebrafish
Hair cells play crucial roles in hearing and balance, and they are variable in morphology and function. To further distinguish the different types of hair cells from the molecular level, we used single-cell RNA-sequencing to analyze the gene expression pattern. Here, the transgenic zebrafish line Tg(Brn3c:mGFP) (Figure 1A) was used as the animal model, and the zebrafish larvae at 6 dpf were dissociated into the single cells by trypsin, and the GFP-positive cells were sorted using the fluorescence activated cell sorting (FACS) method (Figure S1). After high-throughput sequencing based on 10×Genomics system, the gene expression data was obtained. Based on Seurat analysis, the cells were divided into 21 clusters. By analyzing the gene expression pattern of the top genes in each cluster, the four clusters of cells, from the cluster 0, 5, 7, 12, were annotated as hair cells, and the cells from the cluster 2, 3 and 10 were identified as retinal ganglion cells (RGCs). Both of the hair cells and retinal ganglion cells are GFP-expressing cells in the Tg(Brn3c:mGFP) zebrafish. The rest were considered as other cells, which were GFP-negative and brought into the GFP-positive cells due to inevitable technical problems, including red blood cells, lymphocytes, muscle cells and so on (Figure 1B). The number of cells and marker genes detected in each cluster was shown (Figure 1C, D). For each cluster, the top marker genes were listed, and the expression pattern of these genes were also presented (Figure 1E).
The morphological charicteristics of the three types of hair cells in zebrafish
The typical feature of hair cells which differ from other cells is the cilia, including kinocilia and stereocilia, on the surface of the cell body. The cilia of hair cells can detect mechanical vibration by stereocilia bundle deflection, leading to the tension of the tip links, opening of the MET channels, and causing depolarization of hair cells. In zebrafish, except for inner ear, there are hair cells in their lateral line system, and the hair cells can be divided into three types according to their morphology and location, namely crista hair cells, macula hair cells, and neuromast hair cells (Figure 2A). The first two are both located in the inner ear, function importantly in hearing and balance, and the third ones are key component of the lateral line system, which are distributed on the surface of the skin, and help the fish to sense the environmental water. Although the cilia are the common structure of all hair cells, there are also differences among the three types of hair cells. The crista hair cells and macula hair cells have straight kinocilia, which seem inflexible, and the kinocilia of neuromast hair cells can bend themselves to detect the water movement (Figure 2B-D). For the length of kinocilia, taking the zebrafish larva of 3 dpf as an example, the crista hair cells have the longest kinocilia, which are almost 30 µm in length, and the kinocilia of macula hair cells and neuromast hair cells are approximately 10 µm and 20 µm, respectively (Figure 2E). For the size of cell bodies, the macula hair cells are the biggest ones, and the cell bodies of the neuromast hair cells are the smallest among them (Figure 2F).
Validation of the gene expression in the subtypes of hair cells using whole-mount in situ hybridization
Through our analysis, as shown above, the cells from cluster 0, 5, 7, 12 were considered as hair cells. In our subsequent study, we focused on these cells, and further analysis showed that these hair cells can be divided into three subpopulations, namely macula hair cells (cluster 5), crista hair cells (cluster 12) and neuromast hair cells (cluster 0, 7) (Figure 3A). We further analyzed the top marker genes expressed in the four clusters, and we found that some genes were mainly expressed in one of the clusters specifically, for example, tectb in cluster 5, zpld1a in cluster 12, calm1b in cluster 0 and 7 (Figure 3B). Functional enrichment analysis showed that many of the genes expressed in the four clusters of cells above have hair cell-related biological function (Figure 3C-F), which indicated that these cells were hair cells. To further confirm the hair cell clustering and annotation, we performed the whole-mount in situ hybridization (WISH). As shown in Figure 3G, the cells with high tectb gene expression were mainly gathering in the cluster 5, and the tectb gene was proved to be expressed specifically in the macula hair cells, including the utricular hair cells and saccular hair cells, by WISH. The zpld1a gene was mainly expressed in the crista hair cells, which were thought to be located in the cluster 12 according to our analysis (Figure 3H), and this is consistent with previous studies. The cells from the cluster 0 and 7 were considered as neuromast hair cells because of the expression patterns of their marker genes, such as calm1b (Figure 3I). As for cells from cluster 0 and 7, although they are both neuromast hair cells, they are different from each other. The cells from cluster 0 were classified to be mature neuromast hair cells, and the cluster 7 were classified to be young neuromast hair cells, according to the expression of the mature and young hair cell markers, s100s and prox1a, respectively in the two clusters (Figure S2).
