The gene regulatory program of Acrobeloides nanus reveals conservation of phylum-specific expression.

The evolution of development has been studied through the lens of gene regulation by examining either closely related species or extremely distant animals of different phyla. In nematodes, detailed cell- and stage-specific expression analyses are focused on the model Caenorhabditis elegans, in part leading to the view that the developmental expression of gene cascades in this species is archetypic for the phylum. Here, we compared two species of an intermediate evolutionary distance: the nematodes C. elegans (clade V) and Acrobeloides nanus (clade IV). To examine A. nanus molecularly, we sequenced its genome and identified the expression profiles of all genes throughout embryogenesis. In comparison with C. elegans, A. nanus exhibits a much slower embryonic development and has a capacity for regulative compensation of missing early cells. We detected conserved stages between these species at the transcriptome level, as well as a prominent middevelopmental transition, at which point the two species converge in terms of their gene expression. Interestingly, we found that genes originating at the dawn of the Ecdysozoa supergroup show the least expression divergence between these two species. This led us to detect a correlation between the time of expression of a gene and its phylogenetic age: evolutionarily ancient and young genes are enriched for expression in early and late embryogenesis, respectively, whereas Ecdysozoa-specific genes are enriched for expression during the middevelopmental transition. Our results characterize the developmental constraints operating on each individual embryo in terms of developmental stages and genetic evolutionary history.

Genome analysis of Diploscapter coronatus: insights into molecular peculiarities of a nematode with parthenogenetic reproduction.

BACKGROUND: Sexual reproduction involving the fusion of egg and sperm is prevailing among eukaryotes. In contrast, the nematode Diploscapter coronatus, a close relative of the model Caenorhabditis elegans, reproduces parthenogenetically. Neither males nor sperm have been observed and some steps of meiosis are apparently skipped in this species. To uncover the genomic changes associated with the evolution of parthenogenesis in this nematode, we carried out a genome analysis. RESULTS: We obtained a 170 Mbp draft genome in only 511 scaffolds with a N50 length of 1 Mbp. Nearly 90% of these scaffolds constitute homologous pairs with a 5.7% heterozygosity on average and inversions and translocations, meaning that the 170 Mbp sequences correspond to the diploid genome. Fluorescent staining shows that the D. coronatus genome consists of two chromosomes (2n = 2). In our genome annotation, we found orthologs of 59% of the C. elegans genes. However, a number of genes were missing or very divergent. These include genes involved in sex determination (e.g. xol-1, tra-2) and meiosis (e.g. the kleisins rec-8 and coh-3/4) giving a possible explanation for the absence of males and the second meiotic division. The high degree of heterozygosity allowed us to analyze the expression level of individual alleles. Most of the homologous pairs show very similar expression levels but others exhibit a 2-5-fold difference. CONCLUSIONS: Our high-quality draft genome of D. coronatus reveals the peculiarities of the genome of parthenogenesis and provides some clues to the genetic basis for parthenogenetic reproduction. This draft genome should be the basis to elucidate fundamental questions related to parthenogenesis such as its origin and mechanisms through comparative analyses with other nematodes. Furthermore, being the closest outgroup to the genus Caenorhabditis, the draft genome will help to disclose many idiosyncrasies of the model C. elegans and its congeners in future studies.

Developmental variations among Panagrolaimid nematodes indicate developmental system drift within a small taxonomic unit.

Comparative studies of nematode embryogenesis among different clades revealed considerable variations. However, to what extent developmental differences exist between closely related species has mostly remained nebulous. Here, we explore the correlation between phylogenetic neighborhood and developmental variation in a restricted and morphologically particularly uniform taxonomic group (Panagrolaimidae) to determine to what extent (1) morphological and developmental characters go along with molecular data and thus can serve as diagnostic tools for the definition of kinship and (2) developmental system drift (DSD; modifications of developmental patterns without corresponding morphological changes) can be found within a small taxonomic unit. Our molecular approaches firmly support subdivision of Panagrolaimid nematodes into two monophyletic groups. These can be discriminated by distinct peculiarities in early embryonic cell lineages and a mirror-image expression pattern of the gene skn-1. This suggests major changes in the logic of cell specification and the action of DSD in the studied representatives of the two neighboring nematode taxa.

The genome of Romanomermis culicivorax: revealing fundamental changes in the core developmental genetic toolkit in Nematoda.

