Temporal Patterns Of Gene Expression Research Articles

A Synchronized Circadian Clock Enhances Early Chondrogenesis.

Circadian rhythms in cartilage homeostasis are hypothesized to temporally segregate and synchronize the activities of chondrocytes to different times of the day, and thus may provide an efficient mechanism by which articular cartilage can recover following physical activity. While the circadian clock is clearly involved in chondrocyte homeostasis in health and disease, it is unclear as to what roles it may play during early chondrogenesis. The purpose of this study was to determine whether the rhythmic expression of the core circadian clock was detectable at the earliest stages of chondrocyte differentiation, and if so, whether a synchronized expression pattern of chondrogenic transcription factors and developing cartilage matrix constituents was present during cartilage formation. Following serum shock, embryonic limb bud-derived chondrifying micromass cultures exhibited synchronized temporal expression patterns of core clock genes involved in the molecular circadian clock. We also observed that chondrogenic marker genes followed a circadian oscillatory pattern. Clock synchronization significantly enhanced cartilage matrix production and elevated SOX9, ACAN, and COL2A1 gene expression. The observed chondrogenesis-promoting effect of the serum shock was likely attributable to its synchronizing effect on the molecular clockwork, as co-application of small molecule modulators (longdaysin and KL001) abolished the stimulating effects on extracellular matrix production and chondrogenic marker gene expression. Results from this study suggest that a functional molecular clockwork plays a positive role in tissue homeostasis and histogenesis during early chondrogenesis.

Extensive intragenic divergences amongst ancient WRKY transcription factor gene family is largely associated with their functional diversity in plants

Diversity at gene sequence and function level is an evolutionary outcome of a constantly expanding gene family in plants. Genome sequences have provided a platform to comprehend the evolutionary architecture of genes and gene families, especially in plant genera. Apparently, regulatory sequences and the selection of trans-binding proteins have massive impressions of gene expression evolution as compared with a transformation at genic-level itself. Therefore, it becomes essential to comprehensively dissect the genome-wide structure-function evolution of regulatory regions'-binding trans-proteins. WRKY gene family members are developmentally and functionally diverged ancient transcription factors that notably regulates numerous cellular as well as stress responsive phenotypes in plant species. To comprehend the evolutionary consequences of WRKY genes during plant development, a meta-phylogenetic analyses of at least 266 WRKY amino acid sequences of alga, monocot and eudicots plant species was accomplished. Phylogenetic clustering identified assorted groups and sub-groups of WRKY orthologous or paralogous sequences. Paralogous gene divergences of monocot and eudicot species showed strong ‘purifying’ selection during the sequence evolution. Evidently, evolutionary exon-intron structural diversification predominantly arose by ‘exon/intron-gain’ and insertion/deletion during primeval sequence-exonization. The structural DNA-binding domain, and global structures of WRKY proteins are exceedingly divergent and also explicit to the classified phylogenetic clusters. Expression profiling displayed that Group IIe WRKY members are profusely expressed in root tissues of the representative species. Remarkably, the spatial and temporal gene expression patterns of WRKY genes in plants apprise our perception about their contributions in the regulation of cellular functions; and structural-functionalism of these members speculate their probable employment into future crop improvement programs.

Temporal Profile of Brain Gene Expression After Prey Catching Conditioning in an Anuran Amphibian.

A key goal in modern neurobiology is to understand the mechanisms underlying learning and memory. To that end, it is essential to identify the patterns of gene expression and the temporal sequence of molecular events associated with learning and memory processes. It is also important to ascertain if and how these molecular events vary between organisms. In vertebrates, learning and memory processes are characterized by distinct phases of molecular activity involving gene transcription, structural change, and long-term maintenance of such structural change in the nervous system. Utilizing next generation sequencing techniques, we profiled the temporal expression patterns of genes in the brain of the fire-bellied toad Bombina orientalis after prey catching conditioning. The fire-bellied toad is a basal tetrapod whose neural architecture and molecular pathways may help us understand the ancestral state of learning and memory mechanisms in tetrapods. Differential gene expression following conditioning revealed activity in molecular pathways related to immediate early genes (IEG), cytoskeletal modification, axon guidance activity, and apoptotic processes. Conditioning induced early IEG activity coinciding with transcriptional activity and neuron structural modification, followed by axon guidance and cell adhesion activity, and late neuronal pruning. While some of these gene expression patterns are similar to those found in mammals submitted to conditioning, some interesting divergent expression profiles were seen, and differential expression of some well-known learning-related mammalian genes is missing altogether. These results highlight the importance of using a comparative approach in the study of the mechanisms of leaning and memory and provide molecular resources for a novel vertebrate model in the relatively poorly studied Amphibia.

