Developmental regulation of rod photoreceptor number via a light-dependent retrograde pathway from intrinsically photosensitive retinal ganglion cells

Photoreception, a form of sensory experience, is essential for normal development of the mammalian visual system. Detecting photons during development is a prerequisite for visual system function - from shaping visions first synapse and maturation of retinal vascular networks, to transcriptional establishment and maturation of cell types within the visual cortex. Consistent with this theme, we find that the lighting environment regulates developmental rod photoreceptor apoptosis via OPN4-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Using a combination of genetics, sensory environment manipulations, and computational approaches, we establish a molecular pathway in which light-dependent glutamate release from ipRGCs is detected via a transiently expressed kainate receptor (GRIK3) in rod precursors localized to the inner retina. Communication between ipRGCs and nascent inner retinal rods appears to be mediated by transient hybrid neurites projecting from ipRGCs that sense light before eye-opening. These structures, previously referred to as outer retinal dendrites (ORDs), span the ipRGC-rod precursor distance over the first postnatal week and contain the machinery for sensory detection (melanopsin, OPN4) and anterograde neurotransmitter release (Synaptophysin and VGLUT2). Computational and histological assessment of human mid-gestation development reveal conservation of several hallmarks of an ipRGC-to-rod precursor pathway, including displaced rod precursors, transient GRIK3 expression in the rod lineage, and the presence of ipRGCs with putative neurites projecting deep into the developing human retina. Thus, this analysis defines a retinal retrograde signaling pathway that links the sensory environment to rod precursors via ipRGC photoreceptors, allowing the visual system to adapt to distinct lighting environments prior to eye-opening. Opn4+/- x Opn4+/- harem crosses were used to produce wildtype and knockout littermate pups raised in similar lighting environments (~300 lux). On the day of birth (P0), tail and finger clippings were harvested and mice were genotyped prior to determine appropriate genotypes. At P4, mice were sacrificed between ZT6-7 and retina were dissected in ice-cold oxygenated Ames media (95% air, 5% oxygen). Multiple retinas were pooled together from animals of the same genotype (4 retina per sample, 2 animals), and tissue was snap frozen in Trizol (100uL). Post-hoc genotyping was then performed on each sample to confirm genotypes. RNA extraction, library preparation, and sequencing (Illumina HiSeq 2 x 150bp) were all performed at GeneWiz. Samples with >500ng of RNA and a RIN > 8.5 were selected for further processing. The average read depth per sample was 42.7 3.9 million reads. Subsequently, adapters and low-quality reads were trimmed/filtered using Timmomatic v0.39. Reads were pseudoaligned to the mouse reference genome (GRCm38) and counts were quantified using Kallisto v0.46.1. Differential gene expression analysis was performed using Sleuth v0.30.8.光感受作为一类感官体验形式，对哺乳动物视觉系统的正常发育至关重要。发育过程中对光子的检测是视觉系统功能实现的先决条件——从塑造视觉系统首个突触、视网膜血管网络的成熟，到视觉皮层内细胞类型的转录建立与功能成熟。 契合这一研究主题，我们发现光照环境可通过表达视黑素（OPN4）的内在光敏感性视网膜神经节细胞（intrinsically photosensitive retinal ganglion cells，ipRGCs）调控发育中视杆光感受器的细胞凋亡。 本研究结合遗传学操作、感官环境操控策略与计算生物学方法，构建了一条分子通路：ipRGCs依赖光照的谷氨酸释放，可被定位于视网膜内层的视杆前体细胞中瞬时表达的红藻氨酸受体（kainate receptor，GRIK3）所感知。 ipRGCs与新生视网膜内层视杆细胞之间的信号通讯，似乎由眼张开前即可感知光线的ipRGCs伸出的瞬时混合神经突起所介导。这类此前被称为视网膜外层树突（ORDs）的结构，在出生后第一周内即可跨越ipRGC与视杆前体细胞之间的空间距离，且包含感官检测相关功能元件（黑素蛋白、OPN4）与顺行神经递质释放相关蛋白（突触素（Synaptophysin）与囊泡谷氨酸转运体2（VGLUT2））。 对人类妊娠中期发育阶段的计算分析与组织学评估显示，ipRGC至视杆前体细胞信号通路的多项标志性特征具有进化保守性，包括异位分布的视杆前体细胞、视杆细胞谱系中瞬时表达的GRIK3，以及带有疑似突起深入发育中人视网膜的ipRGCs。 因此，本研究明确了一条视网膜逆行信号通路，该通路通过ipRGC光感受器将感官环境与视杆前体细胞关联起来，使视觉系统能够在眼张开前适应不同的光照环境。 本研究采用Opn4+/- × Opn4+/-的单雄多雌交配（harem crosses）繁殖体系，获得野生型与基因敲除同窝幼鼠，并在相似光照环境（约300勒克斯）下统一饲养。在出生当日（P0），采集幼鼠尾尖与指尖组织并进行基因型鉴定，以筛选匹配的基因型分组。在出生后第4天（P4），于ZT6-7时段处死小鼠，在冰冷充氧的Ames培养液（95%空气、5%氧气）中分离视网膜组织。将同基因型动物的视网膜混合（每份样本包含4个视网膜，对应2只小鼠），随后用100μL Trizol试剂将组织快速冷冻保存。后续对每份样本进行事后基因型鉴定，以确认实验分组的基因型正确性。RNA提取、文库制备与高通量测序（Illumina HiSeq 2×150bp）均在GeneWiz公司完成。选取RNA总量＞500ng且RNA完整性指数（RIN）＞8.5的样本开展后续实验。每份样本的平均测序读段深度为42.7±3.9百万条。随后使用Timmomatic v0.39工具对测序读段的接头序列与低质量读段进行修剪与过滤。将过滤后的读段伪比对至小鼠参考基因组（GRCm38），并使用Kallisto v0.46.1进行基因计数定量。采用Sleuth v0.30.8工具完成差异基因表达分析。