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Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila , classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium – such as Caenorhabditis elegans – where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including ‘equivalence-group’ cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.

Studies of gene-expression levels in embryos of Caenorhabditis elegans and of other phyla reveal the timing and location of expression of all genes and support a model in which the endoderm program dates back to the origin of multicellularity while the ectoderm originated as a secondary germ layer freed from ancestral feeding functions. A germ-layer chronology The germ-layer theory — which holds that all the cells and tissues of the body can be grouped into three fundamental layers — goes back to the roots of developmental biology as a discipline 150 years ago. The skin and many external organs are formed of ectoderm; the guts of endoderm, and organs in the middle, such as muscles and bones, form the mesoderm. The mesoderm seems to have been the last of the three layers to have evolved, as Itai Yanai and colleagues confirm with studies on the expression of genes in the embryo of the roundworm Caenorhabditis elegans . But which came first, the ectoderm or the endoderm? Further studies on a range of animals, including the sponge Amphimedon queenslandica , which lacks clear germ layers, show that the endoderm expresses evolutionarily older genes than the ectoderm. The authors speculate that the most primitive animals consisted of what would later become endoderm, with the ectoderm differentiating as cells were freed from the primary function of feeding. The concept of germ layers has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years (refs 1 , 2 , 3 ). Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve 1 , 4 , 5 . The origin of the ectoderm and endoderm germ layers, however, remains unclear, with models supporting the antecedence of each as well as a simultaneous origin 4 , 6 , 7 , 8 , 9 . Here we determine the temporal and spatial components of gene expression spanning embryonic development for all Caenorhabditis elegans genes and use it to determine the evolutionary ages of the germ layers. The gene expression program of the mesoderm is induced after those of the ectoderm and endoderm, thus making it the last germ layer both to evolve and to develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier, and this is also observed in the expression of endoderm orthologues during the embryology of the frog Xenopus tropicalis , the sea anemone Nematostella vectensis and the sponge Amphimedon queenslandica . Querying the phylogenetic ages of specifically expressed genes reveals that the endoderm comprises older genes. Taken together, we propose that the endoderm program dates back to the origin of multicellularity, whereas the ectoderm originated as a secondary germ layer freed from ancestral feeding functions.

Gene expression data holds the potential to shed light on multiple biological processes at once. However, data analysis methods for single cell sequencing mostly focus on finding cell clusters or the principal progression line of the data. Data analysis for spatial transcriptomics mostly addresses clustering and finding spatially variable genes. Existing data analysis methods are effective in finding the main data features, but they might miss less pronounced, albeit significant, processes, possibly involving a subset of the samples. In this work we present SPIRAL: Significant Process InfeRence ALgorithm. SPIRAL is based on Gaussian statistics to detect all statistically significant biological processes in single cell, bulk and spatial transcriptomics data. The algorithm outputs a list of structures, each defined by a set of genes working simultaneously in a specific population of cells. SPIRAL is unique in its flexibility: the structures are constructed by selecting subsets of genes and cells based on statistically significant and consistent differential expression. Every gene and every cell may be part of one structure, more or none. SPIRAL also provides several visual representations of structures and pathway enrichment information. We validated the statistical soundness of SPIRAL on synthetic datasets and applied it to single cell, spatial and bulk RNA-sequencing datasets. SPIRAL is available at https://spiral.technion.ac.il/ .

Long non-coding RNAs (lncRNAs) are pivotal regulators of cellular processes. Here we reveal an interaction between the lncRNA NORAD, noted for its role in DNA stability, and the immune related transcription factor STAT3 in embryonic and differentiated human cells. Results from NORAD knockdown experiments implicate NORAD in facilitating STAT3 nuclear localization and suppressing antiviral gene activation. In NORAD-deficient cells, STAT3 remains cytoplasmic, allowing STAT1 to enhance antiviral activity. Analysis of RNA expression data from in vitro experiments and clinical samples demonstrates reduced NORAD upon viral infection. Additionally, evolutionary conservation analysis suggests that this regulatory function of NORAD is restricted to humans, potentially owing to the introduction of an Alu element in hominoids. Our findings thus suggest that NORAD functions as a modulator of STAT3-mediated immune suppression, adding to the understanding of lncRNAs in immune regulation and evolutionary adaptation in host defense mechanisms. Long non-coding RNA (lncRNA) has diverse functions. Here the authors show that a lncRNA, NORAD, interacts with an immune transcription factor, STAT3, to facilitate STAT3 nuclear import and thereby modulating the intranuclear balance between STAT3 and STAT1 and the downstream activation of anti-viral immunity in vitro.

