Spatial resolution of syncytial nuclear gene regulation

合胞体细胞，共享一个胞质的多核细胞1，是一个转录之谜。原子核是如何 合胞体细胞相互影响，调节和协调转录，并滴定转录以处理 每个合胞体的细胞核数目的变化？骨骼肌，携带肌纤维（肌肉合胞体细胞） 包含数百个细胞核，是体内最丰富的合胞体细胞类型2，3。在未受伤的肌肉中， 肌细胞核沿着细胞的外周沿着长轴定位4。据推测， 肌核的转录输出控制着它周围的确定体积的细胞质，即肌核 domain2，5-9.迄今为止，尚不清楚肌核是转录同步的还是转录不同步的10。 在特定的合胞体细胞核中是否存在转录本的偏倚？如果是这样的话，原子核的空间位置是如何控制 它的转录多样性哪些转录本是空间表达的，哪些是泛表达的？做一些核 超越其他人（hyper- versus hypo-transcribe）？为了解决无数的问题，我将应用一个 激光捕获显微切割（LCM）11-16和单个肌纤维分离17 -19结合 单核RNA测序（snRNA-seq）20，21.本文的发现结果将揭开合胞细胞基因的神秘面纱。 由于核异质性导致的调节，允许生成和可视化计算 描述这些专门的转录网络的模型。模型结合现场 基于杂交的策略，MERFISH 22，23和CODEX 24，将使空间重建的 合胞体内的转录网络。因此，我的目标是采用新的基因组工具， 计算分析，以表征基因调控机制制定的合胞体细胞， vivo.我推测，尽管合胞体细胞核共享基因， 一个共同的细胞质，这提供了区域内的肌纤维空间的核规格。这一努力 我将利用最近的技术发展，主要是由我的赞助商开创的，用于在高含量上分析核 平台20和通过散列合并样本21。这种方法的优势在于合胞体的纯度和规模， 可以从肌纤维中纯化细胞核以进行分析17 - 19，25。使用人类iPSC的平行分析 system 26和选择的人组织样品将补充体内鼠的努力。技术和分析 本研究所建立的方法也适用于研究其他类型的合胞体细胞，如胎盘细胞， 合体滋养层细胞27 -29。本研究的一个主要目的是回答合胞细胞如何代表基因 在细胞核中的表达。总之，这项建议将解决一些核是否专门化， 它们的转录输出，如果是这样，哪些位点是专用的，它们的空间位置，解决了长期存在的 关于合胞体内单个核的同步性的争论，并确定用于空间的生物制剂 区域肌肉结构和功能的异质性，从而为解释 患者特异性病理学。

Syncytial cells, multinucleate cells sharing a cytoplasm1, are a transcriptional enigma. How do nuclei within syncytial cells impact each other, regulate and coordinate transcription, and titrate transcription to deal with variation in the number of nuclei per syncytia? Skeletal muscle, harboring myofibers (muscle syncytial cells) containing hundreds of nuclei, is the most abundant syncytial cell type in the body2,3. In the uninjured muscle, myonuclei are positioned along the periphery of the cell along the long axis4. It has been postulated that each myonucleus’s transcriptional output governs a defined volume of cytoplasm surrounding it, i.e. the myonuclear domain2,5-9. To date, it remains unclear whether myonuclei are transcriptionally synchronous or asynchronous10. Is there a transcript bias across particular syncytial nuclei? If so, how does a nucleus’s spatial position govern its transcriptional diversity? Which transcripts are spatially expressed versus pan-expressed? Do some nuclei outcompete others (hyper- versus hypo-transcribe)? In order to address the myriad of questions, I will apply a combination of laser capture microdissection (LCM)11-16 and single myofiber isolation17-19 in conjunction with single nucleus RNA-sequencing (snRNA-seq)20,21. The results of findings herein will demystify syncytial cell gene regulation resulting from nuclear heterogeneity, allowing for the generation and visualization of computational models describing these specialized transcriptional networks. The models in combination with in situ hybridization-based strategies, MERFISH22,23 and CODEX24, will enable spatial reconstruction of the transcriptional networks within the syncytium. I therefore aim to employ novel genomic tools and computational analysis to characterize the gene regulatory mechanisms enacted in syncytial cells in vivo. I hypothesize that syncytial nuclei are transcriptionally heterogeneous and asynchronous despite sharing a common cytoplasm, and that this affords regional nuclear specifications within the myofiber space. This effort will exploit recent technical developments, largely pioneered by my sponsor, for profiling nuclei on high-content platforms20 and pooling samples by hashing21. A strength of this approach is the purity and scale that syncytial nuclei can be purified from myofibers for profiling17-19,25. Parallel analysis employing the human iPSC system26 and select human tissue samples will complement in vivo murine efforts. Technologies and analysis established in this work will be applicable for studying other syncytial cell types, such as placental syncytiotrophoblast cells27-29. A key objective of this study is to answer of how syncytial cells delegate gene expression across their population of nuclei. Together, this proposal will address whether some nuclei specialize their transcriptional output, and if so, which loci are dedicated and their spatial locale, address the long-standing debate regarding synchrony of individual nuclei within a syncytium, and identify the biologics for spatial heterogeneity underlying regional muscle architecture and function, thus, providing a framework for interpreting patient-specific pathologies.