Expression of genes related to long-chain (C18–22) and very long-chain (>C24) fatty acid biosynthesis in gilthead seabream (Sparus aurata) and Senegalese sole (Solea senegalensis) larvae: investigating early ontogeny and nutritional regulation

Long-chain polyunsaturated fatty acids (LC-PUFA) have been extensively studied in aquaculture due to their importance for survival and development in teleosts. However, very long-chain polyunsaturated fatty acids (VLC-PUFA) have been practically unexplored within the aquaculture scenario. VLC-PUFA, although always present in small amounts, can be pivotal for the correct development and function of tissues such as retina, brain and gonads of vertebrates including fish. This study aimed at determining the temporal expression patterns of genes involved in the biosynthesis of VLC-PUFA (elovl4a, elovl4b) and their precursors, LC-PUFA, (fads2, elovl5) during the early ontogeny of Solea senegalensis and Sparus aurata. Furthermore, we investigated the nutritional regulation of these genes in early life-cycle stages of fish fed low and high LC-PUFA diets consisting of non-enriched and enriched live preys (Brachionus plicatilis and Artemia franciscana), respectively. The effect of dietary LC-PUFA on growth and fatty acid composition was also examined. The results obtained during early development reveal that all genes studied are expressed before the hatching stage. There is a consistency between the timing at which retinogenesis occurs in both species and an increase of the expression of the two elovl4 responsible for the synthesis of VLC-PUFA. The results obtained in nutritional assays for both species suggest that the expression of the two isoforms of elovl4 (isoform a in early larvae, and b in late larvae) can be regulated positively according to the dietary content of LC-PUFA in early stages, which could activate the VLC-PUFA biosynthesis even during short-term feeding periods (seven days). The body part analysis in late larvae of both species revealed that both isoforms of elovl4 are expressed preferentially in the head. This can be associated to their highest presence in the neural and visual tissues.

Heritable temporal gene expression patterns correlate with metabolomic seed content in developing hexaploid oat seed.

SummaryOat ranks sixth in world cereal production and has a higher content of health‐promoting compounds compared with other cereals. However, there is neither a robust oat reference genome nor transcriptome. Using deeply sequenced full‐length mRNA libraries of oat cultivar Ogle‐C, a de novo high‐quality and comprehensive oat seed transcriptome was assembled. With this reference transcriptome and QuantSeq 3′ mRNA sequencing, gene expression was quantified during seed development from 22 diverse lines across six time points. Transcript expression showed higher correlations between adjacent time points. Based on differentially expressed genes, we identified 22 major temporal co‐expression (TCoE) patterns of gene expression and revealed enriched gene ontology biological processes. Within each TCoE set, highly correlated transcripts, putatively commonly affected by genetic background, were clustered and termed genetic co‐expression (GCoE) sets. Seventeen of the 22 TCoE sets had GCoE sets with median heritabilities higher than 0.50, and these heritability estimates were much higher than that estimated from permutation analysis, with no divergence observed in cluster sizes between permutation and non‐permutation analyses. Linear regression between 634 metabolites from mature seeds and the PC1 score of each of the GCoE sets showed significantly lower p‐values than permutation analysis. Temporal expression patterns of oat avenanthramides and lipid biosynthetic genes were concordant with previous studies of avenanthramide biosynthetic enzyme activity and lipid accumulation. This study expands our understanding of physiological processes that occur during oat seed maturation and provides plant breeders the means to change oat seed composition through targeted manipulation of key pathways.

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

SummaryIn multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue‐ or cell‐specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®‐compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue‐specific split luciferase assay for non‐invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap‐compatible split luciferase assay (ETSLA) system to investigate tissue‐specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.

