A transcriptomic examination of encased rotifer embryos reveals the developmental trajectory leading to long-term dormancy; are they "animal seeds"?

BACKGROUND: Organisms from many distinct evolutionary lineages acquired the capacity to enter a dormant state in response to environmental conditions incompatible with maintaining normal life activities. Most studied organisms exhibit seasonal or annual episodes of dormancy, but numerous less studied organisms enter long-term dormancy, lasting decades or even centuries. Intriguingly, many planktonic animals produce encased embryos known as resting eggs or cysts that, like plant seeds, may remain dormant for decades. Herein, we studied a rotifer Brachionus plicatilis as a model planktonic species that forms encased dormant embryos via sexual reproduction and non-dormant embryos via asexual reproduction and raised the following questions: Which genes are expressed at which time points during embryogenesis? How do temporal transcript abundance profiles differ between the two types of embryos? When does the cell cycle arrest? How do dormant embryos manage energy? RESULTS: As the molecular developmental kinetics of encased embryos remain unknown, we employed single embryo RNA sequencing (CEL-seq) of samples collected during dormant and non-dormant embryogenesis. We identified comprehensive and temporal transcript abundance patterns of genes and their associated enriched functional pathways. Striking differences were uncovered between dormant and non-dormant embryos. In early development, the cell cycle-associated pathways were enriched in both embryo types but terminated with fewer nuclei in dormant embryos. As development progressed, the gene transcript abundance profiles became increasingly divergent between dormant and non-dormant embryos. Organogenesis was suspended in dormant embryos, concomitant with low transcript abundance of homeobox genes, and was replaced with an ATP-poor preparatory phase characterized by very high transcript abundance of genes encoding for hallmark dormancy proteins (e.g., LEA proteins, sHSP, and anti-ROS proteins, also found in plant seeds) and proteins involved in dormancy exit. Surprisingly, this period appeared analogous to the late maturation phase of plant seeds. CONCLUSIONS: The study highlights novel divergent temporal transcript abundance patterns between dormant and non-dormant embryos. Remarkably, several convergent functional solutions appear during the development of resting eggs and plant seeds, suggesting a similar preparatory phase for long-term dormancy. This study accentuated the broad novel molecular features of long-term dormancy in encased animal embryos that behave like "animal seeds".

Genomics and transcriptomics of epizoic Seisonidea (Rotifera, syn. Syndermata) reveal strain formation and gradual gene loss with growing ties to the host.

BACKGROUND: Seisonidea (also Seisonacea or Seisonidae) is a group of small animals living on marine crustaceans (Nebalia spec.) with only four species described so far. Its monophyletic origin with mostly free-living wheel animals (Monogononta, Bdelloidea) and endoparasitic thorny-headed worms (Acanthocephala) is widely accepted. However, the phylogenetic relationships inside the Rotifera-Acanthocephala clade (Rotifera sensu lato or Syndermata) are subject to ongoing debate, with consequences for our understanding of how genomes and lifestyles might have evolved. To gain new insights, we analyzed first drafts of the genome and transcriptome of the key taxon Seisonidea. RESULTS: Analyses of gDNA-Seq and mRNA-Seq data uncovered two genetically distinct lineages in Seison nebaliae Grube, 1861 off the French Channel coast. Their mitochondrial haplotypes shared only 82% sequence identity despite identical gene order. In the nuclear genome, distinct linages were reflected in different gene compactness, GC content and codon usage. The haploid nuclear genome spans ca. 46 Mb, of which 96% were reconstructed. According to ~ 23,000 SuperTranscripts, gene number in S. nebaliae should be within the range published for other members of Rotifera-Acanthocephala. Consistent with this, numbers of metazoan core orthologues and ANTP-type transcriptional regulatory genes in the S. nebaliae genome assembly were between the corresponding numbers in the other assemblies analyzed. We additionally provide evidence that a basal branching of Seisonidea within Rotifera-Acanthocephala could reflect attraction to the outgroup. Accordingly, rooting via a reconstructed ancestral sequence led to monophyletic Pararotatoria (Seisonidea+Acanthocephala) within Hemirotifera (Bdelloidea+Pararotatoria). CONCLUSION: Matching genome/transcriptome metrics with the above phylogenetic hypothesis suggests that a haploid nuclear genome of about 50 Mb represents the plesiomorphic state for Rotifera-Acanthocephala. Smaller genome size in S. nebaliae probably results from subsequent reduction. In contrast, genome size should have increased independently in monogononts as well as bdelloid and acanthocephalan stem lines. The present data additionally indicate a decrease in gene repertoire from free-living to epizoic and endoparasitic lifestyles. Potentially, this reflects corresponding steps from the root of Rotifera-Acanthocephala via the last common ancestors of Hemirotifera and Pararotatoria to the one of Acanthocephala. Lastly, rooting via a reconstructed ancestral sequence may prove useful in phylogenetic analyses of other deep splits.