BACKGROUND: The genetics of development in the nematode Caenorhabditis elegans has been described in exquisite detail. The phylum Nematoda has two classes: Chromadorea (which includes C. elegans) and the Enoplea. While the development of many chromadorean species resembles closely that of C. elegans, enoplean nematodes show markedly different patterns of early cell division and cell fate assignment. Embryogenesis of the enoplean Romanomermis culicivorax has been studied in detail, but the genetic circuitry underpinning development in this species has not been explored. RESULTS: We generated a draft genome for R. culicivorax and compared its gene content with that of C. elegans, a second enoplean, the vertebrate parasite Trichinella spiralis, and a representative arthropod, Tribolium castaneum. This comparison revealed that R. culicivorax has retained components of the conserved ecdysozoan developmental gene toolkit lost in C. elegans. T. spiralis has independently lost even more of this toolkit than has C. elegans. However, the C. elegans toolkit is not simply depauperate, as many novel genes essential for embryogenesis in C. elegans are not found in, or have only extremely divergent homologues in R. culicivorax and T. spiralis. Our data imply fundamental differences in the genetic programmes not only for early cell specification but also others such as vulva formation and sex determination. CONCLUSIONS: Despite the apparent morphological conservatism, major differences in the molecular logic of development have evolved within the phylum Nematoda. R. culicivorax serves as a tractable system to contrast C. elegans and understand how divergent genomic and thus regulatory backgrounds nevertheless generate a conserved phenotype. The R. culicivorax draft genome will promote use of this species as a research model.

CRISPR/Cas9 mediated gene editing in non-model nematode Panagrolaimus sp. PS1159.

The phylum Nematoda harbors a huge diversity of species in a broad range of ecosystems and habitats. Nematodes share a largely conserved Bauplan but major differences have been found in early developmental processes. The development of the nematode model organism Caenorhabditis elegans has been studied in great detail for decades. These efforts have provided the community with a large number of protocols and methods. Unfortunately, many of these tools are not easily applicable in non-Caenorhabditis nematodes. In recent years it has become clear that many crucial genes in the C. elegans developmental toolkit are absent in other nematode species. It is thus necessary to study the developmental program of other nematode species in detail to understand evolutionary conservation and novelty in the phylum. Panagrolaimus sp. PS1159 is a non-parasitic nematode exhibiting parthenogenetic reproduction and we are establishing the species to comparatively study evolution, biodiversity, and alternative reproduction and survival strategies. Here, we demonstrate the first successful application of the CRISPR/Cas9 system for genome editing in Panagrolaimus sp. PS1159 and the closely related hermaphroditic species Propanagrolaimus sp. JU765 applying the non-homologous end joining and the homology-directed repair (HDR) mechanisms. Using microinjections and modifying published protocols from C. elegans and P. pacificus we induced mutations in the orthologue of unc-22. This resulted in a visible uncoordinated twitching phenotype. We also compared the HDR efficiency following the delivery of different single-stranded oligodeoxynucleotides (ssODNs). Our work will expand the applicability for a wide range of non-model nematodes from across the tree and facilitate functional analysis into the evolution of parthenogenesis, changes in the developmental program of Nematoda, and cryptobiosis.

Conservation of MAP kinase activity and MSP genes in parthenogenetic nematodes.

BACKGROUND: MAP (mitogen-activated protein) kinase activation is a prerequisite for oocyte maturation, ovulation and fertilisation in many animals. In the hermaphroditic nematode Caenorhabditis elegans, an MSP (major sperm protein) dependent pathway is utilised for MAP kinase activation and successive oocyte maturation with extracellular MSP released from sperm acting as activator. How oocyte-to-embryo transition is triggered in parthenogenetic nematode species that lack sperm, is not known. RESULTS: We investigated two key elements of oocyte-to-embryo transition, MSP expression and MAP kinase signaling, in two parthenogenetic nematodes and their close hermaphroditic relatives. While activated MAP kinase is present in all analysed nematodes irrespective of the reproductive mode, MSP expression differs. In contrast to hermaphroditic or bisexual species, we do not find MSP expression at the protein level in parthenogenetic nematodes. However, genomic sequence analysis indicates that functional MSP genes are present in several parthenogenetic species. CONCLUSIONS: We present three alternative interpretations to explain our findings. (1) MSP has lost its function as a trigger of MAP kinase activation and is not expressed in parthenogenetic nematodes. Activation of the MAP kinase pathway is achieved by another, unknown mechanism. Functional MSP genes are required for occasionally emerging males found in some parthenogenetic species. (2) Because of long-term disadvantages, parthenogenesis is of recent origin. MSP genes remained intact during this short interval although they are useless. As in the first scenario, an unknown mechanism is responsible for MAP kinase activation. (3) The molecular machinery regulating oocyte-to-embryo transition in parthenogenetic nematodes is conserved with respect to C. elegans, thus requiring intact MSP genes. However, MSP expression has been shifted to non-sperm cells and is reduced below the detection limits, but is still sufficient to trigger MAP kinase activation and embryogenesis.