Establishing neuronal diversity in the spinal cord: a time and a place.

The vertebrate spinal cord comprises multiple functionally distinct neuronal cell types arranged in characteristic positions. During development, these different types of neurons differentiate from transcriptionally distinct neural progenitors that are arrayed in discrete domains along the dorsal-ventral and anterior-posterior axes of the embryonic spinal cord. This organization arises in response to morphogen gradients acting upstream of a gene regulatory network, the architecture of which determines the spatial and temporal pattern of gene expression. In recent years, substantial progress has been made in deciphering the regulatory network that underlies the specification of distinct progenitor and neuronal cell identities. In this Review, we outline how distinct neuronal cell identities are established in response to spatial and temporal patterning systems, and outline novel experimental approaches to study the emergence and function of neuronal diversity in the spinal cord.

Distinct enhancer signatures in the mouse gastrula delineate progressive cell fate continuum during embryo development.

Primary germ layers have the potential to form all tissues in the mature organism, and their formation during gastrulation requires precise epigenetic modulation of both proximal and distal regulatory elements. Previous studies indicated that spatial and temporal patterns of gene expression in the gastrula predispose individual regions to distinct cell fates. However, the underlying epigenetic mechanisms remain largely unexplored. Here, we profile the spatiotemporal landscape of the epigenome and transcriptome of the mouse gastrula. We reveal the asynchronous dynamics of proximal chromatin states during germ layer formation as well as unique gastrula-specific epigenomic features of regulatory elements, which have strong usage turnover dynamics and clear germ layer-specific signatures. Importantly, we also find that enhancers around organogenetic genes, which are weakly expressed at the gastrulation stage, are frequently pre-marked by histone H3 lysine 27 acetylation (H3K27ac) in the gastrula. By using the transgenic mice and genome editing system, we demonstrate that a pre-marked enhancer, which is located in the intron of a brain-specific gene 2510009E07Rik, exhibits specific enhancer activity in the ectoderm and future brain tissue, and also executes important function during mouse neural differentiation. Taken together, our study provides the comprehensive epigenetic information for embryonic patterning during mouse gastrulation, demonstrates the importance of gastrula pre-marked enhancers in regulating the correct development of the mouse embryo, and thus broadens the current understanding of mammalian embryonic development and related diseases.

Integrated analysis of transcriptomic and proteomic data from tree peony (P. ostii) seeds reveals key developmental stages and candidate genes related to oil biosynthesis and fatty acid metabolism

Tree peony (Paeonia section Moutan DC.) seeds are an excellent source of beneficial natural compounds that promote health, and they contain high levels of alpha-linolenic acid (ALA). In recent years, tree peony has been emerging as an oil crop. Therefore, combined analysis of the transcriptome and proteome of tree peony (P. ostii) seeds at 25, 32, 39, 53, 67, 81, 88, 95, and 109 days after pollination (DAP) was conducted to better understand the transcriptional and translational regulation of seed development and oil biosynthesis. A total of 38,482 unigenes and 2841 proteins were identified. A total of 26,912 differentially expressed genes (DEGs) and 592 differentially expressed proteins (DEPs) were clustered into three groups corresponding to the rapid growth, seed inclusion enrichment and conversion, and late dehydration and mature stages of seed development. Fifteen lipid metabolism pathways were identified at both the transcriptome and proteome levels. Pathway enrichment analysis revealed that a period of rapid fatty acid biosynthesis occurred at 53–88 DAP. Furthermore, 211 genes and 35 proteins associated with the fatty acid metabolism pathway, 63 genes and 11 proteins associated with the biosynthesis of unsaturated fatty acids (UFAs), and 115 genes and 24 proteins associated with ALA metabolism were identified. Phylogenetic analysis revealed that 16 putative fatty acid desaturase (FAD)-encoding genes clustered into four FAD groups, eight of which exhibited the highest expression at 53 DAP, suggesting that they play an important role in ALA accumulation. RT-qPCR analysis indicated that the temporal expression patterns of oil biosynthesis genes were largely similar to the RNA-seq results. The expression patterns of fatty acid metabolism- and seed development-related proteins determined by MRM were also highly consistent with the results obtained in the proteomic analysis. Correlation analysis indicated significant differences in the number and abundance of DEGs and DEPs but a high level of consistency in expression patterns and metabolic pathways. The results of the present study represent the first combined transcriptomic and proteomic analysis of tree peony seeds and provide insight into tree peony seed development and oil accumulation.

