Single-muscle-cell transcriptome profiling along the planarian anteroposterior axis

How positional information instructs adult tissue maintenance is poorly understood. Planarians undergo whole-body regeneration and tissue turnover, providing a model for adult positional information studies. Genes encoding secreted and transmembrane components of multiple developmental pathways are predominantly expressed in planarian muscle cells. Several of these genes regulate regional identity, consistent with muscle harboring positional information. Here, single-cell RNA-sequencing of 115 muscle cells from distinct anterior-posterior regions identified 44 regionally expressed genes, including multiple Wnt and ndk/FGF receptor-like (ndl/FGFRL) genes. Two distinct FGFRL-Wnt circuits, involving juxtaposed anterior FGFRL and posterior Wnt expression domains, controlled planarian head and trunk patterning. ndl-3 and wntP-2 inhibition expanded the trunk, forming ectopic mouths and secondary pharynges, which independently extended and ingested food. fz5/8-4 inhibition, like that of ndk and wntA, caused posterior brain expansion and ectopic eye formation. Our results suggest that FGFRL-Wnt circuits operate within a body-wide coordinate system to control adult axial positioning. Overall design: Single-cell RNA-seq on cells isolated from 10 distinct anteroposterior (AP) regions and expressed muscle markers was used to identify muscle regionally expressed genes (mRGs). Non-dividing single cells from 10 consecutive regions along the AP axis were isolated by fluorescence activated cell sorting (FACS), and the resulting single-cell cDNA libraries were screened by qRT-PCR for expression of planarian muscle markers troponin or collagen before sequencing. Principal component analysis (PCA) on the 177 single cells sequenced that expressed >1000 unique transcripts (>2 read count) was performed using highly variable transcripts. Two significant principal components that separated cells by expression of muscle markers (PC1<0) and expression of neoblast markers (PC2<0) were identified. PCA and troponin expression confirmed the identity of 115 muscle cells, and these 115 cells were used in all subsequent analyses. Single-cell differential expression (SCDE, (Kharchenko et al. 2014)) analyses of three different anterior-versus-posterior region comparisons (anterior(regions 1, 2, 3; n=23 cells) versus posterior (regions 8, 9, 10; n=38 cells); head (region 1; 11 cells) versus post-pharyngeal (regions 7, 8, 9; n=35 cells); pre-pharyngeal (regions 2, 3, 4; n=22 cells) versus tail (region 10; n=12 cells)) identified transcripts of 99 genes as differentially expressed at p<0.005 (|Z| > 2.58). Whole mount in situ hybridization to visualize gene expression patterns verified 44 genes as regionally expressed (mRGs) (35/44 with p<0.005 in any of anterior-versus-posterior SCDE analyses).