通过产 卵电 穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能
1Division of Histology & Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, College of Medicine,Jinan University, 2Department of Clinical Pathology,First Affiliated Hospital of Jinan University, 3Program in Neuroscience, Department of Biomedical Sciences, College of Medicine,Florida State University
Summary
利用 卵 电穿孔，我们设计了一种选择性转染鸡胚胎中听觉内耳和耳蜗核的方法，以实现在电路组装的离散期间脆性X智力迟钝蛋白的细胞组特异性敲低。
Abstract
脆性X染色体智力低下蛋白（FMRP）是一种调节局部蛋白质翻译的mRNA结合蛋白。FMRP丧失或功能障碍导致脆性X综合征（FXS）的神经元和突触活动异常，其特征是智力障碍，感觉异常和社会沟通问题。FMRP功能和FXS发病机制的研究主要在转基因动物中使用 Fmr1 （编码FMRP的基因）敲除进行。在这里，我们 报告了一种体内 方法，该方法用于使用鸡胚胎确定FMRP在电路组装和突触形成期间的细胞自主功能。该方法采用含有 Fmr1 小发夹RNA（shRNA）和EGFP报告基因的药物诱导载体系统的阶段，位点和方向特异性电穿孔。通过这种方法，我们在听觉神经节（AG）及其脑干靶标之一大细胞核（NM）中实现了选择性FMRP敲低，从而在AG-NM电路中提供了组件特异性操作。此外，转染的镶嵌模式允许动物内对照和邻近神经元/纤维比较，以提高数据分析的可靠性和灵敏度。诱导载体系统提供基因编辑开始的时间控制，以最大限度地减少累积的发育影响。这些策略的组合为剖析FMRP在突触和电路发育中的细胞自主功能提供了一种创新工具。
Introduction
脆性 X 综合征 （FXS） 是一种以智力障碍、感觉异常和自闭症行为为特征的神经发育障碍。在大多数情况下，FXS是由从早期胚胎阶段开始的脆性X智力迟钝蛋白（FMRP;由Fmr1基因编码）的全球损失引起的1。FMRP是一种RNA结合蛋白，通常在大脑中的大多数神经元和神经胶质细胞以及感觉器官中表达2，3，4。在哺乳动物的大脑中，FMRP可能与数百种编码对各种神经活动很重要的蛋白质的mRNA有关5。对常规Fmr1基因敲除动物的研究表明，FMRP表达对突触神经传递的组装和可塑性尤为重要6。一些条件和马赛克敲除模型进一步证明，FMRP作用和信号在几个发育事件中因大脑区域，细胞类型和突触部位而异，包括轴突投影，树突状图案和突触可塑性7，8，9，10，11，12，13，14.通过在脑切片或培养的神经元中细胞内递送抑制性FMRP抗体或FMRP本身来研究FMRP在调节突触传递方面的急性功能15，16，17，18。然而，这些方法不能在开发过程中跟踪FMRP误表达诱导的后果。因此，非常需要开发体内方法来研究FMRP的细胞自主功能，并有望帮助确定FXS患者中报告的异常是相关神经元和回路中FMRP丢失的直接后果，还是发育过程中网络范围变化的继发性后果19。
鸡胚胎的听觉脑干为电路和突触发育中FMRP调节的深入功能分析提供了一个独特的优势模型。易于获得胚胎鸡脑和成熟的用于遗传操作的 卵 电穿孔技术极大地促进了我们对早期胚胎阶段大脑发育的理解。在最近发表的一项研究中，该技术与先进的分子工具相结合，可以对FMRP错误表达进行时间控制20，21。在这里，该方法被推进到分别诱导突触前和突触后神经元的选择性操作。这种方法是在听觉脑干回路中开发的。声音信号由听觉内耳中的毛细胞检测到，然后传递到听神经节（AG;在哺乳动物中也称为螺旋神经节）。AG中的双极神经元通过其外周过程支配毛细胞，进而将中枢投影（听觉神经）发送到脑干，在那里它们终止于两个初级耳蜗核，即大细胞核（NM）和角核（NA）。NM中的神经元在结构和功能上与哺乳动物前腹侧耳蜗核的球形灌丛细胞相当。在NM内，听觉神经纤维（ANF） 突触通过 Hold终端22的大端球在NM神经元的躯体上突触。在发育过程中，NM 神经元起源于后脑23 中的菱形 5 和 6 （r5/6），而 AG 神经元源自耳囊24 中的神经母细胞。在这里，我们分别描述了在突触前AG神经元和突触后NM神经元中选择性敲低FMRP表达的过程。
Protocol
鸡蛋和鸡胚胎按照暨南大学动物护理和使用委员会批准的动物规程进行谨慎和尊重的处理。 1. 卵子和质粒制备 鸡蛋准备从华南农业大学获得新鲜受精鸡蛋（Gallus gallus），并在孵化前储存在16°C。为了获得最佳的活力，请在到达后一周内设置所有种蛋进行孵化。 将鸡蛋水平放置并在38°C下孵育46-48小时，直到汉堡和汉密尔顿（HH）阶段12…
Representative Results
通过在不同部位和不同发育阶段进行 卵 电穿孔，我们在听觉外周或听觉脑干中实现了选择性FMRP敲低。 新墨西哥州的FMRP敲低如前所述，设计针对鸡Fmr1的小发夹RNA（shRNA）并将其克隆到Tet-On载体系统中20。 卵内电穿孔的设置如图1A所示。将带有快速绿色着色的质粒DNA在HH12下以r5 / r6水平注射到神经管中…
Discussion
为了确定FMRP的细胞自主功能，需要操纵其在单个细胞组或细胞类型中的表达。由于FMRP的主要功能之一是调节突触形成和可塑性，因此选择性地操纵某个回路的每个突触组件是充分了解突触通信中FMRP机制的先决条件。在鸡胚胎的卵电穿孔中，我们展示了一种在突触前AG神经元或突触后NM神经元中靶向AG-NM回路中FMRP表达的方法。为了实现这一目标，为电穿孔选择合适的…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究得到以下资助：国家自然科学基金（第32000697号）;广州市科技计划（202102080139）;广东省自然科学基金（2019A1515110625，2021A1515010619）;中央高校基本科研业务费专项资金（11620324）;暨南大学再生医学教育部重点实验室研究经费（No.ZSYXM202107）;中央高校基本科研业务费专项资金（21621054）;中国广东省医学科学研究基金（20191118142729581）。感谢暨南大学医学实验中心。我们感谢Terra Bradley博士对手稿的精心编辑。
