Identification of differentially expressed genes from multipotent epithelia at the onset of an asexual development

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 6 (2016): 27357, doi:10.1038/srep27357.Organisms that have evolved alternative modes of reproduction, complementary to the sexual mode, are found across metazoans. The chordate Botryllus schlosseri is an emerging model for asexual development studies. Botryllus can rebuild its entire body from a portion of adult epithelia in a continuous and stereotyped process called blastogenesis. Anatomy and ontogenies of blastogenesis are well described, however molecular signatures triggering this developmental process are entirely unknown. We isolated tissues at the site of blastogenesis onset and from the same epithelia where this process is never triggered. We linearly amplified an ultra-low amount of mRNA (<10ng) and generated three transcriptome datasets. To provide a conservative landscape of transcripts differentially expressed between blastogenic vs. non-blastogenic epithelia we compared three different mapping and analysis strategies with a de novo assembled transcriptome and partially assembled genome as references, additionally a self-mapping strategy on the dataset. A subset of differentially expressed genes were analyzed and validated by in situ hybridization. The comparison of different analyses allowed us to isolate stringent sets of target genes, including transcripts with potential involvement in the onset of a non-embryonic developmental pathway. The results provide a good entry point to approach regenerative event in a basal chordate.This work was supported by AFM Telethon grant (#16611), IRG Marie Curie grant (#276974), ANR (ANR-14-CE02-0019-01) and IDEX Super (INDIBIO). L.R. was supported by an UPMC-EMREGENCE grant and by a FRM grant (#FDT20140931163). A.C. was supported by a FRM grant (ING 20140129231)

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement

A wnt2 ortholog in the sea urchin Paracentrotus lividus

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Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian

International audienceWe have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at ''oral'' and ''aboral'' poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/b-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes

A minimal molecular toolkit for mineral deposition? Biochemistry and proteomics of the test matrix of adult specimens of the sea urchin Paracentrotus lividus.

12 pagesInternational audienceThe sea urchin endoskeleton consists of a magnesium-rich biocalcite comprising a small amount of occluded organic macromolecules. This structure constitutes a key-model for understanding the mineral - organics interplay, and for conceiving in vitro bio-inspired materials with tailored properties. Here we employed a deep-clean technique to purify the occluded proteins from adult Paracentrotus lividus tests. We characterized them by 1- and 2D-electrophoreses, ELISA and immunoblotting, and using liquid chromatography coupled with Mass Spectrometry (nanoLC-MS/MS), we identified two metalloenzymes (carbonic anhydrase and MMP), a set of MSP130 family members, several C-type lectins (SM29, SM41, PM27) and cytoskeletal proteins. We demonstrate the effect of the protein extract on the crystals, with an in vitro crystallization assay. We suggest that this small set of biomineralization proteins may represent a 'minimal molecular crystallization toolkit'. Biominerals often exhibit superior chemical properties, when compared to their inorganic counterparts. This is due pro parte to the proteins that are occluded in the mineral. However, the limited available studies on biomineralization have not yet succeeded in identifying a minimal set of proteins directly involved in the formation of the biomineral in vivo and sufficiently required for in vitro precipitation. Indeed, the high number of proteins identified by high-throughput screening in the recent years does not encourage the possibility of recreating or tailoring the mineral in vitro. Thus, the identification of biomineralization proteins involved in protein-mineral interactions is highly awaited. In the present study, we used the sea urchin, Paracentrotus lividus (P. lividus), to identify the native proteins directly taking part in protein-mineral interactions. We employed an improved deep-clean technique to extract and purify the native occluded skeletal matrix proteins from the test and identified them by the highly sensitive technique of nanoLC-MS/MS. We show that this minimal set of proteins has a shaping effect on the formation of biocalcite in vitro. This work gives insights on the biomineralization of the sea urchin, while it paves the way for the identification of biomineralization proteins in other biomineralizing systems. Understanding the 'biologically controlled mineralization' will facilitate the in vitro formation and tailoring of biominerals in mild conditions for applications in medicine and materials science

Morpholinos targeting conserved and cnidarian-specific transcripts disrupt development.

