Embryonic development in the acoel Hofstenia miamia.

Acoels are marine worms that belong to the phylum Xenacoelomorpha, a deep-diverging bilaterian lineage. This makes acoels an attractive system for studying the evolution of major bilaterian traits. Thus far, acoel development has not been described in detail at the morphological and transcriptomic levels in a species in which functional genetic studies are possible. We present a set of developmental landmarks for embryogenesis in the highly regenerative acoel Hofstenia miamia. We generated a developmental staging atlas from zygote to hatched worm based on gross morphology, with accompanying bulk transcriptome data. Hofstenia embryos undergo a stereotyped cleavage program known as duet cleavage, which results in two large vegetal pole 'macromeres' and numerous small animal pole 'micromeres'. These macromeres become internalized as micromere progeny proliferate and move vegetally. We also noted a second, previously undescribed, cell-internalization event at the animal pole, following which we detected major body axes and tissues corresponding to all three germ layers. Our work on Hofstenia embryos provides a resource for mechanistic investigations of acoel development, which will yield insights into the evolution of bilaterian development and regeneration.

Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano.

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.

Ins and outs of Spiralian gastrulation.

Gastrulation is a critical stage of metazoan development during which endodermal and mesodermal tissues are internalized, and morphogenesis transforms the early embryo into each animal's unique body-plan. While gastrulation has been studied extensively in classic model systems such as flies, worms, and vertebrates, less is known about gastrulation at a mechanistic level in other taxa. Surprisingly, one particularly neglected group constitutes a major branch of animals: the Spiralia. A unique feature of spiralian development is that taxa with diverse adult body-plans, such as annelids, molluscs, nemerteans and platyhelminths all share a highly stereotyped suite of characters during embryogenesis called spiral cleavage. The spiral cleavage program makes it possible to compare distantly related embryos using not only morphological features, and gene expression patterns, but also homologous cell lineages. Having all three criteria available for comparison is especially critical for understanding the evolution of a complex process like gastrulation. Thus studying gastrulation in spiralians is likely to lead to novel insights about the evolution of body-plans, and the evolution of morphogenesis itself. Here we review relevant literature about gastrulation in spiralians and frame questions for future studies. We describe the internalization of the endoderm, endomesoderm and ectomesoderm; where known, we review data on the cellular and molecular control of those processes. We also discuss several morphogenetic events that are tied to gastrulation including: axial elongation, origins of the mouth and anus, and the fate of the blastopore. Since spiral cleavage is ancestral for a major branch of bilaterians, understanding gastrulation in spiralians will contribute more broadly to ongoing debates about animal body-plan divergence, such as: the origin of the through-gut, the emergence of indirect versus direct development, and the evolution of gene-regulatory networks that specify endomesoderm. We emphasize the fact that spiralian gastrulation provides the unique opportunity to connect well-defined embryonic cell lineages to variation in cell fate and cell behavior, making it an exceptional case study for evo-devo.

Regeneration in spiralians: evolutionary patterns and developmental processes.

Animals differ markedly in their ability to regenerate, yet still little is known about how regeneration evolves. In recent years, important advances have been made in our understanding of animal phylogeny and these provide new insights into the phylogenetic distribution of regeneration. The developmental basis of regeneration is also being investigated in an increasing number of groups, allowing commonalities and differences across groups to become evident. Here, we focus on regeneration in the Spiralia, a group that includes several champions of animal regeneration, as well as many groups with more limited abilities. We review the phylogenetic distribution and developmental processes of regeneration in four major spiralian groups: annelids, nemerteans, platyhelminths, and molluscs. Although comparative data are still limited, this review highlights phylogenetic and developmental patterns that are emerging regarding regeneration in spiralians and identifies important avenues for future research.

Hatching strategies in monogenean (platyhelminth) parasites that facilitate host infection.

