Fig. 1: Multiplex Single Cell Spatial Transcriptomics reveals pluripotent-like cells broadly throughout the neurulating ectoderm.

a Experimental plan and a schematic demonstrating the rise of the neural folds and how the ectoderm is patterned into three domains during neurulation. Dashed squares demonstrate fields of views imaged for scMST. All twelve stages were used for bulkRNAseq and asterisks highlight the four stages that were used for scMST and scRNAseq. The Chick (yellow) image was created using BioRender.com, HH Hamburger Hamilton stage, SS Somite Stage. b The scMST pipeline demonstrates serial hybridization rounds, 3D segmentation of cells and how individual RNA transcripts (white dots) are quantified and analyzed per cell. Then, pooled data of transcript counts in individual cells is pooled into a heatmap followed by back-mapping of cells in the subclusters to visualize their location in the original tissue sections by using pseudo-coloring. c List of genes chosen for scMST. d Pseudo-colored scMST subpopulations at the end of neurulation consists of committed cells as well as separate domains of neural crest and neural stem cells, respectively. n = 4 biological replicates. Scale bar = 30 µm. e scMST results from early neural fold stage highlight broad existence of cells with a pluripotency-like signature throughout the developing ectoderm. Based on unbiased hierarchical clustering, the heatmap shows transcriptionally defined subpopulations labeled with a color. Circle symbols indicate committed cell groups and square symbols indicate stem cell groups. f Each single cell is mapped back into the original position in the embryo image and pseudo-colored according to their representative subpopulation as labeled in the heatmap in e. n = 4 biological replicates. Scale bar = 30 µm. The stem cell groups co-express pluripotency genes together with ectodermal domain markers. Cells in the undecided stem cell population (orange) are situated across the entire ectoderm and demonstrate highest stem cell potential as the future ectodermal domain they will commit to can not be predicted from the transcriptional profile. f’ Higher magnification of committed subpopulations revels that the cells of different groups do not overlap spatially, whereas f”, transitioning stem cell groups show spatial overlap. Source data for scMST are provided as a Source Data file. NNE non neural ectoderm, DNT dorsal neural tube, NT neural tube, NF neural fold, M mesoderm.