Transcriptional Analysis Of Protocadherin Gene Clusters At Single-cell Resolution

Protocadherin(Pcdh)gene clusters are organized into three closely-linked clusters(Pcdh?,? and ?).Pcdh? and ? gene clusters contain variable exons(C-type and non C-type)and constant exons.As previous study shows,the stochastic expression of alternate Pcdh isoforms(non C-type)and the constitutive expression of C-type isoforms coexist in single neuron,but this expression pattern is based on the study of population of one cell type.It must be tested in other types of neurons to determine whether this is a general mechanism.In addition,some cell lines express a specific subset of Pcdh isoforms,whether all of single cells in culture express these Pcdh isoforms are yet to be determined.Finally,the directionality of regulatory elememt HS5-1 is important for expression of Pcdh?,the issue remains unknown as how HS5-1 directionality regulates Pcdh? expression at the single-cell level.Firstly,Single-cell RT-PCR and Single-cell RNA-Seq analyses of Pcdh expression patterns in individual mouse cerebral cortex cells reveal that the C type and non-C type isoforms of Pcdh? and ? were expressed stochastically,and each single cell combinatorially expressed several Pcdh isoforms.Secondly,Single-cell RT-PCR and Single-cell RNA-Seq analysis of Pcdh isoforms in Hec-1-B and SK-N-SH single cells reveals that distinct single cells express different subsets of Pcdh isoforms.Finally,we used single-cell RT-PCR method to investigate the effects of the HS5-1 inversion on Pcdh? isoforms expression.Interestingly,we found that the directionality of HS5-1 plays an important role in differential expression of Pcdh? isoforms at the single-cell level.These investigations on expression patterns of Pcdh gene clusters and functions of HS5-1 directionality shed light into the single-cell diversity generated by the combinatorial expression of Pcdh gene clusters.It will not only help us to fully understand possible roles of Pcdh as neuronal identity molecules in the neural circuit assembly but also has important implications for systematic understanding of the molecular mechanisms of some mental disorders.