Materials
| Egg incubation | |||
| 16 °C refrigerator | MAGAT | Used for fertilized egg storage. | |
| Egg incubator | SHANGHAI BOXUN | GZX-9240MBE | |
| Fertilized eggs | Farm of South China Agricultural University | Eggs must be used in one week for optimal viability. | |
| Plasmid preparation | |||
| Centrifuge | Sigma | 10016 | |
| Fast green | Solarbio | G1661 | Make 0.1% working solution in distilled water and autoclave. |
| Plasmid Maxi-prep kit | QIAGEN | 12162 | Dissolve plasmid DNA in Tris-EDTA (TE) buffer; endotoxin-free preparation kit |
| Sodium Acetate | Sigma-Aldrich | S2889 | Make 7.5M working solution in nuclase-free water. |
| Electroporation and Doxycycline Administration | |||
| Electroporator | BTX | ECM399 | |
| 1 mL / 5 mL Syringe | GUANGZHOU KANGFULAI | ||
| Dissecting microscope | CNOPTEC | SZM-42 | |
| Doxcycline | Sigma-Aldrich | D9891 | Use fresh aliquots for each dose and store at -20 °C. |
| Glass capillary | BEIBOBOMEI | RD0910 | 0.9-1.1 mm*100 mm |
| Laboratory parafilm | PARAFILM | PM996 | transparent film |
| Pipette puller | CHENGDU INSTRUMENT FACTORY | WD-2 | Pulling condition: 500 °C for 15 s |
| Platinum elctrodes | Home made | 0.5 mm diameter, 1.5 mm interval. | |
| Platinum elctrodes | Home made | 0.5 mm diameter, 1.5 mm interval. | |
| Rubber tube | Sigma-Aldrich | A5177 | |
| Tissue Dissection and Fixation | |||
| Forceps | RWD | F11020-11 | Tip size: 0.05*0.01 mm |
| Other surgery tools | RWD | ||
| Paraformaldehyde | Sigma-Aldrich | 158127 | Freshly made 4% PFA solution in phosphate-buffered saline can be stored in 4 °C for up to 1 week. |
| SYLGARD 184 Silicone Elastomer Kit | DOW | 01673921 | For black background plates, food-grade carbon powder is applied. |
| Sectioning | |||
| Cryostat | LEICA | CM1850 | |
| Gelatin | Sigma-Aldrich | G9391 | From bovine skin. |
| Sliding microtome | LEICA | SM2010 | |
| Immunostaining | |||
| Alexa Fluor 488 goat anti-Mouse | Abcam | ab150113 | 1:500 dilution, RRID: AB_2576208 |
| Alexa Fluor 555 goat anti-rabbit | Abcam | ab150078 | 1:500 dilution, RRID: AB_2722519 |
| DAPI | Abcam | ab285390 | 1: 1000 dilution |
| Fluoromount-G mounting medium | Southern Biotech | Sb-0100-01 | |
| FMRP antibody | Y. Wang, Florida State University | #8263 | 1:1000 dilution, RRID: AB_2861242 |
| Islet-1 antibody | DSHB | 39.3F7 | 1:100 dilution, RRID: AB_1157901 |
| Netwell plate | Corning | 3478 | |
| Neurofilament antibody | Sigma-Aldrich | N4142 | 1:1000 dilution, RRID: AB_477272 |
| Parvalbumin antibody | Sigma-Aldrich | P3088 | 1:10000 dilution, RRID: AB_477329 |
| SNAP25 antibody | Abcam | ab66066 | 1:1000 dilution, RRID: AB_2192052 |
| Imaging | |||
| Adobe photoshop | ADOBE | image editing software | |
| Confocal microscope | LEICA | SP8 | |