The genome, transcriptome, and proteome of the fish parasite Pomphorhynchus laevis (Acanthocephala).

Thorny-headed worms (Acanthocephala) are endoparasites exploiting Mandibulata (Arthropoda) and Gnathostomata (Vertebrata). Despite their world-wide occurrence and economic relevance as a pest, genome and transcriptome assemblies have not been published before. However, such data might hold clues for a sustainable control of acanthocephalans in animal production. For this reason, we present the first draft of an acanthocephalan nuclear genome, besides the mitochondrial one, using the fish parasite Pomphorhynchus laevis (Palaeacanthocephala) as a model. Additionally, we have assembled and annotated the transcriptome of this species and the proteins encoded. A hybrid assembly of long and short reads resulted in a near-complete P. laevis draft genome of ca. 260 Mb, comprising a large repetitive portion of ca. 63%. Numbers of transcripts and translated proteins (35,683) were within the range of other members of the Rotifera-Acanthocephala clade. Our data additionally demonstrate a significant reorganization of the acanthocephalan gene repertoire. Thus, more than 20% of the usually conserved metazoan genes were lacking in P. laevis. Ontology analysis of the retained genes revealed many connections to the incorporation of carotinoids. These are probably taken up via the surface together with lipids, thus accounting for the orange coloration of P. laevis. Furthermore, we found transcripts and protein sequences to be more derived in P. laevis than in rotifers from Monogononta and Bdelloidea. This was especially the case in genes involved in energy metabolism, which might reflect the acanthocephalan ability to use the scarce oxygen in the host intestine for respiration and simultaneously carry out fermentation. Increased plasticity of the gene repertoire through the integration of foreign DNA into the nuclear genome seems to be another underpinning factor of the evolutionary success of acanthocephalans. In any case, energy-related genes and their proteins may be considered as candidate targets for the acanthocephalan control.

Rotiferan Hox genes give new insights into the evolution of metazoan bodyplans.

The phylum Rotifera consists of minuscule, nonsegmented animals with a unique body plan and an unresolved phylogenetic position. The presence of pharyngeal articulated jaws supports an inclusion in Gnathifera nested in the Spiralia. Comparison of Hox genes, involved in animal body plan patterning, can be used to infer phylogenetic relationships. Here, we report the expression of five Hox genes during embryogenesis of the rotifer Brachionus manjavacas and show how these genes define different functional components of the nervous system and not the usual bilaterian staggered expression along the anteroposterior axis. Sequence analysis revealed that the lox5-parapeptide, a key signature in lophotrochozoan and platyhelminthean Hox6/lox5 genes, is absent and replaced by different signatures in Rotifera and Chaetognatha, and that the MedPost gene, until now unique to Chaetognatha, is also present in rotifers. Collectively, our results support an inclusion of chaetognaths in gnathiferans and Gnathifera as sister group to the remaining spiralians.Rotifers are microscopic animals with an unusual, nonsegmented body plan consisting of a head, trunk and foot. Here, Fröbius and Funch investigate the role of Hox genes-which are widely used in animal body plan patterning-in rotifer embryogenesis and find non-canonical expression in the nervous system.

