Developmental gene regulation by an ancient intercellular communication system in social amoebae
ABSTRACT: In the social amoebae (Dictyostelia) quorum sensing system mediates aggregation of single cells into multicellular aggregates by chemotactic movement towards gradients of diffusible molecules known as acrasins. The acrasin of P. violaceum is the unusual dipeptide N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam-ethylester, known as glorin. Phylogenetic analysis has indicated that P. violaceum is more related to the most derived group 4 dictyostelids than to the ancient group 2 polysphondylids such as P. pallidum. Nevertheless it has been reported that P. pallidum cells respond to glorin in chemotaxis assays. This has led to the assumption that glorin-based communication may be the most ancient form of intercellular communication that Dictyostelia invented to organize early steps of multicellular development. In this study we show that glorin mediates rapid changes in gene expression at the transition from vegetative growth to aggregation, apparently without pronounced cross-talk with the cyclic AMP-based communication system that coordinates post-aggregation events in this species. We describe glorin-mediated changes in gene expression in the social amoeba Polysphondylium pallidum at the transition from unicellular growth to multicellular development. Comparison of gene expression in growing cells versus cells starving for 2 or 3 hours in the presence or absence of glorin.
Project description:In the social amoebae (Dictyostelia) quorum sensing system mediates aggregation of single cells into multicellular aggregates by chemotactic movement towards gradients of diffusible molecules known as acrasins. The acrasin of P. violaceum is the unusual dipeptide N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam-ethylester, known as glorin. Phylogenetic analysis has indicated that P. violaceum is more related to the most derived group 4 dictyostelids than to the ancient group 2 polysphondylids such as P. pallidum. Nevertheless it has been reported that P. pallidum cells respond to glorin in chemotaxis assays. This has led to the assumption that glorin-based communication may be the most ancient form of intercellular communication that Dictyostelia invented to organize early steps of multicellular development. In this study we show that glorin mediates rapid changes in gene expression at the transition from vegetative growth to aggregation, apparently without pronounced cross-talk with the cyclic AMP-based communication system that coordinates post-aggregation events in this species. We describe glorin-mediated changes in gene expression in the social amoeba Polysphondylium pallidum at the transition from unicellular growth to multicellular development. Overall design: Comparison of gene expression in growing cells versus cells starving for 2 or 3 hours in the presence or absence of glorin.
Project description:The social amoeba Dictyostelium discoideum integrates into a multicellular organism when individual starving cells aggregate and form a mound. The cells then integrate into defined tissues and develop into a fruiting body that consists of a stalk and spores. Aggregation is initially orchestrated by waves of extracellular cyclic adenosine monophosphate (cAMP) and previous theory suggested that cAMP and other field-wide diffusible signals mediate tissue integration and terminal differentiation as well. Cooperation between cells depends on an allorecognition system comprised of the polymorphic adhesion proteins TgrB1 and TgrC1. Binding between compatible TgrB1 and TgrC1 variants ensures that non-matching cells segregate into distinct aggregates prior to terminal development. Here, we have embedded a small number of cells with incompatible allotypes within fields of developing cells with compatible allotypes. We found that compatibility of the allotype encoded by the tgrB1 and tgrC1 genes is required for tissue integration, as manifested in cell polarization, coordinated movement, and differentiation into prestalk and prespore cells. Our results show that the molecules that mediate allorecognition in D. discoideum also control the integration of individual cells into a unified developing organism and this acts as a gating step for multicellularity. Total 12 samples were used for this analysis. 3 developmental timepoints were analyzed with 2 biological replication. And same 3 developmental timepoint of references (5 RNA mix) with 2 biological replication.
