A recent study published on eLife, focusing on deep and bulk transcriptomic analysis of spiral ganglion neurons (SGNs) in mouse inner ear, is performed by researchers in Dr. LIU Zhiyong’s Lab at the Institute of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. Therefore, how to maintain functions of SGN and regenerate them after the damage is one important but tough question in the hearing field.
To provide the mechanistic foundations for future SGN regeneration in the clinic, colleagues from Dr. LIU’s group aimed to address two questions below: 1) Do SGNs express specific genes from early embryonic ages to adult ages? 2) If so, are those specific genes constantly or dynamically expressed? To precisely address these two questions, Dr. LIU’s group used compound genetically modified mice in which the SGNs are specifically labeled with a red fluorescent protein, tdtomato. Following cochlear tissue digestion and manual picking/washing, SGNs were picked from animals at five different ages: E15.5, P1, P8, P14, and P30. Three replicates were included at each age, and ~100 SGNs were picked per replicate. This new strategy was able to obtain transcriptomic profiles 5 times as deep as the previous single-cell RNA-Seq of SGNs. Due to its much deeper coverage, previously unknown SGN specific genes were identified.
Next, Dr. LIU’s group chose two SGN specific genes, Scrt2 and Celf4,and generate two mouse lines: Scrt2-P2A-tdtomato/+ and Celf4-3xHA-P2A-iCreER-T2A-EGFP/+, followed by detailed characterization in the cochlea from E10.5 to adult ages. At single-cell resolution, the transgenic lines also showed that Scrt2 and Celf4 were specifically and highly expressed in SGNs, further validating the quality of the transcroptome database. Those new mouse strains were powerful tools for the inner ear and general neuroscience field, as Scrt2 and Celf4 are expressed in the brain as well.
This work entitled “Comprehensive transcriptome analysis of cochlear spiral ganglion neurons at multiple ages” was published in elife on Jan. 23, 2020. This work was carried out by LI Chao, LI Xiang, BI Zhenghong, Ken Sugino, WANG Guangqin, ZHU Tong, under the supervision of Dr. LIU Zhiyong. It was supported by the grants from National Key R&D Program of China , Strategic Priority Research Program of Chinese Academy of Science , National Natural Science Foundation of China , Shanghai Municipal Science and Technology Major Project , Innovative Research Team of High-Level Local Universities in Shanghai, and the Faculty Research Award from Boehringer Ingelheim (BI) International GmbH . We thank Dr. HU Qian and the Optical Imaging Facility from the Institute of Neuroscience for support with the image analysis. Dr. YANG Hui (Principal Investigator from the Institute of Neuroscience) for sharing the zygote microinjection system to generate the knockin mice, Ms. YING Wenqin (from the laboratory of Dr. YANG Hui at the Institute of Neuroscience) for helping us in transplanting zygotes into pseudopregnant female mice.
(A) Illustration of the experimental procedures. Red SGNs are manually picked under a fluorescence microscope, washed three times, and placed in lysis buffer. (B) 21 SGN-specific and constant genes are identified, which are expressed in SGNs at all 5 ages but not expressed in hair cells (HCs) or glial cells. (C-D) Scrt2 is expressed in SGNs but not HCs or Glia at P1 from Scrt2-P2A-tdTomato/+ mice. Double-labeling for HC marker Myosin-VI and tdTomato in whole-mount (C) and cryosection (D) analysis at P1. a, apical turn; m, middle turn; b: basal turn. Scale bars: 200 μm (C and D) （Image by CEBSIT)