Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants

Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7,868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior versus posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5. At E10.5, we found that anterior PGCs upregulate Nodal transcriptional targets including Lefty1/2 and validated this LEFTY1/2 upregulation via whole-mount immunofluorescence staining. This positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency and epigenetic reprogramming. We pooled sorted GFP+ germ cells and an equal amount of GFP- somatic cells from each timepoint and anatomical location. We loaded counted cells onto a 10X V2 chip to create a single cell emulsion. For E9.5 Anterior, we loaded 3400 cells, and for E9.5 Posterior, 2800 cells. For E10.5 Anterior, we loaded two technical replicates totaling 9300 cells. For E10.5 Posterior, we loaded two technical replicates totaling 11200 cells. For the two E11.5 biological replicates, we loaded 15,000 and 10,000 cells, respectively. Library creation was done in house following 10X ChromiumTM Single Cell 3' Reagent Kits v2 User Guide Rev A. E9.5 and E11.5 libraries were sequenced on HiSeq 4000 (Illumina) with paired-end sequencing parameters: Read1, 98 cycles; Index1, 14 cycles; Index2, 8 cycles; and Read2, 10 cycles. E10.5 libraries were sequenced on Novaseq (Illumina) with paired-end sequencing parameters: Read1, 150 cycles; i7 Index, 8 cycles; i5 index, 0 cycles; and Read2, 150 cycles.

哺乳动物原始生殖细胞（primordial germ cells, PGCs）会通过胚胎后肠与背系膜异步迁移，最终抵达生殖腺。为解析迁移中PGCs及其微环境的转录异质性，我们对迁移阶段（E9.5、E10.5、E11.5）、按前/后位置分选出的13262个小鼠PGCs与7868个周围体细胞进行了单细胞RNA测序，以富集前沿与滞后迁移的细胞群。按位置对PGCs的分析显示，在E9.5时期，前部的快速/早期迁移细胞与后部的慢速/晚期迁移细胞之间存在动态基因表达变化。在E10.5时期，我们发现前部的PGCs会上调Nodal的转录靶基因（包括Lefty1/2），并通过整体免疫荧光染色验证了LEFTY1/2的上调现象。这份小鼠PGCs的位置与时间维度图谱证实：迁移路径上的微环境互作会引发细胞增殖、肌动蛋白动态、多能性及表观重编程相关的改变。我们将每个时间点与解剖位置分选得到的GFP阳性生殖细胞与等量GFP阴性体细胞进行混合，将计数后的细胞加载至10X V2芯片以构建单细胞乳液。其中E9.5前部样本加载3400个细胞，E9.5后部样本加载2800个细胞；E10.5前部样本设置2个技术重复，总计加载9300个细胞；E10.5后部样本同样设置2个技术重复，总计加载11200个细胞；两份E11.5生物学重复样本分别加载15000与10000个细胞。文库构建严格遵循10X Chromium™ 单细胞3'试剂试剂盒v2用户指南Rev A版本在实验室内部完成。E9.5与E11.5文库在Illumina HiSeq 4000平台进行双端测序，测序参数为：Read1 98个循环、Index1 14个循环、Index2 8个循环、Read2 10个循环。E10.5文库则在Illumina NovaSeq平台进行双端测序，测序参数为：Read1 150个循环、i7 Index 8个循环、i5 Index 0个循环、Read2 150个循环。