<p>DIC imaged larvae developed from morpholino-injected eggs after 24hpf and 48hpf development or at the early gastrula stage as indicated. Typical morphology observed at non-toxic morpholino doses are shown in each case, with the oral pole at the top. Similar phenotypes were obtained using a second morpholino in all cases, except FoxQ2c and WegA1, for which no second non-toxic morpholino could be designed. Uninjected embryos (A) and control-MO injected embryos (L) had completed gastrulation at 24hpf with endodermal epithelialisation starting at the aboral pole. By 48hpf uninjected planula larvae (G, Q) had formed with endodermal epithelial layers (black arrows) organized around a central stripe-like cavity and a well-defined lamina layer separating the endoderm and ectoderm (green arrows). WegO1-MO1 embryos showed minor disruption of gastrulation with embryo elongation slightly compromised at 24hpf (B) and the oral half, thin and tapered (asterisk) at 48hpf (H). Morpholinos targeting either Bra1(C, I) or Bra2 (D, J) showed severe delays in gastrulation. By 48 hpf some embryo elongation had occurred but the blastocoel contained only loose, disorganized material. WegA1-MO embryos (F) showed massive cell ingression at the onset of gastrulation, when cell ingression had barely initiated in uninjected embryos cultured in parallel (E). At 48 hpf (K) the endoderm was enlarged and the ectoderm layer was very thin and irregular (black arrowhead). FoxQ2c-MO (M, R) and WegIE2-MO (N, S), showed severely reduced endodermal layer at 48hpf. WegD1-MO and HD02-MO embryos showed severe defects in overall morphology (O, P, T, U), lacking a well defined basal lamina between endoderm and ectoderm. Green arrows indicate the position of this interface. Black arrows indicate the endodermal epithelial layers in G, Q, R and S. Red arrows indicate ingressing cells at the onset of gastrulation in C, D, E and F. Scale bar 50 µm for all panels.</p

Fz1-MO and Stbm-MO have very similar effects on gene expression profiles.

<p>Each set of 3 panels shows typical <i>in situ</i> hybridization patterns obtained for uninjected embryos (left), Fz1-MO embryos (center) and Stbm-MO embryos (right) injected, fixed at the early gastrula stage and processed in parallel. A-B: O-type pattern transcripts (Myb and ZnfO); C-D: IE-type pattern transcripts (FoxA and Znf845); E-F: A-type pattern transcripts (ZnfA and sFRP-A); G-H: D-type pattern transcripts (Botch1 and bZip). Cells with high Botch1 and bZip expression on the blastocoelar face of the ectoderm were discernible (narrows) in Fz1MO and StbmMO embryos. Gene identity is shown in the bottom left of each set of panels. For each transcript the two morpholinos have very similar effects on the expression patterns, confirming the z-score comparisons (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004590#pgen.1004590.s001" target="_blank">File S1</a>). Scale bar 50 µm.</p

Stereotyped modification of gene expression patterns in Wnt3-MO embryos.

<p><i>In situ</i> hybridization analysis of selected transcripts in untreated (left panels) and Wnt3-MO injected (right panels) early gastrulae. Each expression pattern type is represented in the transcripts analyzed, as indicated on the left. A-C: O-type pattern transcripts; D-F: IE-type pattern transcripts, G-I: A-type pattern transcripts; J-L: D-type pattern transcript. For Botch1 (J) and bZip (K), individual cells with high expression on the blastocoelar face of the ectoderm were discernible in the Wnt3-MO embryos. Representative images of the patterns observed in at least three experiments are shown. All control embryos are oriented with the oral pole uppermost. Gene identity is shown in the bottom right of each pair of panels. Scale bar 50 µm.</p

Characteristics of all transcripts analyzed in detail in this study.

<p>Characteristics of all transcripts analyzed in detail in this study.</p