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)

Molecular Phylogeny Restores the Supra-Generic Subdivision of Homoscleromorph Sponges (Porifera, Homoscleromorpha)

Homoscleromorpha is the fourth major sponge lineage, recently recognized to be distinct from the Demospongiae. It contains <100 described species of exclusively marine sponges that have been traditionally subdivided into 7 genera based on morphological characters. Because some of the morphological features of the homoscleromorphs are shared with eumetazoans and are absent in other sponges, the phylogenetic position of the group has been investigated in several recent studies. However, the phylogenetic relationships within the group remain unexplored by modern methods.Here we describe the first molecular phylogeny of Homoscleromorpha based on nuclear (18S and 28S rDNA) and complete mitochondrial DNA sequence data that focuses on inter-generic relationships. Our results revealed two robust clades within this group, one containing the spiculate species (genera Plakina, Plakortis, Plakinastrella and Corticium) and the other containing aspiculate species (genera Oscarella and Pseudocorticium), thus rejecting a close relationship between Pseudocorticium and Corticium. Among the spiculate species, we found affinities between the Plakortis and Plakinastrella genera, and between the Plakina and Corticium. The validity of these clades is furthermore supported by specific morphological characters, notably the type of spicules. Furthermore, the monophyly of the Corticium genus is supported while the monophyly of Plakina is not.As the result of our study we propose to restore the pre-1995 subdivision of Homoscleromorpha into two families: Plakinidae Schulze, 1880 for spiculate species and Oscarellidae Lendenfeld, 1887 for aspiculate species that had been rejected after the description of the genus Pseudocorticium. We also note that the two families of homoscleromorphs exhibit evolutionary stable, but have drastically distinct mitochondrial genome organizations that differ in gene content and gene order

Epithelial morphogenesis : origin and implications for early metazoan evolution

Les premières étapes de l’évolution animale restent obscures mais peuvent toutefois être appréhendées par l’étude comparative du développement des animaux basaux comme les éponges, les cnidaires ou les cténophores. Une des innovations majeures dans l’évolution des formes animales est l’apparition de l’épithélium, classiquement considérée comme une synapomorphie des eumétazoaires. Les homoscléromorphes sont les seules éponges à partager avec Eumetazoa la présence d’un véritable épithélium avec notamment une membrane basale contenant du collagène de type IV. Dans ce clade, la recherche des mécanismes épithéliaux sous-tendant le développement a pour enjeu la meilleure compréhension de leur origine et de leur importance dans l’évolution animale. Le travail de cette thèse a consisté à caractériser chez Oscarella lobularis des outils moléculaires responsables des morphogenèses épithéliales. Trois d’entre eux ont été étudiés chez l’adulte. Le premier, la voie WNT canonique, est capable d’induire l’invagination de l’épithélium externe de l’éponge, ce qui n’est pas sans rappeler ce même rôle dans d’autres contextes épithéliaux d’eumétazoaires. Le deuxième est la voie WNT non canonique ou « voie PCP », qui, quand elle est bloquée, empêche l’invagination initiée par la voie canonique. Enfin, le troisième outil est un membre de la famille des gènes à boîte T, OlTbx qui s’exprime spécifiquement dans l’épithélium après l’invagination sus-mentionnée. Cette expression rappelle des expressions d’autres gènes Tbx dans le feuillet endomésodermique invaginé lors de la gastrulation des eumétazoaires. L’invagination semble utiliser une partie d’un même programme génétique dans la gastrula des Eumetazoa et dans l’adulte des Homoscleromorpha. Mes résultats ouvrent des perspectives intéressantes concernant l’éventuelle reconnaissance d’un stade gastrula chez les éponges, point de discorde de la zoologie classique.The first steps of animal evolution remain obscure but, nevertheless can be better understood by comparative studies of the most basally branching animals, such as sponges, cnidarians and ctenophores. Epithelium is one of the major innovations in the evolution of animal forms and is generally considered as one of the synapomorphies of Eumetazoa. The homoscleromorphs are the only sponges, with Eumetazoa, to have a true epithelium with a basal membrane containing type IV collagen. In this clade, the investigation of epithelial processes underlying development would give insights into their origin and their importance in animal evolution. The aim of my work was to characterize molecular tools involved in epithelial morphogenesis in Oscarella lobularis. I was able to characterize three of those molecular tools. The first one is the canonical WNT pathway inducing invagination of the external epithelium of the sponge, reminiscent of the same function in other epithelial contexts in Eumetazoa. The second one is the non-canonical WNT pathway or “PCP pathway” which blocks invagination when it is inhibited. The third one is a member of the T-box genes family, OlTbx, specifically expressed in the epithelial layer formed by the above-mentioned invagination. Similarly, other Tbx genes are expressed in the endomesodermal layer during eumetazoan gastrulation. Invagination processes involved in both eumetazoan gastrula and homoscleromorph adult tissue seem to share a part (WNT/Tbx) of a common genetical program. My results provide new investigation prospects, in order to answer the difficult question of the origin of gastrulation in sponges

