Visual Neuroscience Elavl2 Regulates Retinal Function Via Modulating the Differentiation of Amacrine Cells Subtype Mengjuan Wu,1 Qinqin Deng,1 Xinlan Lei,1 Yuxin Du,1 and Yin Shen1,2 1Eye Center, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, Hubei, China 2Frontier Science Center for Immunology and Metabolism, Medical Research Institute, School of Medicine, Wuhan University, Wuhan, Hubei, China Correspondence: Yin Shen, Eye PURPOSE. The neuronal ELAV-like proteins (nElavls; Elavl2, Elavl3, Elavl4) have been Center, Renmin Hospital of Wuhan known to regulate neuronal differentiation, maintenance, and axonogenesis in the brain. University, Wuhan University, Wuhan However, the specific role of nElavls in retina remains unclear. Here, we attempted to 430060, Hubei, China; Frontier identify the expression pattern of Elavl2 during retinogenesis and aimed to decipher the Science Center for Immunology and function of Elavl2 in the retina. Metabolism, Medical Research Institute, School of Medicine, Wuhan METHODS. We have used the Cre-loxP system to conditionally inactivate Elavl2 in order University, Wuhan 430071, Hubei, to examine its role in developing retina. Eyes were collected for histology, immuno- China; histochemistry, and TUNEL analysis to identify the structure of retina, and examined [email protected]. by RNA sequencing to analyze the function and pathway enrichment of differentially MW and QD contributed equally to expressed genes in transgenic mice. Moreover, the mechanism by which Elavl2 regulates this work. the differentiation of amacrine cells (ACs) was explored by RNA immunoprecipitation Received: December 16, 2020 assays. Finally, eyes were functionally assessed by whole-cell patch-clamp, electroretinog- Accepted: May 6, 2021 raphy (ERG) and optomotor response. Published: June 1, 2021 RESULTS. Elavl2 was expressed in retinal progenitor cells and retinal ganglion cells (RGCs), Citation: Wu M, Deng Q, Lei X, Du ACs, and horizontal cells. Retina-specific ablation of Elavl2 led to the loss of ACs and the Y, Shen Y. Elavl2 regulates retinal transcription factors involved in ACs differentiation were also downregulated. In addition, function via modulating the the spontaneous activities of RGCs were obviously increased in Elavl2-deficient mice. differentiation of amacrine cells Meanwhile, the loss of ACs that induced by Elavl2 deficiency lead to a decrease in ERG subtype. Invest Ophthalmol Vis responses and visual acuity. Sci. 2021;62(7):1. https://doi.org/10.1167/iovs.62.7.1 CONCLUSIONS. Elavl2 is an intrinsic factor that involved in the differentiation of ACs subtype during retinogenesis, and essential for maintaining the normal retinal function. Keywords: Elavl2, amacrine cells, retinal development, transcription factor, retinal func- tion he neuronal ELAV-like proteins (nElavls) are highly rich more than 60 subtypes of ACs in the retina.9 Despite their T in neurons1 and play an important role in multiple steps broad morphological diversity, ACs are often divided into of neuronal development associated with neuronal differen- three groups, including GABAergic ACs, glycinergic ACs, and tiation, maintenance, axonogenesis, and synaptic plasticity non-GABAergic–non-glycinergic ACs, based on the expres- through their direct binding to the sequence of target mRNA sion of neurotransmitters. Although all types of ACs are in an activity-dependent manner or at a steady-state level.2–4 generated from the multipotent retinal progenitor cells, Elavl2 (ELAV-like neuron-specific RNA-bp2) is a member of they subsequently differentiate into different subtypes after the nElavls family and identified as autoimmune antigens combined action of the intrinsic and extrinsic factors during in a multisystem neurologic disorder in patients with para- retinogenesis.10–12 TFs are commonly used as the critical neoplastic encephalomyelopathy.5 In the embryonic fore- intrinsic components to regulate the cytogenesis, mainte- brain, Elavl2 completely restores the miR-9-dependent gene nance, and cell fate determination of retinal progenitors.10 silencing of Foxg1, a critical transcriptional factor (TF) in the For instance, Foxn4 is required by retinal progenitor cells central nervous system.6 Moreover, the expression of Tbr2, for the amacrine and horizontal precursors at early stages of a marker for intermediate basal neuronal progenitor cells, is retinogenesis.13,14 Ptf1a has been shown to coordinate with partly colabeled with Elavl2 protein during cortical develop- the Prdm13 and together regulate the balance of inhibitory ment, indicating the specific functions of Elavl2 in the divi- and excitatory neurons.15,16 During retinogenesis, deficiency sion of neurons.7 