On the other hand, we analyzed the marker gene of supporting cells, klf17, and found it was mainly expressed in the cells of cluster 1 (Figure S3), which suggests this cluster of cells are supporting cells. Likewise, we also analyzed the genes that were reported to be expressed in mantle cells, such as tnfsf10, ponzr6, pkhd1l1, fat1b, crb3b, cts12, ovgp1, cldne, and found that cells expressing these genes with high level were clustering in cluster 9 (Figure S4). For cluster 14, these cells are closer to the supporting cells (cluster 1) in UMAP clustering (Figure 1B), however, they express some of genes that have be proved to be expressed in hair cells specifically, such as myo6b, myo7aa(Figure S5), which raises a possibility that they are supporting cells that can differentiate into hair cells. Therefore, we concluded that the cells from cluster 1 and 9 were supporting cells and mantle cells, respectively, and the cells from the cluster 14 might be hair cell progenitors.
The molecular properties of the three types of hair cells
Knowing that different types of hair cells were clustering in different populations, we made a comparation among the distinguishable hair cells at the molecular level. As shown in Figure 1D, the number of marker genes detected in the cluster 0, 5, 7, 12 were 2352, 1023, 2029, 928, respectively. The two types of inner ear hair cells, macula hair cells and crista hair cells (cluster 5 and 12), share 568 genes, and 1677 marker genes were expressed in both of the mature and young neuromast hair cells (cluster 0 and 7). However, the neuromast hair cells and inner ear hair cells only share a small amount of marker genes, which reveals their differences on the gene expression level (Figure 4A). Furthermore, the top marker genes expressed in the different clusters were obviously different (Figure 4B). We also performed gene enrichment analysis through gene ontology (GO) term, and found that these different types of hair cells vary dramatically in biological process, molecular function and cellular component. For example, the neuromast hair cells have more energy metabolism-related activity, which raise the possibility that they require more energy to work compared to the inner ear hair cells (Figure 4C-E).
The neuromast hair cells were enriched with MET gene expression
As is known, the MET channels are required for functional hair cells and they are complex comprising of several components, such as TMC1, TMC2, TMIE and LHFPL5, CIB2[2, 28–32]. Hair cells with functional MET channels are crucial for normal hearing and balance. In addition, the tip links play important roles in the stereocilia deflection and MET channel-gating, and it was proved to be composed of two cadherin, CDH23 and PCDH15[33–35]. To determine whether these key molecules are also expressed in zebrafish hair cells and what difference there are among these different hair cells, we analyzed the expression pattern of the genes encoding these important proteins. As illustrated in Figure 5A-F, the orthologs of mammalian MET complex components were expressed in most of the neuromast hair cells, however, only a small proportion of inner ear hair cells can detect these gene expression. The genes encoding the tip link components, cdh23 and pcdh15a, were expressed in a few hair cells (Figure 5G-H).
The zebrafish hair cell scRNA-seq reveals potential hearing loss genes
Hearing is one of the most important sensory functions, and it relies on functional hair cells. Healthy hair cells are the receptors of acoustic signals, therefore, genes responsible for hair cell development, survival and function are also important for normal hearing. There are 119 genes that have been identified as non-syndromic hearing loss (NSHL) genes in humans (https://hereditaryhearingloss.org/). Among these genes, 96 human genes have orthologous genes in zebrafish (Figure 6A), which demonstrates that zebrafish are quite similar with human in terms of NSHL genes. Furthermore, 51 human NSHL genes have 57 orthologous genes which are expressed in zebrafish hair cells (cluster 0, 5, 7, and 12), and the detailed gene information were listed in Figure 6B. Except for the orthologs of identified human NSHL genes, there are still more than 3000 genes highly enriched in the zebrafish hair cells (Figure 6C), and some of these genes are thought to be crucial for hair cell function, and it is quite possible that they are potential hearing loss genes waiting to be confirmed in the future. In other words, this data may provide us clear direction for our scientific research in hearing loss gene identification. Under the guidance of this idea, we randomly selected some genes for further analysis, and found that these genes were specifically expressed in either or both of the zebrafish inner ear and neuromast hair cells (Figure 6D), indicating that these genes function in hair cell-related biological processes, and may even be essential for hearing function.
Functional analysis of the candidate genes involved in hair cell development
Our scRNA-seq analysis revealed genes which are specifically expressed in zebrafish hair cells, some of which are orthologs of human NSHL genes, however, there are many genes that have not been reported to play roles in hair cell development or function. To investigate the function of hair cell-enriched genes, we took the capgb and mb gene as examples, which were expressed in neuromast hair cells and macula hair cells, respectively. Here, we used morpholino-mediated gene knockdown to downregulate the gene expression. As illustrated in Figure 7A, B, the capgb-morphants exhibited decreased hair cells in their lateral line neuromasts compared to the littermate control. Furthermore, the capgb-morphants had less response to the acoustic stimuli in the startle response test (Figure 7C, D), which indicated their hair cells had lost their function to some extent. Likewise, in another experiment, the mb gene knockdown resulted in reduced macula hair cells (Figure 7E, F), and the mb-morphants showed severely abnormal balance ability in the vestibulo-ocular reflex (VOR) test (Figure 7G, H).