Signatures of the Evolution of Parthenogenesis and Cryptobiosis in the Genomes of Panagrolaimid Nematodes.

Most animal species reproduce sexually and fully parthenogenetic lineages are usually short lived in evolution. Still, parthenogenesis may be advantageous as it avoids the cost of sex and permits colonization by single individuals. Panagrolaimid nematodes have colonized environments ranging from arid deserts to Arctic and Antarctic biomes. Many are obligatory meiotic parthenogens, and most have cryptobiotic abilities, being able to survive repeated cycles of complete desiccation and freezing. To identify systems that may contribute to these striking abilities, we sequenced and compared the genomes and transcriptomes of parthenogenetic and outcrossing panagrolaimid species, including cryptobionts and non-cryptobionts. The parthenogens are triploids, most likely originating through hybridization. Adaptation to cryptobiosis shaped the genomes of panagrolaimid nematodes and is associated with the expansion of gene families and signatures of selection on genes involved in cryptobiosis. All panagrolaimids have acquired genes through horizontal gene transfer, some of which are likely to contribute to cryptobiosis.

Differences in the genetic control of early egg development and reproduction between C. elegans and its parthenogenetic relative D. coronatus.

BACKGROUND: The free-living nematode Diploscapter coronatus is the closest known relative of Caenorhabditis elegans with parthenogenetic reproduction. It shows several developmental idiosyncracies, for example concerning the mode of reproduction, embryonic axis formation and early cleavage pattern (Lahl et al. in Int J Dev Biol 50:393-397, 2006). Our recent genome analysis (Hiraki et al. in BMC Genomics 18:478, 2017) provides a solid foundation to better understand the molecular basis of developmental idiosyncrasies in this species in an evolutionary context by comparison with selected other nematodes. Our genomic data also yielded indications for the view that D. coronatus is a product of interspecies hybridization. RESULTS: In a genomic comparison between D. coronatus, C. elegans, other representatives of the genus Caenorhabditis and the more distantly related Pristionchus pacificus and Panagrellus redivivus, certain genes required for central developmental processes in C. elegans like control of meiosis and establishment of embryonic polarity were found to be restricted to the genus Caenorhabditis. The mRNA content of early D. coronatus embryos was sequenced and compared with similar stages in C. elegans and Ascaris suum. We identified 350 gene families transcribed in the early embryo of D. coronatus but not in the other two nematodes. Looking at individual genes transcribed early in D. coronatus but not in C. elegans and A. suum, we found that orthologs of most of these are present in the genomes of the latter species as well, suggesting heterochronic shifts with respect to expression behavior. Considerable genomic heterozygosity and allelic divergence lend further support to the view that D. coronatus may be the result of an interspecies hybridization. Expression analysis of early acting single-copy genes yields no indication for silencing of one parental genome. CONCLUSIONS: Our comparative cellular and molecular studies support the view that the genus Caenorhabditis differs considerably from the other studied nematodes in its control of development and reproduction. The easy-to-culture parthenogenetic D. coronatus, with its high-quality draft genome and only a single chromosome when haploid, offers many new starting points on the cellular, molecular and genomic level to explore alternative routes of nematode development and reproduction.

Analysis of pattern formation during embryonic development of Hydractinia echinata.

Patterning processes during embryonic development of Hydractinia echinata were analysed for alterations in morphology and physiology as well as for changes at the cellular level by means of treatment with proportioning altering factor (PAF). PAF is an endogenous factor known to change body proportions and to stimulate nerve cell differentiation in hydroids (Plickert 1987, 1989). Applied during early embryogenesis, this factor interferes with the proper establishment of polarity in the embryo. Instead of normal shaped planulae with one single anterior and one single posterior end, larvae with multiple termini develop. Preferentially, supernumerary posterior ends, which give rise to polyp head structures during metamorphosis, form while anterior ends are reduced. The formation of such polycaudal larvae coincide with an increase in the number of interstitial cells and their derivatives at the expense of epithelial cells. Treatment of further advanced embryonic stages causes an increase in length, presumably due to the general stimulation of cell proliferation observed in such embryos. Also, the spatial arrangement of cells (i.e. cells in proliferation and RFamide (Arg-Phe-amide immunopositive nerve cells) is altered by PAF. Larvae that develop from treated embryos display altered physiological properties and are remarkably different from normal planulae with respect to their morphogenetic potential: (1) Larvae lose their capacity to regenerate missing anterior parts; isolated posterior larva fragments form regenerates of a bicaudal phenotype. (2) In accordance with the frequently observed reduction of anterior structures, the capacity to respond to metamorphosis-inducing stimuli decreases. (3) The morphogenetic potential to form basal polyp parts is found to be reduced. In contrast, the potential to form head structures during metamorphosis increases, since primary polyps with supernumerary hypostomes and tentacles metamorphose from treated animals.