A Simple Method to Identify Ascidian Brain Lineage Cells at Neural Plate Stages Following In Situ Hybridization.

The technique of in situ hybridization can be used to visualize the spatial and temporal pattern of gene expression during development. Ascidians are invertebrate chordates that develop with a fixed cell cleavage patterninto a tadpole larvae. The knowledge of the cell lineage allows the earliest steps of cell fate specification to be followed at a single cell resolution. This protocol describes preparation of Ciona intestinalis embryos, classical in situ hybridization protocol coupled with nuclear staining, and a guide to identify gene expression in specific precursors of the developing brain at neural plate stages of development.

Profiling Gene Programs in the Blood During Liver Regeneration in Living Liver Donors.

The human liver's capacity to rapidly regenerate to a full-sized functional organ after resection has allowed successful outcomes for living donor liver transplantation (LDLT) procedures. However, the ability to detect and track physiological changes occurring during liver regeneration after resection and throughout the restoration process is still lacking. We performed a comprehensive whole-transcriptome RNA sequencing analysis of liver and circulating blood tissue from 12 healthy LDLT donors to define biomarker signatures for monitoring physiological activities during liver regeneration at 14 time points for up to a 1-year procedural follow-up. LDLT donor liver tissue differentially expressed 1238 coding and noncoding genes after resection, and an additional 1260 genes were selectively regulated after LDLT. A total of 15,011 RNA transcript species were identified in the blood in response to liver resection. The transcripts most highly regulated were sequentially expressed within 3 distinct peaks that correlated with sets of functional genes involved in the induction of liver resection-specific innate immune response (peak 1), activation of the complement system (peak 2), and platelet activation and erythropoiesis (peak 3). Each peak corresponded with progressive phases of extracellular matrix degradation, remodeling, and organization during liver restoration. These processes could be tracked by distinct molecular signatures of up-regulated and down-regulated gene profiles in the blood during phases of liver repair and regeneration. In conclusion, the results establish temporal and dynamic transcriptional patterns of gene expression following surgical liver resection that can be detected in the blood and potentially used as biomarker signatures for monitoring phases of liver regeneration.

Expression Analysis by RNAscope™ In Situ Hybridization.

RNA in situ hybridization has an important place in matrix biology, as the only method that allows for in situ discrimination of precise spatial and temporal patterns of gene expression. Whereas immunohistochemistry shows where a matrix protein localizes, ISH identifies the cell of origin. Thus, these methods provide complementary information for insights on the life cycle of matrix molecules, including ADAMTS proteases. This protocol encompasses the staining of tissue sections to reveal expression of the gene of interest.

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus.

Capsular polysaccharide (CP) biosynthesis in Staphylococcus aureus is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in nongrowing cells. Expression of the corresponding biosynthesis gene cluster is driven by one promoter element (Pcap ). Here, we demonstrate that Pcap contains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat (IR) that is crucial for cap expression. The essentiality of the IR is derived from this region acting as a SigB binding site rather than as an operator site for the proposed cap activators RbsR and MsaB. Furthermore, Pcap contains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for cap repressors such as SaeR, CodY and Rot. Heterogeneous CP synthesis is determined by SigB activity and repressor binding to the upstream region. SigB dependency and regulation by the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even when transcription is rendered constitutive, suggesting additional posttranscriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis.