Formation of body appendages during caudal regeneration in Platynereis dumerilii: adaptation of conserved molecular toolsets.

BACKGROUND: Platynereis and other polychaete annelids with homonomous segmentation are regarded to closely resemble ancestral forms of bilateria. The head region comprises the prostomium, the peristomium, a variable number of cephalized body segments and several appendages, like cirri, antennae and palps. The trunk of such polychaetes shows numerous, nearly identical segments. Each segment bears a parapodium with species-specific morphology on either side. The posterior end of the trunk features a segment proliferation zone and a terminal pygidium with the anus and anal cirri. The removal of a substantial part of the posterior trunk is by no means lethal. Cells at the site of injury dedifferentiate and proliferate forming a blastema to regenerate both the pygidium and the proliferation zone. The pygidium forms new anal cirri, and the proliferation zone generates new segments at a rapid pace. The formation of body appendages like the cirri and the segmental parapodia can thus be studied in the caudal regenerate of Platynereis within only a few days. RESULTS: The development of body appendages in Platynereis is regulated by a network of genes common to polychaetes but also shared by distant taxa. We isolated DNA sequences from P. dumerilii of five genes known to be involved in appendage formation within other groups: Meis/homothorax, Pbx1/extradenticle, Dlx/Distal-less, decapentaplegic and specific protein 1/buttonhead. Analyses of expression patterns during caudal regeneration by in situ hybridization reveal striking similarities related to expression in arthropods and vertebrates. All genes exhibit transient expression during differentiation and growth of segments. As was shown previously in other phyla Pdu-Meis/hth and Pdu-Pbx1/exd are co-expressed, although the expression is not limited to the proximal part of the parapodia. Pdu-Dll is prominent in parapodia but upregulated in the anal cirri. No direct dependence concerning Pdu-Dll and Pdu-sp/btd expression is observed in Platynereis. Pdu-dpp shows an expression pattern not comparable to its expression in other taxa. CONCLUSIONS: The expression patterns observed suggest conserved roles of these genes during appendage formation across different clades, but the underlying mechanisms utilizing this toolset might not be identical. Some genes show broad expression along the proximodistal axis indicating a possible role in proximodistal patterning of body appendages. Other genes exhibit expression patterns limited to specific parts and tissues of the growing parapodia, thus presumably being involved in formation of taxon-specific morphological differences.

Activation of Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii.

The capability of regenerating posterior segments and pygidial structures is ancestral for annelids and has been lost only a few times within this phylum. As one of the three major segmented taxa, annelids enable us to monitor reconstruction of lost tissues and organs. During regeneration, regional identities have to be imprinted onto the newly formed segments. In this study, we show spatial and temporal localization of expression of nine Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Hox genes are homeodomain genes encoding transcriptional regulators of axial patterning in bilaterian animals during development. We demonstrate that five Platynereis Hox genes belonging to paralog groups (PG) 1, 4, 5, 6, and 9-14 are expressed in domains of the regenerating nervous system consistent with providing positional information along the anteroposterior axis of the regenerate. We report that expression in regenerating neuromeres is limited to varying subsets of perikarya, called gangliosomes. Four of nine genes analyzed do not appear to be involved in axial patterning. Two genes, Pdu-Hox2 and Pdu-Hox3, are predominantly expressed in the growth zone region. For some Hox genes expression in newly formed coelomic epithelia can be observed. Platynereis Hox genes do not exhibit temporal or spatial colinearity. Although there are some similarities to previously reported expression patterns during larval and postlarval development in Nereididae (Kulakova et al. 2007), expression patterns observed during caudal regeneration also show unique patterns.

Genomic organization and expression demonstrate spatial and temporal Hox gene colinearity in the lophotrochozoan Capitella sp. I.

