Whole brain analysis of cellular diversity and developmental lineage using single-cell approaches
Bushra Raj1*, Daniel E. Wagner2, James A. Gagnon1, Allon Klein2, and Alexander F. Schier1*
1Dept. of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA; 2Dept. of Systems Biology, Harvard Medical School, Boston, MA, USA
Although studies have begun to characterize the heterogeneity of neurons in the brain, their developmental histories remain largely unknown. We have established a method for combinatorial lineage recording in a synthetic array across two timepoints in zebrafish development using a genomic editing system. We read edits in the array via high throughput sequencing and use patterns of shared edits in the progeny to construct cell lineage relationships. We find that our approach genetically embeds lineage information in progenitor populations at the end of each timepoint such that early and late lineage relationships can be more accurately explored. We have also coupled this technique with droplet microfluidics to simultaneously profile transcriptomes and lineages of many cells. In a pilot run we profiled the transcriptomes of 13,500 single cells from the juvenile zebrafish brain. Using principal component analysis and unsupervised clustering algorithms, we have begun to group cells into clusters based on similarities of gene expression signatures. We identified 56 putative clusters that represent many of the expected cell types including neurons, neural stem/progenitor cells and glial cells, and are characterized by spatial signatures that can be used for silico spatial mapping of clusters to brain regions. Furthermore, we have successfully captured the sequential lineage history of >1000 of these cells. We developed a pipeline to relate lineage records with transcriptional profiles of the same cells, and are using this to investigate the developmental lineage of multiple cell types in the brain. The results will be important for understanding the relationships between diverse cell types during brain development and their maintenance during homeostasis.
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