In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

/pdf/mapping-a-multiplexed-zoo-of-mrna-expression-3ir3qhzxt9.pdf

Mapping a multiplexed zoo of mRNA expression

During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been developed to infer the translational status of a specific mRNA species or to analyze the translatome, i.e. the subset of mRNAs actively translated in a cell. Polysome profiling is especially suitable for emergent model organisms for which genomic data are limited. In this paper, we describe an optimized protocol for the purification of sea urchin polysomes and highlight the critical steps involved in polysome purification. We applied this protocol to obtain experimental results on translational regulation of mRNAs following fertilization. Our protocol should prove useful for integrating the study of the role of translational regulation in gene regulatory networks in any biological model. In addition, we demonstrate how to carry out high-throughput processing of polysome gradient fractions, for the simultaneous screening of multiple biological conditions and large-scale preparation of samples for next-generation sequencing.

https://hal.archives-ouvertes.fr/hal-01389711/document

Analysis of translation using polysome profiling

Developmental genes in metazoan genomes are surrounded by dense clusters of conserved noncoding elements (CNEs). CNEs exhibit unexplained extreme levels of sequence conservation, with many acting as developmental long-range enhancers. Clusters of CNEs define the span of regulatory inputs for many important developmental regulators and have been described previously as genomic regulatory blocks (GRBs). Their function and distribution around important regulatory genes raises the question of how they relate to 3D conformation of these loci. Here, we show that clusters of CNEs strongly coincide with topological organisation, predicting the boundaries of hundreds of topologically associating domains (TADs) in human and Drosophila. The set of TADs that are associated with high levels of noncoding conservation exhibit distinct properties compared to TADs devoid of extreme noncoding conservation. The close correspondence between extreme noncoding conservation and TADs suggests that these TADs are ancient, revealing a regulatory architecture conserved over hundreds of millions of years. Metazoan genomes contain many clusters of conserved noncoding elements. Here, the authors provide evidence that these clusters coincide with distinct topologically associating domains in humans and Drosophila, revealing a conserved regulatory genomic architecture.

Topologically associating domains are ancient features that coincide with Metazoan clusters of extreme noncoding conservation

Background: The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously. Results: Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components. Conclusions: The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins.

/pdf/proteomic-analysis-of-sea-urchin-strongylocentrotus-2onlm1qo5m.pdf

Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix

The RAG1 and RAG2 proteins are essential subunits of the V(D)J recombinase that is required for the generation of the enormous variability of antibodies and T-cell receptors in jawed vertebrates. It was demonstrated previously that the 600-aa catalytic core of RAG1 evolved from the transposase of the Transib superfamily transposons. However, although homologs of RAG1 and RAG2 genes are adjacent in the purple sea urchin genome, a transposon encoding both proteins so far has not been reported. Here we describe such transposons in the genomes of green sea urchin, a starfish and an oyster. Comparison of the domain architectures of the RAG1 homologs in these transposons, denoted TransibSU, and other Transib superfamily transposases provides for reconstruction of the structure of the hypothetical TransibVDJ transposon that gave rise to the VDJ recombinases at the onset of vertebrate evolution some 500 million years ago.

/pdf/evolution-of-the-rag1-rag2-locus-both-proteins-came-from-the-4u2h4c9j1n.pdf

Evolution of the RAG1-RAG2 locus: both proteins came from the same transposon.

Abstract Background Primulina pungentisepala is suitable for use as a potted plant because of its beautiful leaf variegation, which is significantly different in its selfed offspring. However, the mechanism of P. pungentisepala leaf variegation is unclear. In this study, two types of offspring showing the greatest differences were compared in terms of leaf structure, chlorophyll contents, chlorophyll fluorescence parameters and transcriptomes to provide a reference for studying the molecular mechanism of structural leaf variegation. Results Air spaces were found between water storage tissue, and the palisade tissue cells were spherical in the white type. The content of chlorophyll a and total chlorophyll (chlorophyll a + b) was significantly lower in the white type, but there were no significant differences in the content of chlorophyll b, chlorophyll a/b or chlorophyll fluorescence parameters between the white and green types. We performed transcriptomic sequencing to identify differentially expressed genes (DEGs) involved in cell division and differentiation, chlorophyll metabolism and photosynthesis. Among these genes, the expression of the cell division- and differentiation-related leucine-rich repeat receptor-like kinases (LRR-RLKs), xyloglucan endotransglycosylase/hydrolase ( XET/H ), pectinesterase ( PE ), expansin ( EXP ), cellulose synthase-like ( CSL ), VARIEGATED 3 ( VAR3 ), and ZAT10 genes were downregulated in the white type, which might have promoted the development air spaces and variant palisade cells. Chlorophyll biosynthesis-related hydroxymethylbilane synthase ( HEMC ) and the H subunit of magnesium chelatase ( CHLH ) were downregulated, while chlorophyll degradation-related chlorophyllase-2 ( CHL2 ) was upregulated in the white type, which might have led to lower chlorophyll accumulation. Conclusion Leaf variegation in P. pungentisepala was caused by a combination of mechanisms involving structural variegation and low chlorophyll levels. Our research provides significant insights into the molecular mechanisms of structural leaf variegation. 

/pdf/cytological-physiological-and-transcriptomic-analysis-of-3p4wzezy.pdf

Cytological, physiological and transcriptomic analysis of variegated Leaves in Primulina pungentisepala offspring

/pdf/cytological-physiological-and-transcriptomic-analysis-of-qqkk9seq.pdf

SpBase is a system of databases focused on the genomic information from sea urchins and related echinoderms. It is exposed to the public through a web site served with open source software (http://spbase.org/). The enterprise was undertaken to provide an easily used collection of information to directly support experimental work on these useful research models in cell and developmental biology. The information served from the databases emerges from the draft genomic sequence of the purple sea urchin, Strongylocentrotus purpuratus and includes sequence data and genomic resource descriptions for other members of the echinoderm clade which in total span 540 million years of evolutionary time. This version of the system contains two assemblies of the purple sea urchin genome, associated expressed sequences, gene annotations and accessory resources. Search mechanisms for the sequences and the gene annotations are provided. Because the system is maintained along with the Sea Urchin Genome resource, a database of sequenced clones is also provided.

/pdf/spbase-the-sea-urchin-genome-database-and-web-site-4suh0yaxdi.pdf

SpBase: the sea urchin genome database and web site

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Cytological, physiological and transcriptomic analysis of variegated Leaves in Primulina pungentisepala offspring

Cytological, physiological and transcriptomic analysis of variegated Leaves in Primulina pungentisepala offspring

SpBase: the sea urchin genome database and web site