Computations in the mammalian cortex are carried out by glutamatergic and γ-aminobutyric acid-releasing (GABAergic) neurons forming specialized circuits and areas. Here we asked how these neurons and areas evolved in amniotes. We built a gene expression atlas of the pallium of two reptilian species using large-scale single-cell messenger RNA sequencing. The transcriptomic signature of glutamatergic neurons in reptilian cortex suggests that mammalian neocortical layers are made of new cell types generated by diversification of ancestral gene-regulatory programs. By contrast, the diversity of reptilian cortical GABAergic neurons indicates that the interneuron classes known in mammals already existed in the common ancestor of all amniotes.
Removal of the germ cells of C. elegans extends lifespan in part because signals from the somatic reproductive tissues activate the nuclear hormone receptor DAF-12.
The two parts of the Caenorhabditis elegans reproductive system, the germ cells and the somatic reproductive tissues, each influence the life span of the animal. Removing the germ cells increases longevity, and this life span extension requires the somatic gonad. Here we show that the somatic gonad and the germ cells make distinct contributions to life span determination. The life span increase produced by loss of the germ cells requires the DAF-16/FOXO transcription factor. In response to germ-cell removal, DAF-16 accumulates in nuclei. We find that the somatic gonad is not required for DAF-16 nuclear accumulation or for the increased stress resistance that is produced by germ-cell removal. The somatic gonad is required, however, for expression of specific DAF-16 target genes. DAF-16 is known to be activated by reduced insulin/IGF-1 signaling in C. elegans. In certain insulin/IGF-1-pathway mutants, the somatic gonad is not required for germ-cell removal to extend life span. Our genetic experiments suggest that these mutations reduce insulin/IGF-1 signaling below a critical threshold level. At these low levels of insulin/IGF-1 signaling, factors normally provided by the somatic gonad are no longer needed for germ-cell removal to increase the expression of DAF-16 target genes.
Background
Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation.
Results
Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures.
Conclusion
Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.
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