Expression pattern of ABCDE model genes in floral organs of bolting garlic clone

A correct understanding of the ABCDE model genes expression, especially genes involved in the development of the reproductive floral organs, in bolting and fertile garlic clones improves the process of its fertilization and breeding programs. Therefore, Real-Time PCR was employed to evaluate the temporal and spatial expression patterns of some floral organ identity genes in the inflorescence and different floret organs in the two stages of green and purple florets of bolting garlic clone. Relative expression of the studied genes, except AsSTK, in the mature inflorescence increased significantly during the early stages of initiation and differentiation of floral organs. Relative expression of the AsAP1 in the tepal and carpel, and AsAP2 in the tepal, stamen and carpel increased significantly. The highest relative expression levels of the AsAP1 and AsAP2 were found in the tepal of green florets and in the carpel of purple florets, respectively. AsAP3 and AsPI expression increased significantly in the stamen and carpel, and the highest relative expression of these two genes were observed in the green floret tepal. Relative expression of the AsAG increased significantly only in the reproductive floral organs and decreased significantly both in the carpel and stamen at floret maturity. The AsSEP1, 3 were expressed in all floral organs, but the AsSTK was only expressed in the carpel and its relative expression increased significantly at floret maturity. Finally, since considerable expression levels of the above genes were observed in the floral organs, these genes seem to be influential in the formation of floral organs in bolting garlic.

Luteal peptides and their genes as important markers of ovarian differentiation

Secreted peptide hormones and components of the steroidogenic machinery are molecules that are expressed usually in high amounts and in a time- and cell-specific fashion within the cells that give rise to the bovine corpus luteum. They thus serve as useful markers for the events occurring within the nuclei of these cells that result in differentiation and the expression of the specific luteal phenotype. We have studied the bovine genes of three such luteal products: oxytocin, the new relaxin-like factor (RLF), and the steroidogenic acute regulatory protein (StAR). The oxytocin gene is expressed in the granulosal cells of the preovulatory follicle and in the large luteal cells of the immediately resulting early corpus luteum. The RLF gene is a major thecal cell product in antral and atretic follicles. It is also transcribed in luteal cells, but only in the mid- to late ovarian cycle and in pregnancy, following a temporal pattern of expression very similar to that of relaxin in pigs. The StAR gene appears to be upregulated only in the mid- to late ovarian cycle, several days after the increase in steroidogenic enzymes associated with luteinization and progesterone production. All three genes make use of the transcription factor SF-1 (Ad4BP) and, although they all respond to LH activation of adenylate cyclase, none utilize CRE-linked systems. Specific transcriptional activation must involve other factors to encode the information for the widely diverse temporal and cellular patterns of gene expression for these three genes.

Characterization and Evolution of Dishevelled Genes in Paralichthys olivaceus

The study examined the key gene Dishevelled (Dvl or Dsh) in Wnt (Wingless and INT-1) signaling pathways. The gene (Dvl) was characterized in the flat fish Paralichthys olivaceus for its expression pattern structure and phylogenetics at the Ocean University of China in Qingdao, China in 2018. Three gene paralogues (Dvl1, Dvl2 and Dvl3) of the Dvl family were cloned in P. olivaceus and a N-terminal DAX domain, a central PDZ domain and a C-terminal DEP domain were discovered in all three protein paralogues. Phylogenetic analysis revealed that Dvl genes in P. olivaceus are most closely related to those in marine teleosts Larimichthys crocea and Stegastes partitus, followed by those in Cynoglossus semilaevis. For each Dvl gene, the genes in teleosts fall into a clade independent from the ones in other vertebrates, suggesting that the duplication of Dvl genes occurred prior to the divergence of vertebrates. The temporal expression patterns of the three Dvl genes were characterized during the embryonic development of teleosts. In P. olivaceus, all three Dvl genes remain at low expression levels during the early stages of development until gastrula stage, when the expression of Dvl1 was significantly up-regulated. The research revealed vastly different temporal expression patterns of Dvl genes and suggested that the structure of Dvl proteins is conserved, but the expression patterns of Dvl genes vary significantly among different classes.

Spatial clustering and common regulatory elements correlate with coordinated gene expression.

Many cellular responses to surrounding cues require temporally concerted transcriptional regulation of multiple genes. In prokaryotic cells, a single-input-module motif with one transcription factor regulating multiple target genes can generate coordinated gene expression. In eukaryotic cells, transcriptional activity of a gene is affected by not only transcription factors but also the epigenetic modifications and three-dimensional chromosome structure of the gene. To examine how local gene environment and transcription factor regulation are coupled, we performed a combined analysis of time-course RNA-seq data of TGF-β treated MCF10A cells and related epigenomic and Hi-C data. Using Dynamic Regulatory Events Miner (DREM), we clustered differentially expressed genes based on gene expression profiles and associated transcription factors. Genes in each class have similar temporal gene expression patterns and share common transcription factors. Next, we defined a set of linear and radial distribution functions, as used in statistical physics, to measure the distributions of genes within a class both spatially and linearly along the genomic sequence. Remarkably, genes within the same class despite sometimes being separated by tens of million bases (Mb) along genomic sequence show a significantly higher tendency to be spatially close despite sometimes being separated by tens of Mb along the genomic sequence than those belonging to different classes do. Analyses extended to the process of mouse nervous system development arrived at similar conclusions. Future studies will be able to test whether this spatial organization of chromosomes contributes to concerted gene expression.