Hox genes define regional identities along the anterior-posterior axis in many animals. In a number of species, Hox genes are clustered in the genome, and the relative order of genes corresponds with position of expression in the body. Previous Hox gene studies in lophotrochozoans have reported expression for only a subset of the Hox gene complement and/or lack detailed genomic organization information, limiting interpretations of spatial and temporal colinearity in this diverse animal clade. We studied expression and genomic organization of the single Hox gene complement in the segmented polychaete annelid Capitella sp. I. Total genome searches identified 11 Hox genes in Capitella, representing 11 distinct paralog groups thought to represent the ancestral lophotrochozoan complement. At least 8 of the 11 Capitella Hox genes are genomically linked in a single cluster, have the same transcriptional orientation, and lack interspersed non-Hox genes. Studying their expression by situ hybridization, we find that the 11 Capitella Hox genes generally exhibit spatial and temporal colinearity. With the exception of CapI-Post1, Capitella Hox genes are all expressed in broad ectodermal domains during larval development, consistent with providing positional information along the anterior-posterior axis. The anterior genes CapI-lab, CapI-pb, and CapI-Hox3 initiate expression prior to the appearance of segments, while more posterior genes appear at or soon after segments appear. Many of the Capitella Hox genes have either an anterior or posterior expression boundary coinciding with the thoracic-abdomen transition, a major body tagma boundary. Following metamorphosis, several expression patterns change, including appearance of distinct posterior boundaries and restriction to the central nervous system. Capitella Hox genes have maintained a clustered organization, are expressed in the canonical anterior-posterior order found in other metazoans, and exhibit spatial and temporal colinearity, reflecting Hox gene characteristics that likely existed in the protostome-deuterostome ancestor.

Capitella sp. I homeobrain-like, the first lophotrochozoan member of a novel paired-like homeobox gene family.

The paired-like class of homeobox genes contains numerous distinct families, many of which have been implicated in a variety of developmental functions. We report the isolation and expression of a gene with high similarity to Drosophila melanogaster homeobrain from the polychaete annelid Capitella sp. I. The homeobrain-like (hbnl) gene is a paired-like gene that contains a conserved homeodomain, octapeptide region, alanine stretches, and an OAR domain. Gene orthology analyses of the homeodomain from CapI-hbnl places this gene in a new family of paired-like homeodomain genes that includes D. melanogaster homeobrain (hbn) and representatives from all major bilaterian clades as well as a cnidarian gene. CapI-hbnl expression is largely restricted to subsets of cells in the brain and eyes during larval development in Capitella sp. I. The earliest expression of CapI-hbnl is in small discrete cell clusters in the cerebral ganglia. This expression persists through late larval developmental stages whereas expression is absent in postmetamorphic juveniles. Outside the brain, expression is present on the ventral side of the larva in two small cell clusters, at the brain/pharyngeal border and in the anterior-most segment. CapI-hbnl shares features of brain expression with hbn, although in contrast to hbn, which is expressed along the length of the ventral nerve cord, CapI-hbnl has a restricted anterior expression pattern. CapI-hbnl represents an important neural marker for characterization of the annelid nervous system.

ParaHox gene expression in the polychaete annelid Capitella sp. I.

Hox and ParaHox genes are transcriptional regulators vital for many aspects of embryonic development in bilaterian animals and are considered to have originated from one ancestral proto-Hox/ParaHox cluster. Hox genes are clustered in the genome of both protostomes and deuterostomes, and there is a specific relationship between the position of a gene in the cluster and the position of its expression along the animal body axis (colinearity). It is not clear whether the ParaHox genes Gsx, Xlox, and, Cdx generally exhibit a similar phenomenon since developmental expression for all three ParaHox genes within a single species has not yet been described for any protostome animal. Here we show the spatial and temporal localization for all three ParaHox genes in the polychaete Capitella sp. I, a member of one of the morphologically most diverse and understudied groups within the Metazoa, the Lophotrochozoa. Our data demonstrate that although both CapI-Xlox and CapI-Cdx are regionally expressed in the gut, the three Capitella sp. I ParaHox genes as a group do not perfectly fit predictions of temporal or spatial colinearity. Instead, there is a conservation of expression across species associated with development of particular tissues, and the relative order of initiation of ParaHox gene expression likely reflects the relative order of species-specific tissue development during ontogenesis.

Expression of developmental genes during early embryogenesis of Hydra.

Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning.