Speaker
Description
Primary cilia (PCs) are single-copy organelles functioning as important signaling hubs for multimodal stimuli. Dysregulation of PCs leads to syndromic phenotypes (including hindbrain malformations) and developmental disorders referred to as primary ciliopathies. Aberrant activation of ciliary pathways is a major driver in the formation of brainstem and cerebellar tumors. It is well known that the structure, function, and composition of PCs is tightly regulated by cell cycle, but it is becoming increasingly clear that other factors also regulate ciliary dynamics. However, little is known about the transcriptional profiles associated with such ciliary dynamics at a single-cell resolution in intact tissue environments. We use the chicken cerebellum and brainstem in combination with scRNA-seq and spatial transcriptomics to investigate the ciliary transcriptional landscape across cerebellar development. Classical in-situ hybridization of candidate primary cilia genes across cerebellar and spinal cord development revealed differential expression patterns in both space and time. Using scRNA-seq, we were able to identify 827 chicken homologs of the 956 genes listed in the CiliaCarta (a validated compendium of ciliary genes) across different cerebellar cell types and states of differentiation. Unsupervised clustering based on the CiliaCarta homologs identifies distinct ciliary transcriptional clusters. We found that ciliary transcriptional clusters map closely to general cell type clusters. To investigate the spatial distribution and relationships between these ciliary transcriptional clusters, we have established a BARseq2 pipeline. BARseq2 allows for highly-multiplexed spatial transcriptomic readouts by combining nucleotide barcodes with Illumina in-situ sequencing. Initial small-scale pilot experiments have shown the BARseq2 pipeline to be compatible with chicken embryonic cerebella. We are scaling up the BARseq2 panel to > 100 genes to investigate spatially-resolved co-expression at single-cell resolution in an intact cerebellar environment. By ultimately collecting data across different stages of cerebellar development, we will be able to map ciliary transcriptional clusters across space and time.
| Author(s) | Reto B. Cola*, Esther T. Stoeckli |
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| Affiliation(s) | University of Zurich |
