A single-cell transcriptomic atlas of inner ear morphogenesis in zebrafish

The inner ear constitutes different cell types next to one another: the sensory patches whose hair cells synapse with neurons, the thin channels of three semicircular canals whose perpendicular organization enables detection of directional head rotation, and the endolymphatic duct and sac whose conditional epithelial barrier relieves excess pressure and promotes fluid pressure homeostasis. How the ear’s component cell states are established during development has remained unknown. We use single-cell RNA sequencing to distinguish cell states within the developing ear with wild-type zebrafish embryos and lmx1bb mutants that exhibit defects in canal and sac morphogenesis. We identify the earliest marker for the semicircular canal-genesis zone (ccn1l1), unexpected genes in the endolymphatic sac that suggest a role for tissue contraction in its function (smtnb), parallel gene sets for sensory patches in the neuromast and ear, and a conserved role for cell-cycle pausing (cdkn1bb expression in the canals and sac as previously observed in the developing mouse ear). This atlas provides the most comprehensive transcriptional profiling of the developing inner ear, identifying new molecular leads to understand ear morphogenesis. Blocks of tissue enriched for the inner ear were dissected and collected from wild-type zebrafish at 40, 48, 60, and 72 hours post fertilization (hpf) were collected, and from lmx1bb mutant zebrafish at 60 and 7 hpf. Single-cells were captured and transcriptome libraries were prepared by the single-cell core at Harvard Medical School with established protocols (Klein et al. 2015; Wagner et al. 2018). Libraries were pooled in a manner to evenly distribute samples and prevent overlapping of barcodes. Assembled libraries were then sequenced with paired end reads using 21 flow cells of 75 cycles on an Illumina NextSeq 500. As described previously, cDNA reads were mapped using the GRCz11 zebrafish genome assembly (Wagner et al. 2018). We provide the raw data (.fastq files) and the entire pooled and filtered data after removing doublets as EarAtlas_FullProcessed.h5ad. Information about pooling libraries and library indices is provided in Swinburne_EarAtlas_PoolingSamples.xlsx.