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. Overall design: 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.