van Oosten, E. | Search bioRxiv

bioRxiv2021

Functional single cell selection and annotated profiling of dynamically changing cancer cells

A method connecting single cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, would be transformative for single cell and cancer biology. Here, we present fSCS: functional single cell selection. This technology combines a custom-built ultrawide field-of-view optical screening microscope, fast automated image analysis and a new photolabeling method, phototagging, using a newly synthesized visible-light-photoactivatable dye. Using fSCS, we screen, selectively photolabel and isolate cells of interest from large heterogeneous populations based on functional dynamics like fast migration, morphological variation, small molecule uptake or cell division. We combined fSCS with single cell RNA sequencing for functionally annotated transcriptomic profiling of fast migrating and spindle-shaped MCF10A cells with or without TGF{beta} induction. We identified critical genes and pathways driving aggressive migration as well as mesenchymal-like morphology that could not be detected with state-of-the-art single cell transcriptomic analysis. fSCS provides a crucial upstream selection paradigm for single cell sequencing independent of biomarkers, allows enrichment of rare cells and can facilitate the identification and understanding of molecular mechanisms underlying functional phenotypes.

bioengineering↗

bioRxiv2021

Plagl1 is part of the mammalian retinal injury response and a critical regulator of Muller glial cell quiescence

Retinal damage triggers reactive gliosis in Muller glia across vertebrate species, but only in regenerative animals, such as teleost fish, do Muller glia initiate repair; proliferating and undergoing neurogenesis to replace lost cells. By mining scRNA-seq and bulk RNA-seq datasets, we found that Plagl1, a maternally imprinted gene, is dynamically regulated in reactive Muller glia post-insult, with transcript levels transiently increasing before stably declining. To study Plagl1 retinal function, we examined Plagl1+/-pat null mutants postnatally, revealing defects in retinal architecture, visual signal processing and a reactive gliotic phenotype. Plagl1+/-pat Muller glia proliferate ectopically and give rise to inner retinal neurons and photoreceptors. Transcriptomic and ATAC-seq profiles revealed similarities between Plagl1+/-pat retinas and neurodegenerative and injury models, including an upregulation of pro-gliogenic and pro-proliferative pathways, such as Notch, not observed in wild-type retinas Plagl1 is thus an essential component of the transcriptional regulatory networks that retain mammalian Muller glia in quiescence.

neuroscience↗