| Fluorescent stereomicroscope | OLYMPUS | MVX10 | |
| Olympus Image-Pro Plus 7.0 | OlYMPUS | commercial image processing software package |
Referências
- Hagerman, R. J., et al. Fragile X syndrome. Nature Reviews Disease Primers. 3, 17065 (2017).
- Hinds, H. L., et al. Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome. Nature Genetics. 3 (1), 36-43 (1993).
- Frederikse, P. H., Nandanoor, A., Kasinathan, C. Fragile X Syndrome FMRP co-localizes with regulatory targets PSD-95, GABA receptors, CaMKIIalpha, and mGluR5 at fiber cell membranes in the eye lens. Neurochemical Research. 40 (11), 2167-2176 (2015).
- Zorio, D. A., Jackson, C. M., Liu, Y., Rubel, E. W., Wang, Y. Cellular distribution of the fragile X mental retardation protein in the mouse brain. Journal of Comparative Neurology. 525 (4), 818-849 (2017).
- Darnell, J. C., et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell. 146 (2), 247-261 (2011).
- Bakker, C. E., Oostra, B. A. Understanding fragile X syndrome: insights from animal models. Cytogenetic and Genome Research. 100 (1-4), 111-123 (2003).
- Hanson, J. E., Madison, D. V. Presynaptic FMR1 genotype influences the degree of synaptic connectivity in a mosaic mouse model of fragile X syndrome. Journal of Neuroscience. 27 (15), 4014-4018 (2007).
- Deng, P. Y., Sojka, D., Klyachko, V. A. Abnormal presynaptic short-term plasticity and information processing in a mouse model of fragile X syndrome. Journal of Neuroscience. 31 (30), 10971-10982 (2011).
- Patel, A. B., Hays, S. A., Bureau, I., Huber, K. M., Gibson, J. R. A target cell-specific role for presynaptic Fmr1 in regulating glutamate release onto neocortical fast-spiking inhibitory neurons. Journal of Neuroscience. 33 (6), 2593-2604 (2013).
- Patel, A. B., Loerwald, K. W., Huber, K. M., Gibson, J. R. Postsynaptic FMRP promotes the pruning of cell-to-cell connections among pyramidal neurons in the L5A neocortical network. Journal of Neuroscience. 34 (9), 3413-3418 (2014).
- Higashimori, H., et al. Selective deletion of astroglial FMRP dysregulates glutamate transporter GLT1 and contributes to Fragile X syndrome phenotypes in vivo. Journal of Neuroscience. 36 (27), 7079-7094 (2016).
- Hodges, J. L., et al. Astrocytic contributions to synaptic and learning abnormalities in a mouse model of Fragile X syndrome. Biological Psychiatry. 82 (2), 139-149 (2017).
- Gonzalez, D., et al. Audiogenic seizures in the Fmr1 knock-out mouse are induced by Fmr1 deletion in subcortical, VGlut2-expressing excitatory neurons and require deletion in the inferior colliculus. Journal of Neuroscience. 39 (49), 9852-9863 (2019).
- Bland, K. M., et al. FMRP regulates the subcellular distribution of cortical dendritic spine density in a non-cell-autonomous manner. Neurobiology of Disease. 150, 105253 (2021).