Coenzyme A and protein CoAlation levels are regulated in response to oxidative stress and during morphogenesis in Dictyostelium discoideum

Dictyostelium discoideum (D. discoideum) is a simple eukaryote with a unique life cycle in which it differentiates from unicellular amoebae into a fruiting body upon starvation. Reactive oxygen species (ROS) have been associated with bacterial predation, as well as regulatory events during D. discoideum development and differentiation. Coenzyme A (CoA) is a key metabolic integrator in all living cells. A novel function of CoA in redox regulation, mediated by covalent attachment of CoA to cellular proteins in response to oxidative or metabolic stress, has been recently discovered and termed protein CoAlation. In this study, we report that the level of CoA and protein CoAlation in D. discoideum are developmentally regulated, and correlate with the temporal expression pattern of genes implicated in CoA biosynthesis during morphogenesis. Furthermore, treatment of growing D. discoideum cells with oxidising agents results in a dose-dependent increase of protein CoAlation. However, much higher concentrations were required when compared to mammalian cells and bacteria. Increased resistance of D. discoideum to oxidative stress induced by H2O2 has previously been attributed to high levels of catalase activity. In support of this notion, we found that H2O2-induced protein CoAlation is significantly increased in CatA-deficient D. discoideum cells. Collectively, this study provides insights into the role of CoA and protein CoAlation in the maintenance of redox homeostasis in amoeba and during D. discoideum morphogenesis.

Spatial and temporal patterns of gene expression during neurogenesis in the sea urchin Lytechinus variegatus

BackgroundThe sea urchin is a basal deuterostome that is more closely related to vertebrates than many organisms traditionally used to study neurogenesis. This phylogenetic position means that the sea urchin can provide insights into the evolution of the nervous system by helping resolve which developmental processes are deuterostome innovations, which are innovations in other clades, and which are ancestral. However, the nervous system of echinoderms is one of the least understood of all major metazoan phyla. To gain insights into echinoderm neurogenesis, spatial and temporal gene expression data are essential. Then, functional data will enable the building of a detailed gene regulatory network for neurogenesis in the sea urchin that can be compared across metazoans to resolve questions about how nervous systems evolved.ResultsHere, we analyze spatiotemporal gene expression during sea urchin neurogenesis for genes that have been shown to be neurogenic in one or more species. We report the expression of 21 genes expressed in areas of neurogenesis in the sea urchin embryo from blastula stage (just before neural progenitors begin their specification sequence) through pluteus larval stage (when much of the nervous system has been patterned). Among those 21 gene expression patterns, we report expression of 11 transcription factors and 2 axon guidance genes, each expressed in discrete domains in the neuroectoderm or in the endoderm. Most of these genes are expressed in and around the ciliary band. Some including the transcription factors Lv-mbx, Lv-dmrt, Lv-islet, and Lv-atbf1, the nuclear protein Lv-prohibitin, and the guidance molecule Lv-semaa are expressed in the endoderm where they are presumably involved in neurogenesis in the gut.ConclusionsThis study builds a foundation to study how neurons are specified and evolved by analyzing spatial and temporal gene expression during neurogenesis in a basal deuterostome. With these expression patterns, we will be able to understand what genes are required for neural development in the sea urchin. These data can be used as a starting point to (1) build a spatial gene regulatory network for sea urchin neurogenesis, (2) identify how subtypes of neurons are specified, (3) perform comparative studies with the sea urchin, protostome, and vertebrate organisms.

Identification of the Regulatory Elements and Target Genes of Megakaryopoietic Transcription Factor MEF2C.

Megakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.