Single cell next generation sequencing of wild type and Dnmt1 conditional knock in E18.5 embryonic lung. Single cell next generation sequencing of wild type and Dnmt1 conditional knock in E18.5 embryonic lung

Background DNA methylation is an epigenetic mark that restricts chromatin accessibility and serves to modulate temporal and spatial gene expression during organogenesis. Dnmt1 is one of the most studied DNA methyltransferases, and it is primarily involved in preserving the DNA methylation pattern in cells undergoing mitotic division. While the role of Dnmt1 in the embryonic lung endoderm has been revealed, its role in expansion, maintence and differentiation of the embryonic lung mesoderm is mostly unknown. Here, we present the first evidence showing that Dnmt1 is necessary for the proper development of the embryonic lung mesoderm. By selectively deleting Dnmt1 in the embryonic lung mesoderm we found that Dnmt1 is critical for the development of embryonic vasculature and fully commitment of mesoderm towards the lung mesenchymal cell lineages. Results Dnmt1 deletion in the embryonic lung mesenchyme at the E7.5 stage led to a lethal phenotype which was characterized by bilateral lung hypoplasia, impaired lung branching morphogenesis, and widespread hemorrhages in the parenchyma. The genesis of the hemorrhages was likely a result of halted development of lung vasculature, specifically the ontogeny of pericytes, which was detected in the mutant lungs. However, despite the severe mesenchyme alteration, differentiation of the lung endoderm appeared normal. When Dnmt1 was deleted at later developmental periods (E13.5), the lung matured normally and mutant pups were viable, although reduced differentiation of Pdgfr- myofibroblasts and alveolar simplification were observed in all mutant animals as soon as 7 days after birth. Gene expression profiling studies revealed that deletion of Dnmt1 induced the ectopic expression of genes specific for testis, placenta, and ovary, such as Tex10.1, Tex19.1 and Pet2 which suggested a loss of commitment of the lung mesoderm toward the pulmonary cell lineages. Conclusions Taken together our findings have shown that dnmt1 expression in the mesenchyme of the developing lung is crucial for restricting the differentiation capacity of the pulmonary mesoderm and, thus, ensuring the adequate differentiation of vasculature cells and myofibroblasts in the fetal and postnatal lung. Overall design: Examination of gene expression changes at the single cell level in wild type and dnmt1-null embryonic murine lungs.

**背景** DNA甲基化（DNA methylation）是一类表观遗传标记（epigenetic mark），可限制染色质可及性（chromatin accessibility），并在器官发生（organogenesis）过程中调控基因的时空表达模式。Dnmt1是研究最为广泛的DNA甲基转移酶（DNA methyltransferase）之一，其主要功能是维持处于有丝分裂（mitotic division）阶段的细胞中的DNA甲基化谱式。尽管Dnmt1在胚胎肺内胚层（embryonic lung endoderm）中的作用已被阐明，但其在胚胎肺中胚层（embryonic lung mesoderm）的扩增、维持与分化过程中的功能仍尚不明确。本研究首次提供证据表明，Dnmt1对于胚胎肺中胚层的正常发育不可或缺。通过特异性敲除胚胎肺中胚层中的Dnmt1，我们发现其对于胚胎脉管系统（vasculature）的发育，以及中胚层向肺间充质细胞谱系（lung mesenchymal cell lineages）的完全定向分化至关重要。

**结果** 在胚胎发育E7.5阶段敲除胚胎肺间充质中的Dnmt1，会引发致死表型，具体表现为双侧肺发育不全、肺分支形态发生受损，以及肺实质组织广泛出血。出血现象的产生大概率源于肺脉管系统发育停滞，尤其是突变肺组织中检测到的周细胞（pericytes）发生受阻。尽管间充质出现严重异常，但肺内胚层的分化却未受明显影响。当在发育后期（E13.5阶段）敲除Dnmt1时，肺可正常成熟，突变幼崽可存活，但所有突变个体在出生后7天内均出现了Pdgfr-α阳性肌成纤维细胞（myofibroblasts）分化受损以及肺泡简化现象。基因表达谱分析（gene expression profiling）研究显示，敲除Dnmt1会诱导睾丸、胎盘与卵巢特异性基因的异位表达（ectopic expression），例如Tex10.1、Tex19.1及Pet2，这表明肺中胚层向肺细胞谱系的定向分化能力出现了缺失。

**结论** 综合本研究所有结果可知，发育中肺的间充质内dnmt1的表达对于限制肺中胚层的分化潜能至关重要，从而确保胎儿期与出生后肺内脉管系统细胞及肌成纤维细胞的正常分化。

**整体实验设计**：对野生型与dnmt1敲除型胚胎小鼠肺组织开展单细胞水平的基因表达变化检测。