In parasites, environmental cues may influence hatching of eggs and enhance the success of infections. The two major endoparasitic groups of parasitic platyhelminths, cestodes (tapeworms) and digeneans (flukes), typically have high fecundity, infect more than one host species, and transmit trophically. Monogeneans are parasitic flatworms that are among the most host specific of all parasites. Most are ectoparasites with relatively low fecundity and direct life cycles tied to water. They infect a single host species, usually a fish, although some are endoparasites of amphibians and aquatic chelonian reptiles. Monogenean eggs have strong shells and mostly release ciliated larvae, which, against all odds, must find, identify, and infect a suitable specific host. Some monogeneans increase their chances of finding a host by greatly extending the hatching period (possible bet-hedging). Others respond to cues for hatching such as shadows, chemicals, mechanical disturbance, and osmotic changes, most of which may be generated by the host. Hatching may be rhythmical, larvae emerging at times when the host is more vulnerable to invasion, and this may be combined with responses to other environmental cues. Different monogenean species that infect the same host species may adopt different strategies of hatching, indicating that tactics may be more complex than first thought. Control of egg assembly and egg-laying, possibly by host hormones, has permitted colonization of frogs and toads by polystomatid monogeneans. Some monogeneans further improve the chances of infection by attaching eggs to the host or by retaining eggs on, or in, the body of the parasite. The latter adaptation has led ultimately to viviparity in gyrodactylid monogeneans.

Environmental constraints influencing survival of an African parasite in a north temperate habitat: effects of temperature on egg development.

Factors affecting survival of parasites introduced to new geographical regions include changes in environmental temperature. Protopolystoma xenopodis is a monogenean introduced with the amphibian Xenopus laevis from South Africa to Wales (probably in the 1960s) where low water temperatures impose major constraints on life-cycle processes. Effects were quantified by maintenance of eggs from infections in Wales under controlled conditions at 10, 12, 15, 18, 20 and 25°C. The threshold for egg viability/ development was 15°C. Mean times to hatching were 22 days at 25°C, 32 days at 20°C, extending to 66 days at 15°C. Field temperature records provided calibration of transmission schedules. Although egg production continues year-round, all eggs produced during >8 months/ year die without hatching. Output contributing significantly to transmission is restricted to 10 weeks (May-mid-July). Host infection, beginning after a time lag of 8 weeks for egg development, is also restricted to 10 weeks (July-September). Habitat temperatures (mean 15·5°C in summer 2008) allow only a narrow margin for life-cycle progress: even small temperature increases, predicted with 'global warming', enhance infection. This system provides empirical data on the metrics of transmission permitting long-term persistence of isolated parasite populations in limiting environments.

Left-right asymmetries in Spiralia.

In spite of the great diversity of forms found in nature, most metazoans are organized along an anterior-posterior and a dorsoventral axis. These two orthogonal axes define the left and right sides of animals. While most animals show overall bilateral symmetry, in some cases, specific features or organs display consistent left and right asymmetries that can be critical for their normal functioning. Descriptions of these asymmetries and the mechanisms behind them are available mainly for deuterostomes, whereas information for other groups like spiralians is rather scarce. Here I review previous and recent data, highlighting the relevance of the cleavage program in establishing the dorsoventral axis and therefore the left and right sides of spirally cleaving embryos of groups like platyhelminthes, nemerteans, mollusks, and annelids. In addition, I summarize morphological asymmetries detected throughout embryogenesis and in the adult forms of these groups as well as the mechanisms involved in these processes. This information contributes to our understanding of how bilateral asymmetries are regulated in spiralians and provides additional insights into the evolution of left-right asymmetries through the evolutionary history of bilaterians.

Melav2, an elav-like gene, is essential for spermatid differentiation in the flatworm Macrostomum lignano.

BACKGROUND: Failure of sperm differentiation is one of the major causes of male sterility. During spermiogenesis, spermatids undergo a complex metamorphosis, including chromatin condensation and cell elongation. Although the resulting sperm morphology and property can vary depending on the species, these processes are fundamental in many organisms. Studying genes involved in such processes can thus provide important information for a better understanding of spermatogenesis, which might be universally applied to many other organisms. RESULTS: In a screen for genes that have gonad-specific expression we isolated an elav-like gene, melav2, from Macrostomum lignano, containing the three RNA recognition motifs characteristic of elav-like genes. We found that melav2 mRNA was expressed exclusively in the testis, as opposed to the known elav genes, which are expressed in the nervous system. The RNAi phenotype of melav2 was characterized by an aberrant spermatid morphology, where sperm elongation often failed, and an empty seminal vesicle. Melav2 RNAi treated worms were thus male-sterile. Further analysis revealed that in melav2 RNAi treated worms precocious chromatin condensation occurred during spermatid differentiation, resulting in an abnormally tightly condensed chromatin and large vacuoles in round spermatids. In addition, immunostaining using an early-spermatid specific antibody revealed that melav2 RNAi treated worms had a larger amount of signal positive cells, suggesting that many cells failed the transition from early spermatid stage. CONCLUSION: We characterize a new function for elav-like genes, showing that melav2 plays a crucial role during spermatid differentiation, especially in the regulation of chromatin condensation and/or cell elongation.