Necessity of protein synthesis for metamorphosis in the marine hydroidHydractinia echinata.

In the marine colonial hydroidHydractinia echinata metamorphosis from the larval to the adult (polyp) stage is induced by various agents, including CsCI and dioctanoylglycerol (diC8). Induction is prevented when the inhibitors of protein synthesis cycloheximide or ementine were applied simultaneously with the metamorphosis-inducing agents. With diC8 treatment, the inhibitors caused most animals to transform into mosaics consisting of larval and polyp body parts instead of normal shaped polyps. In contrast, treatment with cycloheximide or ementine just before or after incubation with the metamorphosis-inducing agents did not prevent larvae from metamorphosis. No substantial quantitative changes in protein synthesis occur during induction of metamorphosis, however, the protein pattern is changed upon induction. The most prominent new polypeptides (25 and 73 kD) were observed when CsCI was used to trigger metamorphosis. In addition, both in CsCl- and in diC8-treated larvae, the synthesis of a new 23 kD protein occurred, whilst synthesis of others ceased (41 and 44 kD).

Heat shock as inducer of metamorphosis in marine invertebrates.

In most sessile marine invertebrates, metamorphosis is dependent on environmental cues. Here we report that heat stress is capable of inducing metamorphosis in the hydroid Hydractinia echinata. The onset of heat-induced metamorphosis is correlated with the appearance of heat-shock proteins. Larvae treated with the metamorphosis-inducing agents Cs+ or NH4+ also synthesize heat-shock proteins. In heat-shocked larvae, the internal NH4+-concentration increases. This fits the hypothesis that methylation plays a central role in control of metamorphosis. In the tunicate Ciona intestinalis, a heat shock is able to induce metamorphosis too.

Pattern of cell proliferation in embryogenesis and planula development ofHydractinia echinata predicts the postmetamorphic body pattern.

During embryogenesis and planula development of the colonial hydroidHydractinia echinata cell proliferation decreases in a distinct spatio-temporal pattern. Arrest in S-phase activity appears first in cells localized at the posterior and then subsequently at the anterior pole of the elongating embryo. These areas do not resume S-phase activity, even during the metamorphosis of the planula larva into the primary polyp. Tissue containing the quiescent cells gives rise to the terminal structures of the polyp. The posterior area of the larva becomes the hypostome and tentacles, while the anterior part of the larva develops into the basal plate and stolon tips. In mature planulae only a very few cells continue to proliferate. These cells are found in the middle part of the larva. Labelling experiments indicate that the prospective material of the postmetamorphic tentacles and stolon tips originates from cells which have exited from the cell cycle in embryogenesis or early in planula development. Precursor cells of the nematocytes which appear in the tentacles of the polyp following metamorphosis appear to have ceased cycling before the 38th hour of embryonic development. The vast majority of the cells that constitute the stolon tips of the primary polyp leave the cell cycle not later than 58 h after the beginning of development. We also report the identification of a cell type which differentiates in the polyp without passing through a post-metamorphic S-phase. The cell type appears to be neural in origin, based upon the identification of a neuropeptide of the FMRFamide type.

Signal transmission and covert prepattern in the metamorphosis of Hydractinia echinata (Hydrozoa).

Planulae are simply structured larvae lacking an overt longitudinal organization. In the course of a rapid metamorphosis, however, they transform into polyps, which display striking structural patterns. Metamorphosis takes place only in response to external stimuli. Surgical removal and transplantation of larval parts reveal that external stimuli, including artificial inducers such as cesium ions, tumor promoters and diacylglycerol, act on the anterior quarter of the larva where sensory cells containing Arg-Phe-amide-like peptides are located. The external stimuli initiate the release of an internal signal, which is transmitted to the posterior end causing the successive transformation of larval into adult tissue. The transformation front moves from the anterior to the posterior quarter in 60 min. The internal signal can be released or bypassed by a transitory lowering of the Mg2+ content of the seawater. By using this procedure, or by administering an extract containing the putative internal signal substance, each isolated part of the larva can be induced to metamorphose separately. Provided there is no time for regeneration after cutting before metamorphosis is initiated, the most anterior fragment forms only stolons, the most posterior fragment forms only a head. The overt pattern of the polyp is, therefore, generated under the influence of a covert anterior-posterior prepattern of the larva.