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.

Embryos in a particular phylum of the animal kingdom tend to most resemble one another at a stage in the middle of embryogenesis known as the phylotypic period; a transcriptional analysis of embryogenesis from single embryos of ten different phyla reveals that the transcripts expressed at the phylotypic stage (or mid-developmental transition) differ greatly between phyla, and a ‘phylum’ may be defined as a set of species sharing the same signals and transcription factor networks during the mid-developmental transition. Stage set for defining a phylum Embryos in a particular phylum tend to resemble one another closely at some point in the middle of embryogeny. This is known as the phylotypic stage, and it has been established that embryos at this stage tend to express a conserved set of genes that are evolutionarily older than the genes expressed before and after. This, however, only applies within a phylum, as Yanai and colleagues demonstrate in an analysis of transcriptomes from individual embryos of ten disparate phyla. Considered across the whole animal kingdom, the transcripts expressed at the phylotypic stage differ greatly between phyla and could be said to define the characters of a particular phylum. This work also provides an operational definition for a phylum as a set of species — with a common ancestor — that share the same molecular mechanisms at the phylotypic stage. Animals are grouped into ~35 ‘phyla’ based upon the notion of distinct body plans 1 , 2 , 3 , 4 . Morphological and molecular analyses have revealed that a stage in the middle of development—known as the phylotypic period—is conserved among species within some phyla 5 , 6 , 7 , 8 , 9 . Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals 10 . Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent ‘mid-developmental transition’ that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

A focus on sea anemones throws the classic concept of germ layer homology on its head, as cnidarians are found to possess the gene expression programmes for three, rather than two, germ layers.

Background Organisms from many distinct evolutionary lineages acquired the capacity to enter a dormant state in response to environmental conditions incompatible with maintaining normal life activities. Most studied organisms exhibit seasonal or annual episodes of dormancy, but numerous less studied organisms enter long-term dormancy, lasting decades or even centuries. Intriguingly, many planktonic animals produce encased embryos known as resting eggs or cysts that, like plant seeds, may remain dormant for decades. Herein, we studied a rotifer Brachionus plicatilis as a model planktonic species that forms encased dormant embryos via sexual reproduction and non-dormant embryos via asexual reproduction and raised the following questions: Which genes are expressed at which time points during embryogenesis? How do temporal transcript abundance profiles differ between the two types of embryos? When does the cell cycle arrest? How do dormant embryos manage energy? Results As the molecular developmental kinetics of encased embryos remain unknown, we employed single embryo RNA sequencing (CEL-seq) of samples collected during dormant and non-dormant embryogenesis. We identified comprehensive and temporal transcript abundance patterns of genes and their associated enriched functional pathways. Striking differences were uncovered between dormant and non-dormant embryos. In early development, the cell cycle-associated pathways were enriched in both embryo types but terminated with fewer nuclei in dormant embryos. As development progressed, the gene transcript abundance profiles became increasingly divergent between dormant and non-dormant embryos. Organogenesis was suspended in dormant embryos, concomitant with low transcript abundance of homeobox genes, and was replaced with an ATP-poor preparatory phase characterized by very high transcript abundance of genes encoding for hallmark dormancy proteins (e.g., LEA proteins, sHSP, and anti-ROS proteins, also found in plant seeds) and proteins involved in dormancy exit. Surprisingly, this period appeared analogous to the late maturation phase of plant seeds. Conclusions The study highlights novel divergent temporal transcript abundance patterns between dormant and non-dormant embryos. Remarkably, several convergent functional solutions appear during the development of resting eggs and plant seeds, suggesting a similar preparatory phase for long-term dormancy. This study accentuated the broad novel molecular features of long-term dormancy in encased animal embryos that behave like “animal seeds”.

DNA methylation inhibits gene expression in animal cells, probably by affecting chromatin structure. Biochemical studies suggest that this process may be mediated by methyl-specific binding proteins that recruit enzymatic machinery capable of locally altering histone modification 1 . To test whether DNA methylation actually has a role in the assembly of chromatin during normal development, we used cell transfection and a transgene construct genetically programmed to be either methylated or unmethylated in all cell types of the mouse 2 . Chromatin immunoprecipitation (ChIP) analysis shows that the presence of DNA methylation brings about the deacetylation of histone H4 and methylation of Lys9 of histone H3 (H3 Lys9) and prevents methylation of Lys4 of histone H3 (H3 Lys4), thus generating a structure identical to that of methylated sequences in the genome. These results indicate that the methylation pattern established in early embryogenesis is profoundly important in setting up the structural profile of the genome.

In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.