Faculty Bibliography

Single-cell RNA sequencing has revolutionized the way we look at cell populations. Of the methods available, CEL-Seq was the first to use linear RNA amplification. With early barcoding and 3' sequencing, it is sensitive, cost-effective and easy to perform. Here we describe a protocol for performing CEL-Seq2 on sorted cells, which can be performed without any special equipment.

Developmental programs are executed by tightly controlled gene regulatory pathways. Here, we combined the unique sample retrieval capacity afforded by laser capture microscopy with analysis of mRNA abundance by CEL-Seq, to generate a spatiotemporal gene expression map of the C. elegans syncytial germline from adult hermaphrodites and males. We found that over 6,000 genes exhibit spatiotemporal dynamic expression patterns throughout the hermaphrodite germline, with two dominant groups of genes exhibiting reciprocal shifts in expression at late pachytene during meiotic prophase I. We found a strong correlation between restricted spatiotemporal expression and known developmental and cellular processes, indicating that these gene expression changes may be an important driver of germ cell progression. Analysis of the male gonad revealed a shift in gene expression at early pachytene, and upregulation of subsets of genes following the meiotic divisions, specifically in early and late spermatids, mostly transcribed from the X chromosome. We observed that while the X chromosome is silenced throughout the first half of the gonad, some genes escape this control and are highly expressed throughout the germline. Although we found a strong correlation between the expression of genes corresponding to CSR-1-interacting 22G-RNAs during germ cell progression, we also found that a large fraction of genes may bypass the need for CSR-1-mediated germline licensing. Taken together, these findings suggest the existence of mechanisms that enable a shift in gene expression during prophase I to promote germ cell progression.

The evolution of development has been studied through the lens of gene regulation by examining either closely related species or extremely distant animals of different phyla. In nematodes, detailed cell- and stage-specific expression analyses are focused on the modelCaenorhabditis elegans, in part leading to the view that the developmental expression of gene cascades in this species is archetypic for the phylum. Here, we compared two species of an intermediate evolutionary distance: the nematodesC. elegans(clade V) andAcrobeloides nanus(clade IV). To examineA. nanusmolecularly, we sequenced its genome and identified the expression profiles of all genes throughout embryogenesis. In comparison withC. elegans,A. nanusexhibits a much slower embryonic development and has a capacity for regulative compensation of missing early cells. We detected conserved stages between these species at the transcriptome level, as well as a prominent middevelopmental transition, at which point the two species converge in terms of their gene expression. Interestingly, we found that genes originating at the dawn of the Ecdysozoa supergroup show the least expression divergence between these two species. This led us to detect a correlation between the time of expression of a gene and its phylogenetic age: evolutionarily ancient and young genes are enriched for expression in early and late embryogenesis, respectively, whereas Ecdysozoa-specific genes are enriched for expression during the middevelopmental transition. Our results characterize the developmental constraints operating on each individual embryo in terms of developmental stages and genetic evolutionary history.

miRNAs play essential roles in the mechanics of gene regulation, however, on an organismal-scale, the processes in which they are deployed are not well understood. Here, we adopt an evolutionary developmental approach to study miRNA function by examining their expression throughout embryogenesis in both C. elegans and D. melanogaster. We find that, in both species, miRNA transcriptomic shifts in a punctuated fashion during the mid-developmental transition, specifying two dominant modes of early and late expression profiles. Strikingly, late-expressed miRNAs are enriched for phylogenetic conservation and function by fine-tuning the expression of their targets, implicating a role in the canalization of cell-types during differentiation. In contrast, early-expressed miRNAs are inversely expressed with their targets suggesting strong target-inhibition. Taken together, our work exposes a bimodal role for miRNA function during animal development, involving late-expressed physiological roles and early-expressed repressive roles.

Evolution and development are two inherently intertwined processes. As the embryo develops it does so in ways that both reflect past constraints and bias the future evolution of the species. While research exploiting this insight typically studies individual genes, transcriptomic analyses have sparked a new wave of discoveries. In this opinion piece, I review the evidence arising from transcriptomics on the topics of the evolution of germ layers, the phylotypic stage, and developmental constraints. The spatiotemporal pattern of gene expression across germ layers provides evidence that the endoderm was the first germ layer to evolve. Comparing transcriptome dynamics throughout developmental time across distant species reveals a mid-developmental transition under strong developmental constraints. These studies highlight the efficiency of exploratory data analysis using computational tools and comparative approaches for discovery.

The recent shift of computational biologists from bioinformatics service providers to leaders of cutting-edge programs highlights the accompanying cultural and conceptual changes that should be implemented by funding bodies and academic institutions.

The interaction between a pathogen and a host is a highly dynamic process in which both agents activate complex programs. Here, we introduce a single-cell RNA-sequencing method, scDual-Seq, that simultaneously captures both host and pathogen transcriptomes. We use it to study the process of infection of individual mouse macrophages with the intracellular pathogen Salmonella typhimurium. Among the infected macrophages, we find three subpopulations and we show evidence for a linear progression through these subpopulations, supporting a model in which these three states correspond to consecutive stages of infection.

New methods for simultaneously quantifying protein and gene expression at the single-cell level have the power to identify cell types and to classify cell populations.

Evolutionary theory assumes that genetic variation is uniform and gradual in nature, yet morphological and gene expression studies have revealed that different life-stages exhibit distinct levels of cross-species conservation. In particular, a stage in mid-embryogenesis is highly conserved across species of the same phylum, suggesting that this stage is subject to developmental constraints, either by increased purifying selection or by a strong mutational bias. An alternative explanation, however, holds that the same 'hourglass' pattern of variation may result from increased positive selection at the earlier and later stages of development. To distinguish between these scenarios, we examined gene expression variation in a population of the nematode Caenorhabditis elegans using an experimental design that eliminated the influence of positive selection. By measuring gene expression for all genes throughout development in 20 strains, we found that variations were highly uneven throughout development, with a significant depletion during mid-embryogenesis. In particular, the family of homeodomain transcription factors, whose expression generally coincides with mid-embryogenesis, evolved under high constraint. Our data further show that genes responsible for the integration of germ layers during morphogenesis are the most constrained class of genes. Together, these results provide strong evidence for developmental constraints as the mechanism underlying the hourglass model of animal evolution. Understanding the pattern and mechanism of developmental constraints provides a framework to understand how evolutionary processes have interacted with embryogenesis and led to the diversity of animal life on Earth.