Spatial resolution of syncytial nuclear gene regulation

合体细胞，即共用一个细胞质的多核细胞，是一个转录之谜。核内的核是如何 合体细胞相互影响，调节和协调转录，并滴定转录以处理 每个合胞体的核数量是否存在差异？骨骼肌，含有肌纤维(肌肉合胞细胞) 含有数百个细胞核，是体内含量最丰富的合体细胞类型2，3.在未损伤的肌肉中， 肌核位于细胞的外围，沿着长轴4。据推测，每一个 肌核的转录输出控制着它周围一定体积的细胞质，即肌核 2，5-9域。到目前为止，还不清楚肌核在转录上是同步的还是异步的。 在特定的合体细胞核中是否存在转录偏向？如果是这样的话，原子核的空间位置如何决定 它的转录多样性？哪些转录本是空间表达的，而不是泛表达的？做一些原子核 比其他人更具竞争力(高转录与低转录)？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和单肌纤维分离17-19联合应用 单核RNA测序(SnRNA-seq)20，21。这些发现的结果将揭开合体细胞基因的神秘面纱。 由核异质性引起的调节，允许产生和可视化计算 描述这些专门转录网络的模型。与现场相结合的模型 基于杂交的策略，MERFISH2，23和CODEX24，将使空间重建成为可能 合胞体内的转录网络。因此我的目标是使用新的基因组工具和 用计算机分析研究合体细胞中的基因调控机制 活着。我假设合胞体的核在转录上是异质性和异步性的，尽管共享 一个共同的细胞质，这提供了肌纤维间隙内的区域核规格。这一努力 我将利用最近的技术发展，主要是由我的赞助人开创的，用来分析高含量的核 平台20和通过hashing21汇集样本。这种方法的一个优点是合胞体的纯度和规模 可以从肌纤维中提纯细胞核，以形成17-19，25的轮廓。利用人的IPSC进行平行分析 系统26和精选的人类组织样本将补充体内小鼠的努力。技术和分析 这项工作的建立将适用于研究其他合体细胞类型，如胎盘 合体滋养层细胞27-29。这项研究的一个关键目标是回答合体细胞如何代表基因 在它们的核群中表达。总而言之，这项提议将解决一些原子核是否专门化 他们的转录输出，如果是的话，哪些基因座是专用的，以及他们的空间区域，解决了长期存在的 关于合胞体内单个核的同步性的辩论，并确定空间上的生物学成分 区域肌肉结构和功能的异质性，从而为解释提供了一个框架 特定于患者的病理。

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.