Project description:Development of the soil amoeba Dictyostelium discoideum is triggered by starvation. When placed on a solid substrate, the starving solitary amoebae cease growth, communicate via extracellular cAMP, aggregate by tens of thousands and develop into fully integrated multicellular organisms. Early phases of the developmental program are often studied in cells starved in suspension while cAMP is provided exogenously. Previous studies revealed massive shifts in the transcriptome under these developmental conditions and a close relationship between gene expression and morphogenesis, but were limited by the sampling frequency and the resolution of the methods. Here, we combine the superior depth and specificity of RNA-seq with high frequency sampling during filter development and cAMP pulsing in suspension. We found that the developmental transcriptome exhibits mostly gradual changes interspersed by a few instances of large changes. Considering the whole transcriptome as a single phenotype, we treated each time point as an independent entity and were able to characterize development as groups of similar time points separated by gaps. The grouped time points represent gradual changes in mRNA abundance and the gaps represent times during which many genes are differentially expressed rapidly. Comparing development on solid substrate to development in suspension revealed that gene expression in filter developed cells lagged behind cells treated with exogenous cAMP in suspension. The high sampling frequency revealed many genes whose regulation is reproducibly more complex than indicated by previous studies. Gene Ontology enrichment analysis showed that the transition to multicellularity at the mound stage coincided with rapid accumulation of transcripts associated with mitosis and DNA replicative processes. Later development included the up-regulation of organic signaling molecules and co-factor biosynthesis. We observed multiple instances of enrichment of oxidation-reduction and reactive oxygen related terms. Our analysis also demonstrated a high level of synchrony between the developing structures throughout development. Our data describe D. discoideum development as a series of coordinated cellular and multicellular activities. Coordination occurred within fields of aggregating cells and between multicellular bodies, such as mounds or migratory slugs that experience both cell-cell contact and various soluble signaling regimes. These time courses, sampled at the highest temporal resolution to date in this system, provide a comprehensive resource for future studies of developmental gene expression. Dictyosteloium discoideum gene expression (mRNA) profiles from two experimental time courses: development on filters and cAMP signaling in suspension. Filter development consists of 2 replicates of 19 time point samples, while cAMP in suspension contains 3 replicates of 10, 10 and 9 samples each.
Project description:Biological oscillations are observed at many levels of cellular organization. In the social amoebae Dictyostelium discoideum, starvation-triggered multicellular development is organized by periodic cAMP waves, which provide both chemoattractant gradients and developmental signals. We report that GtaC, a GATA transcription factor, exhibits rapid nucleocytoplasmic shuttling in response to cAMP waves. This behavior requires coordinated action of a nuclear localization signal and reversible G protein-coupled receptor (GPCR)-mediated phosphorylation. While both are required for developmental gene expression, receptor occupancy promotes nuclear exit of GtaC, which leads to a transient burst of transcription at each cAMP cycle. We demonstrate that this biological circuit, like an “edge trigger”, filters out high frequency signals and counts those admitted, thereby enabling cells to modulate gene expression according to the dynamic pattern of the external stimuli. Transcriptional profiling during early development of wild-type, gtaC, GFP-GtaC/gtaC, and NLSex-GFP-GtaC/gtaC strains
Project description:In many developmental systems, morphogenesis is coupled with dramatic changes in spatiotemporal gene expression, often orchestrated by the coordinated action of transcription factors. Development of the social soil amoebae Dictyostelium discoideum proceeds through a sequence of morphological and transcriptional changes, but the role of transcription factors in development is not well understood. GtaC, a GATA-type zinc-finger transcription factor, is essential for Dictyostelium development. It decodes pulsatile extracellular cAMP signals during early development and mediates cell-type differentiation at later stages. Here, we studied the developmental regulatory roles of GtaC through the concerted analysis of temporal ChIP- and RNA-sequencing data from strains that carry different alleles of gtaC. We show that GtaC exhibits temporally distinctive DNA-binding patterns throughout early development, accompanied by largely cotemporaneous expression of its target genes. We also show that GtaC binds DNA in two modes. One of these modes exhibits binding preferences for canonical GATA-like sequences, the regulatory consequences accompanying which is predominantly up-regulation of target gene expression. The other binding mode is mostly associated with down-regulation. Among its targets we find transcription factors that are essential for development as well as genes involved in cAMP signaling and cell-type specification. Our results suggest that GtaC is a master regulator that regulates multiple physiological processes during early development, when Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism. Cotemporaneous transcriptional profiling and ChIP sequencing during early Dictyostelium development
