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Deciphering the principles and mechanisms by which gene activity orchestrates complex cellular arrangements in multicellular organisms has far-reaching implications for research in the life sciences. Recent technological advancements in next-generation sequencing-based and imaging based approaches have established the potential of spatial transcriptomics to measure expression levels of all or most genes systematically throughout tissue space, and have been adopted to generate biological insight in neuroscience, development, plant biology, and a range of diseases including cancer. Similar to datasets made possible by genomic sequencing and population health surveys, the large-scale atlases generated by this technology lend themselves to exploratory data analysis for hypothesis generation. Here, we review spatial transcriptomic technologies and describe the repertoire of operations available for paths of analysis of the resulting data. Spatial transcriptomics can also be deployed for hypothesis testing using experimental designs comparing timepoints or conditions -including genetic or environmental perturbations. Finally, spatial transcriptomic data is naturally amenable to integration with other data modalities providing an expandable framework for insight into tissue organization.Many of the notable discoveries in the life sciences followed from the recognition that cellular organization within tissues is intimately linked to biological function. In developmental biology, central topics such as symmetry-breaking between daughter cells and cell fate decisions are based on spatial relationships between cells 1 . In clinical settings, histopathology is often used as a conclusive diagnostic, precisely because diseases are characterized by abnormal spatial organization within tissues 2 . Infectious and inflammatory processes can drastically change the cellular organization of tissues 3 . These discoveries were supported by methods in molecular biology -including in situ hybridization 4 (ISH) and immunohistochemistry 5 -that provided the ability to visualize biological processes more directly by mapping DNA, RNA and protein within tissues. However, these methods limit analysis to at most a handful of genes or proteins at a time.

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Abstract:A long-standing question in molecular biology relates to why the testes express the largest number of genes relative to all other organs. Here, we report a detailed gene expression map of human spermatogenesis using single-cell RNA-Seq. Surprisingly, we found that 20 spermatogenesis-expressed genes contain significantly fewer germline mutations than unexpressed genes, with the lowest mutation rates on the transcribed DNA strands. These results suggest a model of 'transcriptional scanning' to reduce germline mutations by correcting DNA damage. This model also explains the rapid evolution in sensory-and immune-defense related genes, as well as in male reproduction genes. Collectively, our results indicate that widespread 25 expression in the testes achieves a dual mechanism for maintaining the DNA integrity of most genes, while selectively promoting variation of other genes.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/282129 doi: bioRxiv preprint first posted online Mar. 14, 2018; 2 Main Text:Human tissues and organs are distinguished by the genes that they express and those that they do not 1,2 . Tissues have transcriptomes of different complexities in terms of uniquely-expressed genes, as well as those genes expressed at differential levels [3][4][5][6] . One overarching goal in the life sciences is to characterize the specific transcriptomic signatures of all human tissues, and 5 ultimately each different cell type at the single-cell level 7 .In males, the testis is unique in comparison with somatic tissues in that it contains germ cells which pass the genetic information on to the next generation 8 . Interestingly, it has been known for many years that the testis stands out as having the most complex transcriptome with the highest number of expressed genes [9][10][11][12] . Widespread transcription in the testes has been 10 reported to account for an amazing expression of over 80% of all our protein-coding genes 10,11,13 , as well as across many other mammals 3,10 .Several hypotheses have been proposed to explain this observation. Widespread expression may represent a functional requirement for the gene-products in question 12 .However, other more complex organs such as the brain do not exhibit a corresponding number of 15 expressed genes despite the fact that they consist of a substantially greater number of distinct cell types 3,10,14-16 . Moreover, recent animal studies have shown that many testis-enriched and evolutionarily-conserved genes are not required for male fertility in mice 17 . A second hypothesis implicates leaky transcription during the massive chromatin remodeling that occurs throughout spermatogenesis 12,18,19 . However, this model predicts more expression during later stages of 20 spermatogenesis -when the genome is undergoing the most chromatin changes -contradicting ...

Highlights d Genes expressed in the testis have reduced germline mutation rates d Germline mutational signature is tuned by spermatogenesisgene expression levels d Genes not expressed during spermatogenesis are enriched for fast-evolving functions d A germline mutational signature generated by TCR follows a ''3 0 -pyrimidine rule''