L'origine des morphogenèses épithéliales et leurs implications concernant l'évolution précoce des métazoaires

Les premières étapes de l évolution animale restent obscures mais peuvent toutefois être appréhendées par l étude comparative du développement des animaux basaux comme les éponges, les cnidaires ou les cténophores. Une des innovations majeures dans l évolution des formes animales est l apparition de l épithélium, classiquement considérée comme une synapomorphie des eumétazoaires. Les homoscléromorphes sont les seules éponges à partager avec Eumetazoa la présence d un véritable épithélium avec notamment une membrane basale contenant du collagène de type IV. Dans ce clade, la recherche des mécanismes épithéliaux sous-tendant le développement a pour enjeu la meilleure compréhension de leur origine et de leur importance dans l évolution animale. Le travail de cette thèse a consisté à caractériser chez Oscarella lobularis des outils moléculaires responsables des morphogenèses épithéliales. Trois d entre eux ont été étudiés chez l adulte. Le premier, la voie WNT canonique, est capable d induire l invagination de l épithélium externe de l éponge, ce qui n est pas sans rappeler ce même rôle dans d autres contextes épithéliaux d eumétazoaires. Le deuxième est la voie WNT non canonique ou voie PCP , qui, quand elle est bloquée, empêche l invagination initiée par la voie canonique. Enfin, le troisième outil est un membre de la famille des gènes à boîte T, OlTbx qui s exprime spécifiquement dans l épithélium après l invagination sus-mentionnée. Cette expression rappelle des expressions d autres gènes Tbx dans le feuillet endomésodermique invaginé lors de la gastrulation des eumétazoaires. L invagination semble utiliser une partie d un même programme génétique dans la gastrula des Eumetazoa et dans l adulte des Homoscleromorpha. Mes résultats ouvrent des perspectives intéressantes concernant l éventuelle reconnaissance d un stade gastrula chez les éponges, point de discorde de la zoologie classique.The first steps of animal evolution remain obscure but, nevertheless can be better understood by comparative studies of the most basally branching animals, such as sponges, cnidarians and ctenophores. Epithelium is one of the major innovations in the evolution of animal forms and is generally considered as one of the synapomorphies of Eumetazoa. The homoscleromorphs are the only sponges, with Eumetazoa, to have a true epithelium with a basal membrane containing type IV collagen. In this clade, the investigation of epithelial processes underlying development would give insights into their origin and their importance in animal evolution. The aim of my work was to characterize molecular tools involved in epithelial morphogenesis in Oscarella lobularis. I was able to characterize three of those molecular tools. The first one is the canonical WNT pathway inducing invagination of the external epithelium of the sponge, reminiscent of the same function in other epithelial contexts in Eumetazoa. The second one is the non-canonical WNT pathway or PCP pathway which blocks invagination when it is inhibited. The third one is a member of the T-box genes family, OlTbx, specifically expressed in the epithelial layer formed by the above-mentioned invagination. Similarly, other Tbx genes are expressed in the endomesodermal layer during eumetazoan gastrulation. Invagination processes involved in both eumetazoan gastrula and homoscleromorph adult tissue seem to share a part (WNT/Tbx) of a common genetical program. My results provide new investigation prospects, in order to answer the difficult question of the origin of gastrulation in sponges.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Comparative metagenomics of cellulose- and poplar hydrolysate-degrading microcosms from gut microflora of the Canadian Beaver (Castor canadensis) and North American moose (Alces americanus) after long-term enrichment

To identify carbohydrate-active enzymes (CAZymes) that might be particularly relevant for wood fiber processing, we performed a comparative metagenomic analysis of digestive systems from Canadian beaver (Castor canadensis) and North American moose (Alces americanus) following 3 years of enrichment on either microcrystalline cellulose or poplar hydrolysate. In total, 9,386 genes encoding CAZymes and carbohydrate-binding modules (CBMs) were identified, with up to half predicted to originate from Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria phyla, and up to 17% from unknown phyla. Both PCA and hierarchical cluster analysis distinguished the annotated glycoside hydrolase (GH) distributions identified herein, from those previously reported for grass-feeding mammals and herbivorous foragers. The CAZyme profile of moose rumen enrichments also differed from a recently reported moose rumen metagenome, most notably by the absence of GH13-appended dockerins. Consistent with substrate-driven convergence, CAZyme profiles from both poplar hydrolysate-fed cultures differed from cellulose-fed cultures, most notably by increased numbers of unique sequences belonging to families GH3, GH5, GH43, GH53, and CE1. Moreover, pairwise comparisons of moose rumen enrichments further revealed higher counts of GH127 and CE15 families in cultures fed with poplar hydrolysate. To expand our scope to lesser known carbohydrate-active proteins, we identified and compared multi-domain proteins comprising both a CBM and domain of unknown function (DUF) as well as proteins with unknown function within the 416 predicted polysaccharide utilization loci (PULs). Interestingly, DUF362, identified in iron-sulfur proteins, was consistently appended to CBM9; on the other hand, proteins with unknown function from PULs shared little identity unless from identical PULs. Overall, this study sheds new light on the lignocellulose degrading capabilities of microbes originating from digestive systems of mammals known for fiber-rich diets, and highlights the value of enrichment to select new CAZymes from metagenome sequences for future biochemical characterization.Peer reviewe