- Pfeiffer, B. E., Huber, K. M. Fragile X mental retardation protein induces synapse loss through acute postsynaptic translational regulation. Journal of Neuroscience. 27 (12), 3120-3130 (2007).
- Pfeiffer, B. E., et al. Fragile X mental retardation protein is required for synapse elimination by the activity-dependent transcription factor MEF2. Neuron. 66 (2), 191-197 (2010).
- Deng, P. Y., et al. FMRP regulates neurotransmitter release and synaptic information transmission by modulating action potential duration via BK channels. Neuron. 77 (4), 696-711 (2013).
- Yang, Y. M., et al. Identification of a molecular locus for normalizing dysregulated GABA release from interneurons in the Fragile X brain. Molecular Psychiatry. 25 (9), 2017-2035 (2020).
- Razak, K. A., Dominick, K. C., Erickson, C. A. Developmental studies in fragile X syndrome. Journal of Neurodevelopmental Disorders. 12 (1), 13 (2020).
- Wang, X., Zorio, D. A. R., Schecterson, L., Lu, Y., Wang, Y. Postsynaptic FMRP regulates synaptogenesis in vivo in the developing cochlear nucleus. Journal of Neuroscience. 38 (29), 6445-6460 (2018).
- Wang, X., et al. Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit. Development. 147 (21), (2020).
- Rubel, E. W., Fritzsch, B. Auditory system development: primary auditory neurons and their targets. Annual Review of Neuroscience. 25, 51-101 (2002).
- Cramer, K. S., Fraser, S. E., Rubel, E. W. Embryonic origins of auditory brain-stem nuclei in the chick hindbrain. Biologia do Desenvolvimento. 224 (2), 138-151 (2000).
- Chervenak, A. P., Hakim, I. S., Barald, K. F. Spatiotemporal expression of Zic genes during vertebrate inner ear development. Developmental Dynamics. 242 (7), 897-908 (2013).
- Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chick embryo. Journal of Morphology. 88 (1), 49-92 (1951).
- Lu, T., Cohen, A. L., Sanchez, J. T. In ovo electroporation in the chicken auditory brainstem. Journal of Visualized Experiments. (124), e56628 (2017).
- Evsen, L., Doetzlhofer, A. Gene transfer into the chicken auditory organ by in ovo micro-electroporation. Journal of Visualized Experiments. (110), e53864 (2016).
- Schecterson, L. C., Sanchez, J. T., Rubel, E. W., Bothwell, M. TrkB downregulation is required for dendrite retraction in developing neurons of chicken nucleus magnocellularis. Journal of Neuroscience. 32 (40), 14000-14009 (2012).
- Wang, X. Y., et al. High glucose environment inhibits cranial neural crest survival by activating excessive autophagy in the chick embryo. Scientific Reports. 5, 18321 (2015).
- Yu, X., Wang, X., Sakano, H., Zorio, D. A. R., Wang, Y. Dynamics of the fragile X mental retardation protein correlates with cellular and synaptic properties in primary auditory neurons following afferent deprivation. Journal of Comparative Neurology. 529 (3), 481-500 (2021).
- Li, H., et al. Islet-1 expression in the developing chicken inner ear. Journal of Comparative Neurology. 477 (1), 1-10 (2004).
- Carr, C. E., Boudreau, R. E. Central projections of auditory nerve fibers in the barn owl. Journal of Comparative Neurology. 314 (2), 306-318 (1991).
- Sandell, L. L., Butler Tjaden, N. E., Barlow, A. J., Trainor, P. A. Cochleovestibular nerve development is integrated with migratory neural crest cells. Biologia do Desenvolvimento. 385 (2), 200-210 (2014).
- Cramer, K. S., Bermingham-McDonogh, O., Krull, C. E., Rubel, E. W. EphA4 signaling promotes axon segregation in the developing auditory system. Biologia do Desenvolvimento. 269 (1), 26-35 (2004).
- Evsen, L., Sugahara, S., Uchikawa, M., Kondoh, H., Wu, D. K. Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1. Journal of Neuroscience. 33 (9), 3879-3890 (2013).
- Curnow, E., Wang, Y. New animal models for understanding FMRP functions and FXS pathology. Cells. 11 (10), 1628 (2022).
Citar este artigo
Fan, Q., Zhang, X., Wang, Y., Wang, X. Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation. J. Vis. Exp. (185), e64187, doi:10.3791/64187 (2022).