Phylogenetic reconstruction of the intestinal epithelium via evolving barcodes

项目摘要 肠道是人体内周转率最高的组织，使其成为成体干细胞的极好模型 生物学不断的更新使肠道容易患结肠直肠癌和损伤，如辐射毒性。 肠干细胞（ISCs）是肠道更新的组成部分，因此也是肠道中众多的干细胞中的许多。 病理学尽管ISCs的临床意义，两个不一致的模型，他们的身份和行为， 存在.分层模型认为存在两种不同的ISC类型-罕见的静止干细胞（QSC）和 更丰富，快速分裂的隐窝基底柱状干细胞（CBCs）。CBCs和QSC都可以自我更新， QSC可以是不同的，但QSC只能来自其他QSC。另一方面，连续统模型认为， CBCs是唯一真正的干细胞，它们可以以连续的状态存在，包括静止。 为解决这两种模式所作的广泛努力都是不成功和相互矛盾的。主要来源 混淆的原因是过度依赖所谓的细胞类型特异性启动子，它们标记重叠和 异质细胞群。 需要一种新颖、公正且精确的方法来探索不同QSC的存在 人口考虑到这一点，我们将联合收割机系统发育推断与CRISPR/Cas9基因组编辑相结合， 在不使用偏向性启动子的情况下彻底绘制肠细胞分裂树。我们将产生 含有CRISPR/Cas9系统元件以及短合成DNA的转基因小鼠 Cas9靶向的条形码序列。当肠细胞分裂时，它们的条形码被切割和修复， 非同源末端连接（NHEJ）的突变倾向途径，产生了巨大的多样性， 遗传突变然后从单细胞中对条形码进行测序，沿着转录组， 细胞谱系历史和身份的完整图片。 通过将该系统应用于肠道，我们假设我们将观察到与肠道匹配的谱系树。 分层模型的预测，即所有其他细胞分支的连续QSC谱系。的 连续的QSC谱系的存在将在面对复制和 代谢压力，这对致癌和衰老生物学有影响。更广泛地说，我们的方法 将作为一个模板，探测组织的发展，维护，损伤，和肿瘤在各种 前所未有的深度。

Project Summary The intestine is the highest turnover tissue in the body, making it an excellent model of adult stem cell biology. Constant renewal makes the gut susceptible to colorectal cancer and injury, such as radiation toxicity. Intestinal stem cells (ISCs) are integral to the renewal of the intestine and hence to many of its numerous pathologies. Despite the clinical significance of ISCs, two unreconciled models of their identity and behavior exist. The hierarchical model argues that two distinct ISC types exist – rare quiescent stem cells (QSCs) and more abundant, rapidly-dividing crypt base columnar stem cells (CBCs). Both CBCs and QSCs self-renew and differentiate, but QSCs can only come from other QSCs. On the other hand, the continuum model argues that CBCs are the only true stem cells and that they can exist in a continuum of states, including quiescence. Extensive efforts to resolve these two models have been unsuccessful and contradictory. The primary source of confusion has been an overreliance on purported cell type-specific promoters, which mark overlapping and heterogeneous cell populations. A novel, unbiased, and precision approach is required to probe the existence of a distinct QSC population. With this in mind, we will combine phylogenetic inference with CRISPR/Cas9 genome editing to thoroughly map the intestinal cellular division tree without the use of biased promoters. We will generate transgenic mice that contain the elements of the CRISPR/Cas9 system, as well as a short synthetic DNA barcode sequence that is targeted by Cas9. As intestinal cells divide, their barcodes are cut and repaired by the mutation-prone pathway of nonhomologous end-joining (NHEJ), producing an enormous diversity of heritable mutations. Barcodes are then sequenced from single cells, along with the transcriptome, providing a complete picture of both cellular lineage history and identity. By applying this system to the intestine, we hypothesize that we will observe lineage trees matching the predictions of the hierarchical model, namely a continuous QSC lineage that all other cells branch from. The existence of a continuous QSC lineage would act to maintain genome integrity in the face of replicative and metabolic stress, which carries implications for carcinogenesis and aging biology. More broadly, our approach will serve as a template for probing tissue development, maintenance, injury, and neoplasia across a variety of tissues with unprecedented depth.