Comparative Metagenomics of Cellulose- and Poplar Hydrolysate-Degrading Microcosms from Gut Microflora of the Canadian Beaver (Castor canadensis) and North American Moose (Alces americanus) after Long-Term Enrichment

To identify carbohydrate-active enzymes (CAZymes) that might be particularly relevant for wood fiber processing, we performed a comparative metagenomic analysis of digestive systems from Canadian beaver (Castor canadensis) and North American moose (Alces americanus) following 3 years of enrichment on either microcrystalline cellulose or poplar hydrolysate. In total, 9,386 genes encoding CAZymes and carbohydrate-binding modules (CBMs) were identified, with up to half predicted to originate from Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria phyla, and up to 17% from unknown phyla. Both PCA and hierarchical cluster analysis distinguished the annotated glycoside hydrolase (GH) distributions identified herein, from those previously reported for grass-feeding mammals and herbivorous foragers. The CAZyme profile of moose rumen enrichments also differed from a recently reported moose rumen metagenome, most notably by the absence of GH13-appended dockerins. Consistent with substrate-driven convergence, CAZyme profiles from both poplar hydrolysate-fed cultures differed from cellulose-fed cultures, most notably by increased numbers of unique sequences belonging to families GH3, GH5, GH43, GH53, and CE1. Moreover, pairwise comparisons of moose rumen enrichments further revealed higher counts of GH127 and CE15 families in cultures fed with poplar hydrolysate. To expand our scope to lesser known carbohydrate-active proteins, we identified and compared multi-domain proteins comprising both a CBM and domain of unknown function (DUF) as well as proteins with unknown function within the 416 predicted polysaccharide utilization loci (PULs). Interestingly, DUF362, identified in iron–sulfur proteins, was consistently appended to CBM9; on the other hand, proteins with unknown function from PULs shared little identity unless from identical PULs. Overall, this study sheds new light on the lignocellulose degrading capabilities of microbes originating from digestive systems of mammals known for fiber-rich diets, and highlights the value of enrichment to select new CAZymes from metagenome sequences for future biochemical characterization

DataSheet_1_Taxonomic composition and carbohydrate-active enzyme content in microbial enrichments from pulp mill anaerobic granules after cultivation on lignocellulosic substrates.zip

Metagenomes of lignocellulose-degrading microbial communities are reservoirs of carbohydrate-active enzymes relevant to biomass processing. Whereas several metagenomes of natural digestive systems have been sequenced, the current study analyses metagenomes originating from an industrial anaerobic digester that processes effluent from a cellulose pulp mill. Both 16S ribosomal DNA and metagenome sequences were obtained following anaerobic cultivation of the digester inoculum on cellulose and pretreated (steam exploded) poplar wood chips. The community composition and profile of predicted carbohydrate-active enzymes were then analyzed in detail. Recognized lignocellulose degraders were abundant in the resulting cultures, including populations belonging to Clostridiales and Bacteroidales orders. Poorly defined taxonomic lineages previously identified in other lignocellulose-degrading communities were also detected, including the uncultivated Firmicutes lineage OPB54 which represented nearly 10% of the cellulose-fed enrichment even though it was not detected in the bioreactor inoculum. In total, 3580 genes encoding carbohydrate-active enzymes were identified through metagenome sequencing. Similar to earlier enrichments of animal digestive systems, the profile encoded by the bioreactor inoculum following enrichment on pretreated wood was distinguished from the cellulose counterpart by a higher occurrence of enzymes predicted to act on pectin. The majority (> 93%) of carbohydrate-active enzymes predicted to act on plant polysaccharides were identified in the metagenome assembled genomes, permitting taxonomic assignment. The taxonomic assignment revealed that only a small selection of organisms directly participates in plant polysaccharide deconstruction and supports the rest of the community.</p

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection.

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ∼75% of the genus-level bacterial and archaeal taxa present in the rumen

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B-12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents similar to 75% of the genus-level bacterial and archaeal taxa present in the rumen