Coordinated spatial and temporal expression of Hox genes during embryogenesis in the acoel Convolutriloba longifissura.

BACKGROUND: Hox genes are critical for patterning the bilaterian anterior-posterior axis. The evolution of their clustered genomic arrangement and ancestral function has been debated since their discovery. As acoels appear to represent the sister group to the remaining Bilateria (Nephrozoa), investigating Hox gene expression will provide an insight into the ancestral features of the Hox genes in metazoan evolution. RESULTS: We describe the expression of anterior, central and posterior class Hox genes and the ParaHox ortholog Cdx in the acoel Convolutriloba longifissura. Expression of all three Hox genes begins contemporaneously after gastrulation and then resolves into staggered domains along the anterior-posterior axis, suggesting that the spatial coordination of Hox gene expression was present in the bilaterian ancestor. After early surface ectodermal expression, the anterior and central class genes are expressed in small domains of putative neural precursor cells co-expressing ClSoxB1, suggesting an evolutionary early function of Hox genes in patterning parts of the nervous system. In contrast, the expression of the posterior Hox gene is found in all three germ layers in a much broader posterior region of the embryo. CONCLUSION: Our results suggest that the ancestral set of Hox genes was involved in the anterior-posterior patterning of the nervous system of the last common bilaterian ancestor and were later co-opted for patterning in diverse tissues in the bilaterian radiation. The lack of temporal colinearity of Hox expression in acoels may be due to a loss of genomic clustering in this clade or, alternatively, temporal colinearity may have arisen in conjunction with the expansion of the Hox cluster in the Nephrozoa.

Embryonic muscle development in direct and indirect developing marine flatworms (Platyhelminthes, Polycladida).

We compared embryonic myogenesis of the direct-developing acotylean polyclad Melloplana ferruginea with that of Maritigrella crozieri, a cotylean that develops via a larval stage. Fluorescently labeled F-actin was visualized with laser confocal microscopy. Developmental times are reported as percentages of the time from oviposition to hatching: 7 days for M. crozieri and 22 days for M. ferruginea. The epithelium began to form at 30% development in M. crozieri and at 15% development in M. ferruginea. Random myoblasts appeared in peripheral areas of the embryo at 36% and 22-30% development in M. crozeri and M. ferruginea, respectively. Circular and longitudinal muscle bands formed synchronously at 37-44% development in M. crozieri; yolk obscured observations of early myogenesis in M. ferruginea. An orthogonal muscle grid was established by 45-50% development in both species. Diagonal muscles developed in M. ferruginea at 60-71% development. Hence, juveniles of this species hatch with the same basic body-wall musculature as adults. Larvae of M. crozieri did not hatch with diagonal muscles; these muscles are acquired postmetamorphosis. Additionally, a specialized musculature developed in the larval lobes of M. crozieri. Oral musculature was complex and established by 72% development in both species. Our results are comparable to the muscle differentiation reported for other indirect-developing polyclads and for direct-developing species of macrostomid flatworms. Furthermore, they provide additional support that the orthogonal muscle pattern of circular and longitudinal muscles is a symplesiomorphy of Spiralia.

Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola) / Desenvolvimento embrionário de Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola)

The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

O desenvolvimento embrionário dos tricladidos é conhecido, fundamentalmente, por estudos realizados em espécies de Dendrocoelum, Planaria, Polycelis e, mais recentemente, Schmidtea. O presente estudo descreve o desenvolvimento embrionário de Girardia tigrina (Girard, 1850), a partir de análises realizadas em cortes histológicos seriados e semifinos de glicol-metacrilato, ao microscópio óptico. Noventa e quatro casulos foram coletados no período entre a postura e a eclosão, em intervalos de até vinte e quatro horas. A seqüência das modificações morfológicas no embrião permitiu a identificação de nove estágios embrionários. Na postura dos casulos, envoltos por uma cápsula rígida, observam-se numerosos embriões dispersos entre grande quantidade de células vitelinas. No estágio I (aproximadamente até 6 horas após a postura), as células vitelinas e as embrionárias mostram uma distribuição aleatória. O estágio II (entre 12 e 24 horas após a postura) caracteriza-se pela formação de agrupamentos de blastômeros, os quais posteriormente formam uma enteroblástula. Aproximadamente dois dias após a postura (estágio III), ocorre a formação da epiderme e do sistema digestivo embrionário, sendo que este último degenera no estágio seguinte. O estágio V (até o quarto dia após a postura) caracteriza-se pela formação da epiderme definitiva. Entre o quarto e o sexto dia posteriores à postura, começa a organogênese dos órgãos internos definitivos (estágio VI). Aproximadamente catorze dias após a postura (estágio IX), completa-se a formação do sistema nervoso. Neste estágio, o embrião já apresenta características similares aos espécimes juvenis. A eclosão de Girardia tigrina ocorre entre doze e vinte e dois dias após a postura dos casulos.

Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola).

The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

Free-living flatworms under the knife: past and present.

Traditionally, regeneration research has been closely tied to flatworm research, as flatworms (Plathelminthes) were among the first animals where the phenomenon of regeneration was discovered. Since then, the main focus of flatworm regeneration research was on triclads, for which various phenomena were observed and a number of theories developed. However, free-living flatworms encompass a number of other taxa where regeneration was found to be possible. This review aims to display and to compare regeneration in all major free-living flatworm taxa, with special focus on a new player in the field of regeneration, Macrostomum lignano (Macrostomorpha). Findings on the regeneration capacity of this organism provide clues for links between regeneration and (post-)embryonic development, starvation, and asexual reproduction. The role of the nervous system and especially the brain for regeneration is discussed, and similarities as well as particularities in regeneration among free-living flatworms are pointed out.

The Macrostomum lignano EST database as a molecular resource for studying platyhelminth development and phylogeny.

We report the development of an Expressed Sequence Tag (EST) resource for the flatworm Macrostomum lignano. This taxon is of interest due to its basal placement within the flatworms. As such, it provides a useful comparative model for understanding the development of neural and sensory organization. It was anticipated on the basis of previous studies [e.g., Sánchez-Alvarado et al., Development, 129:5659-5665, (2002)] that a wide range of developmental markers would be expressed in later-stage macrostomids, and this proved to be the case, permitting recovery of a range of gene sequences important in development. To this end, an adult Macrostomum cDNA library was generated and 7,680 Macrostomum ESTs were sequenced from the 5' end. In addition, 1,536 of these aforementioned sequences were sequenced from the 3' end. Of the roughly 5,416 non-redundant sequences identified, 68% are similar to previously reported genes of known function. In addition, nearly 100 specific clones were obtained with potential neural and sensory function. From these data, an annotated searchable database of the Macrostomum EST collection has been made available on the web. A major objective was to obtain genes that would allow reconstruction of embryogenesis, and in particular neurogenesis, in a basal platyhelminth. The sequences recovered will serve as probes with which the origin and morphogenesis of lineages and tissues can be followed. To this end, we demonstrate a protocol for combined immunohistochemistry and in situ hybridization labeling in juvenile Macrostomum, employing homologs of lin11/lim1 and six3/optix. Expression of these genes is shown in the context of the neuropile/muscle system.

Phosphatized polar lobe-forming embryos from the Precambrian of southwest China.

In developing embryos of some extant spiralian animals, polar lobe formation is one of the symmetry-breaking mechanisms for segregation of maternal cytoplasmic substances to certain blastomeres and not others. Polar lobe formation leads to unique early cleavage morphologies that include trilobed, J-shaped, and five-lobed structures. Fossil embryos similar to modern lobeforming embryos are recognized from the Precambrian Doushantuo Formation phosphates, Weng'an, Guizhou Province, China. These embryos are abundant and form a developmental sequence comparable to different developing stages observed in lobe-forming embryos of extant spiralians. These data imply that lobe formation is an evolutionarily ancient process of embryonic specification.

