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Ethanol Causes Cell Death and Neuronal Differentiation Defect During Initial Neurogenesis of the Neural Retina by Disrupting Calcium Signaling in Human Retinal Organoids

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Abstract

Fetal Alcohol Syndrome (FAS) affects a significant proportion, exceeding 90%, of afflicted children, leading to severe ocular aberrations such as microphthalmia and optic nerve hypoplasia. During the early stages of pregnancy, the commencement of neural retina neurogenesis represents a critical period for human eye development, concurrently exposing the developing retinal structures to the highest risk of prenatal ethanol exposure due to a lack of awareness. Despite the paramount importance of this period, the precise influence and underlying mechanisms of short-term ethanol exposure on the developmental process of the human neural retina have remained largely elusive. In this study, we utilize the human embryonic stem cells derived retinal organoids (hROs) to recapitulate the initial retinal neurogenesis and find that 1% (v/v) ethanol slows the growth of hROs by inducing robust cell death and retinal ganglion cell differentiation defect. Bulk RNA-seq analysis and two-photon microscope live calcium imaging reveal altered calcium signaling dynamics derived from ethanol-induced down-regulation of RYR1 and CACNA1S. Moreover, the calcium-binding protein RET, one of the downstream effector genes of the calcium signaling pathway, synergistically integrates ethanol and calcium signals to abort neuron differentiation and cause cell death. To sum up, our study illustrates the effect and molecular mechanism of ethanol on the initial neurogenesis of the human embryonic neural retina, providing a novel interpretation of the ocular phenotype of FAS and potentially informing preventative measures for susceptible populations.

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Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This study was supported by funding from the National Key Research and Development Program of China grants 2021YFA1101203; the National Natural Science Foundation of China grants 31930068; General Project of Chongqing Natural Science Foundation CSTB2022NSCQ-MSX0065; Chongqing Medical University Program for Youth Innovation in Future Medicine W0158. The funding bodies had no role in study design, in the collection, analysis, or interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.

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  1. Yu Gong and Lingling Ge contributed equally to this work.

Authors and Affiliations

  1. Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, People’s Republic of China

    Yu Gong, Lingling Ge, Qiyou Li, Jing Gong, Min Chen, Hui Gao, Jiahui Kang & Haiwei Xu

  2. Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, People’s Republic of China

    Yu Gong, Lingling Ge, Qiyou Li, Jing Gong, Min Chen, Hui Gao, Jiahui Kang & Haiwei Xu

  3. Department of Ophthalmology, University-Town Hospital of Chongqing Medical University, Chongqing, China

    Yu Gong & Jiawen Li

  4. Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China

    Jing Gong

  5. Department of Clinical Laboratory, The 89th Hospital of The People’s Liberation Army, Weifang, People’s Republic of China

    Ting Yu

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  1. Yu Gong
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Contributions

YG, LG, QL, JG, MC, HG, JK, and TY performed the experiments and analyzed the results. JL and HX designed the project and directed the research. YG and LG wrote the manuscript.

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Correspondence to Jiawen Li or Haiwei Xu.

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Video 1. Clones of hESC-GCaMP5G visualized with GCaMP5G and two-photon microscopy. (Framerate 60/min; fast forward 5x) (MP4 41.3 MB)

Video 2. PBS-treated hROs-GCaMP5G at D30 imaged with two-photon microscopy at room temperature. (Total duration:1000 s; framerate 60/min; fast forward 5x) (AVI 37.4 MB)

Video 3. 1% EtOH treated hROs-GCaMP5G at D30 imaged with two-photon microscopy at room temperature. (Total duration:1000 s; framerate 60/min; fast forward 5x) (AVI 63.8 MB)

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Gong, Y., Ge, L., Li, Q. et al. Ethanol Causes Cell Death and Neuronal Differentiation Defect During Initial Neurogenesis of the Neural Retina by Disrupting Calcium Signaling in Human Retinal Organoids. Stem Cell Rev and Rep 19, 2790–2806 (2023). https://doi.org/10.1007/s12015-023-10604-3

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