# Developmental scRNASeq Trajectories in Gene- and Cell-State Space--The Flatworm Example
## 3 Results
### The Transcriptome Landscape of Planarian  
Next, we further characterized the topology of SOM expression landscape and its functional context in detail. In total, we segmented the 3600 metagenes used for the SOM training into 32 expression modules by applying k-means clustering to the metagene's expression profiles. The modules were labeled by capital letters \"A-F1\" (Figure 3A). A summary of the top expressed marker genes in each of the modules, enriched functional gene sets, and samples showing upregulation are provided in Table 1. The modules are divided into 11 highly variant expression clusters (A, D, F, J, L, N, R, B1, D1, E1, F1) and into 21 less variant clusters. The former ones form \"spot-like\" red areas along the periphery of the SOM which specifically upregulate in the different tissues (Figure 3C). The low variance clusters were found in the middle and right lower part of the map. About 65% of all genes are virtually invariant and accumulate in cluster A1 evident as blue area in the summary and variance maps (Figure 3B). Hence, the topology of the SOM splits into one developmental source spot-collecting genes overexpressed in stem cells, into a series of developmental sink spots-collecting genes upregulated in differentiated tissues and into an intermediate transition region in between. Further, SOM identified an area collecting virtually invariant genes and spot R of unknown origin. Presumably, it can be attributed to methodical effects systematically disturbing parts of the expression patterns. The landscape is governed by the amplitudes and particular shapes of the gene expression profiles. They are shown around the map for selected modules together with gene maps indicating the positions of gene referring to specific Gene Ontology (GO) terms (Figure 3A). For example, genes of the GO-term \"Muscle structure development\" accumulate in and near the \"muscle\" module L upregulated in muscle tissue this way confirming its functional impact. The heatmap in Figure 3C visualizes the profiles of the less variant clusters. Overall, these profiles split into three major strata either upregulated in stem cells (marked with grey color), progenitor cells (light apricot), or differentiated cells (apricot), respectively (Figure 3A-C). The progenitor cell clusters can be further divided into \"earlier progenitors\" activated still in stem cells and into \"later progenitor cells\" not showing activation in stem cells. Marker genes of differentiation between the different planarian cell types (Table S1 and [44]) map into or near the modules upregulated in the respective cell types (Figure 3D). For example, the epidermal module J includes the epidermal gene markers prog-1 or agat-1 while the stemness module N contains the stem cell marker Smedwi-1 and tub-\(\alpha\)1. Overexpression analysis of selected reference sets of co-regulated genes also well agreed with our modules. They further specify functional interpretation, e.g., in terms of activated cell cycle phases within the stem cell compartment (see Figure S1B-E). Note that spots and underlying cellular programs are activated in different cell types (see the profiles in Figure 3A). For example, cell-cycle-related transcription is virtually active in all cell types however to a different extent (Figure S1D). These genes locate mostly in spots W and V near the stemness module N. Other common programs are more selective: genes related to morphogenesis accumulate in spot F upregulated in epidermal and muscle tissues. Such genes activated in several differentiated tissues overall accumulate in a stripe-like area near the lower left corner of the map including spots F, Y, and M (Figure 3A,D). Gene set enrichment analysis using the database planMine for mining flatworm genomes and transcriptomes [43] provided detailed functional information associated with each cell type (Table 1).