[The criteria of identification of "critical" populations in aquatic radiochemoecology].

Data on chromosome mutagenesis levels in populations of aquatic organisms in the Black and the Aegean Seas, the Danube and the Dnieper Rivers, the 30-km zone of ChNPP are presented. The highest level of mutagenesis was observed in hydrobionts populations in the 10-km zone of the ChNPP. The obvious damaged effects of ionizing radiation were noted only in these populations. The comparison of the adaptation rate of aquatic crustaceans and worms populations with different reproduction modes was made. It is found that the studied species with sexual reproduction have higher rate of adaptation to the pollution in comparison with species with prevalent asexual reproduction. Hypothetic mechanisms of population adaptation are discussed. On the basis of species and populations characteristics, the criteria for the identification of "critical" populations (species) and an algoritm of ecological risk assessment for them are proposed.

Effects of temperature, salinity, desiccation and chemical treatments on egg embryonation and hatching success of Benedenia seriolae (Monogenea: Capsalidae), a parasite of farmed Seriola spp.

The effects of temperature and salinity on the embryonation period and hatching success of eggs of Benedenia seriolae were investigated. Temperature strongly influenced embryonation period; eggs first hatched 5 days after laying at 28 degrees C and 16 days after laying at 14 degrees C. The relationship between temperature and embryonation period is described by quadratic regression equations for time to first and last hatching. Hatching success was >70% for B. seriolae eggs incubated at temperatures from 14 to 28 degrees C. However, no B. seriolae eggs embryonated and hatched at 30 degrees C and <2% of eggs hatched when incubated at 24 degrees C after transfer to 30 degrees C for 48 h. Embryonation period was similar for eggs incubated in sea water at 25, 30 and 35 per thousand salinity, but increased for eggs incubated at higher or lower salinities. When incubated at salinities ranging from 25 to 45 per thousand, more than 70% of B. seriolae eggs embryonated and hatched. Hatching success was lower at 20 and 50 per thousand salinity and few or no eggs hatched at 10 and 15 per thousand. Hatching of B. seriolae eggs can be prevented by desiccation for 3 min, by immersion in water at 50 degrees C for 30 s or by treatment with 25% ethanol for 3 min.

The embryonic development of the flatworm Macrostomum sp.

Macrostomid flatworms represent a group of basal bilaterians with primitive developmental and morphological characteristics. The species Macrostomum sp., raised under laboratory conditions, has a short generation time of about 2-3 weeks and produces a large number of eggs year round. Using live observation, histology, electron microscopy and immunohistochemistry we have carried out a developmental analysis of Macrostomum sp. Cleavage (stages 1-2) of this species follows a modified spiral pattern and results in a solid embryonic primordium surrounded by an external yolk layer. During stage 3, cells at the anterior and lateral periphery of the embryo evolve into the somatic primordium which gives rise to the body wall and nervous system. Cells in the center form the large yolk-rich gut primordium. During stage 4, the brain primordium and the pharynx primordium appear as symmetric densities anterior-ventrally within the somatic primordium. Organ differentiation commences during stage 5 when the neurons of the brain primordium extend axons that form a central neuropile, and the outer cell layer of the somatic primordium turns into a ciliated epidermal epithelium. Cilia also appear in the lumen of the pharynx primordium, in the protonephridial system and, slightly later, in the lumen of the gut. Ultrastructurally, these differentiating cells show the hallmarks of platyhelminth epithelia, with a pronounced apical assembly of microfilaments (terminal web) inserting at the zonula adherens, and a wide band of septate junctions underneath the zonula. Terminal web and zonula adherens are particularly well observed in the epidermis. During stage 6, the somatic primordium extends around the surface dorsally and ventrally to form a complete body wall. Muscle precursors extend myofilaments that are organized into a highly regular orthogonal network of circular, diagonal and longitudinal fibers. Neurons of the brain primordium differentiate a commissural neuropile that extends a single pair of ventro-lateral nerve trunks (the main longitudinal cords) posteriorly. The primordial pharynx lumen fuses with the ventral epidermis anteriorly and the gut posteriorly, thereby generating a continuous digestive tract. The embryo adopts its final shape during stages 7 and 8, characterized by the morphallactic lengthening of the body into a U-shaped form and the condensation of the nervous system.