Illumina HiSeq 2500 sequencing; GSM2892790: HH11_Trailblazer_1; Gallus gallus; RNA-Seq

HH11_Trailblazer_1

Neural crest cells migrate throughout the embryo, but how cells move in a directed and collective manner has remained unclear. Here, we perform the first single-cell transcriptome analysis of cranial neural crest cell migration at three progressive stages in chick and identify and establish hierarchical relationships between cell position and time-specific transcriptional signatures. We determine a novel transcriptional signature of the most invasive neural crest Trailblazer cells that is consistent during migration and enriched for approximately 900 genes. Knockdown of several Trailblazer genes shows significant but modest changes to total distance migrated. However, in vivo expression analysis by RNAscope and immunohistochemistry reveals some salt and pepper patterns that include strong individual Trailblazer gene expression in cells within other subregions of the migratory stream. These data provide new insights into the molecular diversity and dynamics within a neural crest cell migratory stream that underlie complex directed and collective cell behaviors. Overall design: 501 single cells analyzed from various parts of the migrating neural crest during various timepoints in development. 96 cells from HH11 Trailblazers, 50 cells from HH13 Trailblazers, 50 cells from HH13 Leaders, 60 cells from HH13 Trailers, 50 each from HH15 Trailblazers, Leaders, and Trailers. For definitions, see the extract protocol. Neural crest from a neural tube placed in culture were also analyzed.

Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions [scRNA-seq]

Submitted on 20-DEC-2017

For scRNA-seq, the NC r4 stream was isolated by manual dissection at HHSt11, 13 and 15. HHSt11 r4 streams containing few migrating NC were pooled. For HHSt13 and 15 embryos, the r4 NC stream was further subdivided by manual dissection into three spatially distinct, non-overlapping portions: the most invasive front (5% of the stream, termed 'Front'), the leading portion of the stream (25% of stream proximal of the most invasive front, termed 'Lead') and the proximal-most trailing portion of the stream (70% of the stream, termed 'Trail'). Developmental stage- and stream position-matched tissues were pooled and dissociated before cytometric isolation. Cells were isolated by FACS, which included forward scatter, side scatter, pulse width, live/dead stain and YFP gates. Cells were sorted directly into 6 ul of Clontech lysis solution containing 0.05% RNAse inhibitor. Following lysis for 5 min at room temperature, lysates were immediately frozen on dry ice and stored at −80C. Single-cell lysates were thawed on ice. cDNA synthesis and library preparation were performed with SMART-seq v4 Ultra Low Input RNA-seq (634892, Takara, Kusatsu, Shiga, Japan) and Nextera XT DNA dual indexing library preparation kits as recommended by the manufacturer (FC-131–1096, Illumina). Despite more cost-effective scRNAseq strategies, SMARTseq v4 was employed in order to detect the greatest number of genes per single cell, which is critical for distinguishing highly similar cell subpopulations (Ziegenhain et al., 2017; Dueck et al., 2016). 

Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions [scRNA-seq]

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