of Prdm13 leads to a significant decrease in the number of The retina is a part of the central nervous system CALBs+ GABAergic or glycinergic ACs,17 together with the derived from the forebrain neural ectoderm.8 Amacrine cells Ebf3+ ACs.18 In addition, Barhl2 has been verified to play an (ACs) are the important inhibitory intermediate neurons in important role in specifying both glycinergic and GABAer- the retina that could regulate visual information to RGCs, gic ACs19,20; meanwhile, Nr4a2 and Pax6 are involved in enabling elaborate visual functions. At present, there are specifying GABAergic and glycinergic ACs, respectively.21,22 Copyright 2021 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/27/2021 Elavl2 Regulates the Development of Amacrine Cells IOVS |June2021|Vol.62|No.7|Article1|2 Disruption in the balance of TFs including Six6, Vax1, Vsx2 Real-Time Quantitative Reverse Transcription or Otx2, could result in aberrant retinal development and (qRT)-PCR retinal dysfunction, as well as microphthalmia.23 Currently, the molecular mechanism of governing the specification or After euthanizing mice, total RNA was quickly extracted differentiation of ACs remains elusive. from different genotypes retinas using the TRIzol reagent Here, we characterize the expression profile and local- (Invitrogen, Carlsbad, CA, USA). The gene transcription ization of Elavl2, and explore the role of it in the develop- was quantified by qRT-PCR with SYBR Green I fluores- ing and adult mouse retina. Furthermore, our studies reveal cent dye (Vazyme Biotech Co., Ltd, Nanjing, China). All the differences of the generation of ACs in Elavl2-deficient values were calibrated relative to the endogenous levels of retina, and their effects on the RGCs activities and normal Gapdh and normalized to that of the control group. The visual functions. On the basis of these observations, our find- primers used for qRT-PCR were described in Supplementary ings highlight a novel role for Elavl2 signaling in the retina. Table S2. METHODS TUNEL Assay Animals Terminal deoxynucleotidyl transferase dUTP nick end label- ing (TUNEL) in situ labeling was performed on retinal All procedures were performed according to the National cryosections using One Step TUNEL Apoptosis Assay Kit Institutes of Health guidelines and approved by the Wuhan (Beyotime Biotechnology, Jiangsu, China) according to the University Laboratory Animal Center and compiled with manufacturer’s instruction. Briefly, retinal sections were the ARVO statement for the Use of Animals in Ophthalmic incubated with 0.5% Triton-X-100 in PBS for five minutes at and Vision Research. To generate a floxed Elavl2 allele, RT after washed with PBS, and then incubated one hour at we inserted loxP sites in introns 5 and 6 by homologous 37°C with TUNEL detection solution, followed by DAPI for recombination (Fig. 3A). Conditional knock-out mice were nuclear staining and imaging under the Olympus FV1000 obtained by crossing floxed Elavl2 allele mice with the Six3- confocal laser scanning microscope. Cre transgenic line to delete exon 5 of Elavl2 in the devel- oping retina to produce the Elavl2fl/fl , Elavl2fl/+; Six3-Cre and Elavl2fl/fl; Six3-Cre (Elavl2 fl/ fl) mice, and maintained RNA Immunoprecipitation (RIP) Assay by breeding with C57BL6/J (wild type) mice. For RIP assay, protein A/G magnetic beads (Thermo Fisher Scientific, USA) were incubated with antibody against Elavl2 Immunohistochemistry or normal IgG for two hours and followed by the incubated with retina extracts at 4°C overnight. After washing, 200 μL Retinal sections were blocked with a mixture of 2% bovine TE buffer containing 10 mM DTT was used to elute protein serum albumin (BSA) and 0.1% Triton-X-100 in PBS for and RNA complexes for 30 minutes at 37°C. The enriched one hour at room temperature (RT). At the end of the RNAs were then extracted by TRIzol and analyzed by qRT- blocking, the sections were incubated overnight at 4°C with PCR. primary antibodies (Supplementary Table S1) and secondary antibodies for one hour, followed by 4’,6-diamidino-2- phenylindole (DAPI) for nuclear staining and imaging under Electroretinogram (ERG) the confocal Olympus FV1000 laser scanning microscope (Olympus, Tokyo, Japan). ERG responses were recorded in P30, P120 and P270 Elavl2fl/fl and the control mice using the RetiMINER IV system (IRC Medical Equipment, Chongqing, China) just as RNA Sequencing (RNA-seq) Analysis performed previously.28 After adaption to darkness, scotopic ERGs were recorded at stimulus intensities levels of 0.003, Total RNA was isolated from Postnatal (P) 6 retinas of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10 cd. s/m2, with 30-second inter- Elavl2fl/fl