Project description:Sexual reproduction is nearly universal among multicellular animals, but sex can be determined by cues including sex chromosomes, temperature, social status, and photoperiod. DMRT transcription factors are key regulators of sex in animals that use diverse sex-determining strategies. These proteins are related to the sexual regulators Doublesex (Dsx) and Male abnormal-3 (MAB-3) of insects and nematodes, respectively. DMRT proteins share the DM DNA binding domain, comprised of a unique intertwined double zinc-binding module flanked by a C-terminal recognition helix that binds to a pseudopalindromic target DNA. Despite the central role of DMRT proteins in metazoan sexual development, how they recognize target DNA sequences is poorly understood. Here we find that DMRT proteins employ multiple DNA binding modes due to surprising versatility in how specific base contacts are made. Human DMRT1 can bind as a dimer, trimer or tetramer, in each case using paired antiparallel recognition helices that together insert into a widened DNA major groove to make base-specific contacts. Insertion of two helices in a single major groove is, to our knowledge, a DNA binding interaction unique to DMRT proteins. High resolution in vivo DNA binding analysis (ChIP-Exo) indicates that multiple DNA binding modes also are used in the mouse testis. Finally, we show that mutations affecting amino acid residues crucial for DNA recognition are associated with sex reversal in flies and also, for the first time, with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction that underlies much of metazoan sexual development. Overall design: ChIP-Seq (2 samples) and Chip-Exo (1 sample) in 8 week mouse Testis, ChIP-Seq in adult Human Testis (1 sample).
Project description:Social insects frequently engage in oral fluid exchange – trophallaxis – between adults, and between adults and larvae. Although trophallaxis is normally considered a simple food-sharing mechanism, we hypothesized that endogenous components of this fluid might underlie a novel means of chemical communication that directly influences colony organization. Through protein and small-molecule mass spectrometry and RNA sequencing in the ant Camponotus floridanus, we found that trophallactic fluid contains a set of specific proteins, hydrocarbons, microRNAs, and Juvenile Hormone, an important insect growth regulator. Comparison of trophallactic fluid proteins across social insect species (ants Camponotus fellah and Solenopsis invictis, honeybees Apis mellifera) revealed that many are regulators of growth and development. These results raise the possibility that, in addition to its role in food transfer, trophallaxis is a mode of communication that enables communal control of colony phenotypes.
Project description:A major challenge in biology is to determine how evolutionarily novel characters originate, however, mechanistic explanations for the origin of novelties are almost completely unknown. The evolution of mammalian pregnancy is an excellent system in which to study the origin of novelties because extant mammals preserve major stages in the transition from egg-laying to live-birth. To determine the molecular bases of this transition we characterized the pregnant/gravid uterine transcriptome from tetrapods, including species in the three major mammalian lineages, and used ancestral transcriptome reconstruction to trace the evolutionary history of uterine gene expression. We show that thousands of genes evolved endometrial expression during the origins of mammalian pregnancy, including numerous genes that mediate maternal-fetal communication and immunotolerance.Furthermore we show that thousands of regulatory elements active in decidualized human endometrial stromal cells are derived from ancient mammalian transposable elements which provided binding sites for transcription factors that mediate decidualization and endometrial cell-type identity. Our results indicate that one of the defining mammalian novelties evolved via domestication of ancient mammalian transposable elements into hormone-responsive regulatory elements throughout the genome. Examination of histone modification and DNAse hypersensitivity in decidualized dESC
Project description:Autism spectrum disorder (ASD) affects these core domains: ritualistic-repetitive behaviors, impaired social interaction, and deficits in non-verbal communication/language development. Although ASD is heritable, its extreme heterogeneity defies genetic analyses. A number of ASD susceptibility genes have been identified. The Lebanese population is ideal for uncovering recessive genes because of a high rate of shared ancestry, consanguineous marriages, and high number of offspring per family. We recruited 36 ASD families (ASD: 37, unaffected parents: 36, unaffected siblings: 33). Cytogenetics 2.7M Microarrays/CytoScan™ HD arrays allowed mapping of homozygous regions of the genome.