Characterisation of transgenic lines labelling reticulospinal neurons in larval zebrafish

Pre-print Characterisation of transgenic lines labelling reticulospinal neurons in larval zebrafish. For more information see pre-print here. Abstract From lamprey to monkeys, the organization of the descending control of locomotion is conserved across vertebrates. Reticulospinal neurons (RSNs) form a bottleneck for descending commands, receiving innervation from diencephalic and mesencephalic locomotor centres and providing locomotor drive to spinal motor circuits. Given their optical accessibility in early development, larval zebrafish offer a unique opportunity to study reticulospinal circuitry. In fish, RSNs are few, highly stereotyped, uniquely identifiable, large neurons spanning from the midbrain to the medulla. Classically labelled by tracer dye injections into the spinal cord, recent advances in genetic tools have facilitated the targeted expression of transgenes in diverse brainstem neurons of larval zebrafish. Here, we provide a comparative characterization of four existing and three newly established transgenic lines in larval zebrafish. We determine which identified neurons are consistently labelled and offer projection-specific genetic access to subpopulations of RSNs. We showcase transgenic lines that label most or all RSNs (nefma, adcyap1b_ccu96Et) or subsets of RSNs, including ipsilateral (vsx2, calca_ccu75Et) contralateral (pcp4a_ccu97Tg) or all (tiam2a_y264Et) components of the Mauthner array, or midbrain-only RSNs (s1171tEt). In addition to RSNs, selected transgenic lines (nefma, s1171tEt, calca_ccu75Et) labelled other potential neurons of interest in the brainstem. For those, we performed in situ hybridisation to show expression patterns of several excitatory and inhibitory neurotransmitters at larval stages as well as glutamatergic expression patterns in juvenile fish. We provide an overview of transgene expression in the brainstem of larval zebrafish that serves to lay a foundation for future studies in the supraspinal control of locomotion. Data We provide a stack for an example fish from each transgenic line, as well as a ROI file that labels reticulospinal neurons in the nefma fish to be used in the ROI manager in ImageJ. First channel (green) is the transgenic line, second channel (grey) is the tERK counter-stain, third channel (magenta) is the reticulospinal backfill.Pre-print 斑马鱼幼鱼中标记网状脊髓神经元（reticulospinal neurons, RSNs）的转基因品系特征分析。更多详情可参见此处的预印本。 摘要 从七鳃鳗到灵长类动物，运动下行控制的组织模式在脊椎动物中高度保守。网状脊髓神经元（RSNs）是下行运动指令的关键瓶颈：它们接收来自间脑和中脑运动中枢的神经支配，并为脊髓运动环路提供运动驱动信号。鉴于斑马鱼幼鱼在发育早期具有光学可及性，其为研究网状脊髓环路提供了独特的实验模型。在鱼类中，RSNs数量稀少、高度定型且可唯一识别，是从中脑延伸至延髓的大型神经元。传统上通过向脊髓注射示踪染料来标记RSNs，而近年来遗传工具的进步使得在斑马鱼幼鱼的多样化脑干神经元中靶向表达转基因成为可能。本研究对斑马鱼幼鱼中4种已有的和3种新构建的转基因品系进行了比较性特征分析，明确了可被稳定标记的神经元类型，并实现了对RSNs亚群按投射特性进行遗传靶向操控的能力。我们展示了可标记绝大多数或全部RSNs的转基因品系（nefma、adcyap1b_ccu96Et），以及可标记RSNs亚群的品系：包括同侧投射亚群（vsx2、calca_ccu75Et）、对侧投射亚群（pcp4a_ccu97Tg）、马氏神经元群的全部组分（tiam2a_y264Et），以及仅标记中脑RSNs的品系（s1171tEt）。除RSNs外，部分选定的转基因品系（nefma、s1171tEt、calca_ccu75Et）还可标记脑干中其他具有研究价值的神经元。针对这些神经元，我们通过原位杂交技术展示了幼鱼阶段多种兴奋性和抑制性神经递质的表达模式，以及幼鱼的谷氨酸能神经元表达模式。本研究对斑马鱼幼鱼脑干中的转基因表达情况进行了系统性梳理，为未来开展运动的脊髓上控制相关研究奠定了基础。 数据集 本数据集提供了每个转基因品系对应的单条示例斑马鱼成像堆叠文件，同时附带了可在ImageJ的ROI管理器中使用的、用于标记nefma品系斑马鱼网状脊髓神经元的ROI文件。图像第一通道（绿色）为转基因标记信号，第二通道（灰色）为tERK复染信号，第三通道（品红色）为网状脊髓神经元逆行标记信号。