Embryonic organizer specification in the mud snail Ilyanassa obsoleta depends on intercellular signaling.

In early embryos of the caenogastropod snail Ilyanassa obsoleta, cytoplasmic segregation of a polar lobe is required for establishment of the D quadrant founder cell, empowering its great-granddaughter macromere 3D to act as a single-celled organizer that induces ectodermal pattern along the secondary body axis of the embryo. We present evidence that polar lobe inheritance is not sufficient to specify 3D potential, but rather makes the D macromere lineage responsive to some intercellular signal(s) required for normal expression of 3D-specific phenotypes. Experimental removal of multiple micromeres resulted in loss of organizer-linked MAPK activation, complete and specific defects of organizer-dependent larval organs, and progressive cell cycle retardation, leading to equalization of the normally accelerated division schedule of 3D (relative to the third-order macromeres of the A, B and C quadrants). Ablation of the second-quartet micromere 2d greatly potentiated the effects of first micromere quartet ablation. Our findings link organizer activation in I. obsoleta to the putative ancestral spiralian mechanism in which a signal from micromeres leads to specification of 3D among four initially equivalent macromeres.

Embryonic organizer specification in the mud snail Ilyanassa obsoleta depends on intercellular signaling.

In early embryos of the caenogastropod snail Ilyanassa obsoleta, cytoplasmic segregation of a polar lobe is required for establishment of the D quadrant founder cell, empowering its great-granddaughter macromere 3D to act as a single-celled organizer that induces ectodermal pattern along the secondary body axis of the embryo. We present evidence that polar lobe inheritance is not sufficient to specify 3D potential, but rather makes the D macromere lineage responsive to some intercellular signal(s) required for normal expression of 3D-specific phenotypes. Experimental removal of multiple micromeres resulted in loss of organizer-linked MAPK activation, complete and specific defects of organizer-dependent larval organs, and progressive cell cycle retardation, leading to equalization of the normally accelerated division schedule of 3D (relative to the third-order macromeres of the A, B and C quadrants). Ablation of the second-quartet micromere 2d greatly potentiated the effects of first micromere quartet ablation. Our findings link organizer activation in I. obsoleta to the putative ancestral spiralian mechanism in which a signal from micromeres leads to specification of 3D among four initially equivalent macromeres.

Functional Divergence of the Tribolium castaneum engrailed and invected Paralogs.

Engrailed (en) and invected (inv) encode paralogous transcription factors found as a closely linked tandem duplication within holometabolous insects. Drosophila en mutants segment normally, then fail to maintain their segments. Loss of Drosophila inv is viable, while loss of both genes results in asegmental larvae. Surprisingly, the knockdown of Oncopeltus inv can result in the loss or fusion of the entire abdomen and en knockdowns in Tribolium show variable degrees of segmental loss. The consequence of losing or knocking down both paralogs on embryogenesis has not been studied beyond Drosophila. To further investigate the relative functions of each paralog and the mechanism behind the segmental loss, Tribolium double and single knockdowns of en and inv were analyzed. The most common cuticular phenotype of the double knockdowns was small, limbless, and open dorsally, with all but a single, segmentally iterated row of bristles. Less severe knockdowns had fused segments and reduced appendages. The Tribolium paralogs appear to act synergistically: the knockdown of either Tribolium gene alone was typically less severe, with all limbs present, whereas the most extreme single knockdowns mimic the most severe double knockdown phenotype. Morphological abnormalities unique to either single gene knockdown were not found. inv expression was not affected in the Tribolium en knockdowns, but hh expression was unexpectedly increased midway through development. Thus, while the segmental expression of en/inv is broadly conserved within insects, the functions of en and inv are evolving independently in different lineages.

Elongation during segmentation shows axial variability, low mitotic rates, and synchronized cell cycle domains in the crustacean, Thamnocephalus platyurus.

BACKGROUND: Segmentation in arthropods typically occurs by sequential addition of segments from a posterior growth zone. However, the amount of tissue required for growth and the cell behaviors producing posterior elongation are sparsely documented. RESULTS: Using precisely staged larvae of the crustacean, Thamnocephalus platyurus, we systematically examine cell division patterns and morphometric changes associated with posterior elongation during segmentation. We show that cell division occurs during normal elongation but that cells in the growth zone need only divide ~ 1.5 times to meet growth estimates; correspondingly, direct measures of cell division in the growth zone are low. Morphometric measurements of the growth zone and of newly formed segments suggest tagma-specific features of segment generation. Using methods for detecting two different phases in the cell cycle, we show distinct domains of synchronized cells in the posterior trunk. Borders of cell cycle domains correlate with domains of segmental gene expression, suggesting an intimate link between segment generation and cell cycle regulation. CONCLUSIONS: Emerging measures of cellular dynamics underlying posterior elongation already show a number of intriguing characteristics that may be widespread among sequentially segmenting arthropods and are likely a source of evolutionary variability. These characteristics include: the low rates of posterior mitosis, the apparently tight regulation of cell cycle at the growth zone/new segment border, and a correlation between changes in elongation and tagma boundaries.

Dynamics of growth zone patterning in the milkweed bug Oncopeltus fasciatus.

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.

Linking gene regulation to cell behaviors in the posterior growth zone of sequentially segmenting arthropods.

Virtually all arthropods all arthropods add their body segments sequentially, one by one in an anterior to posterior progression. That process requires not only segment specification but typically growth and elongation. Here we review the functions of some of the key genes that regulate segmentation: Wnt, caudal, Notch pathway, and pair-rule genes, and discuss what can be inferred about their evolution. We focus on how these regulatory factors are integrated with growth and elongation and discuss the importance and challenges of baseline measures of growth and elongation. We emphasize a perspective that integrates the genetic regulation of segment patterning with the cellular mechanisms of growth and elongation.

Wnt repertoire and developmental expression patterns in the crustacean Thamnocephalus platyurus.

Wnt genes are a family of conserved glycoprotein ligands that play a role in a wide variety of cell and developmental processes, from cell proliferation to axis elongation. There are 13 Wnt subfamilies found among metazoans. Eleven of these appear conserved in arthropods with a pattern of loss during evolution of as many as six subfamilies among hexapods. Here we report on Wnt genes in the branchiopod crustacean, Thamnocephalus platyurus, including the first documentation of the expression of the complete Wnt gene family in a crustacean. Our results suggest fewer Wnt genes were retained in Thamnocephalus than in the related crustacean, Daphnia, although the Thamnocephalus Wnt repertoire is larger than that found in insects. We also find an intriguing pattern of staggered expression of Wnts-an anterior-posterior stagger within the posterior growth zone and a dorsal-ventral stagger in the developing segments-suggesting a potential for subfunctionalization of Wnts in these regions.

Composition and genomic organization of arthropod Hox clusters.

BACKGROUND: The ancestral arthropod is believed to have had a clustered arrangement of ten Hox genes. Within arthropods, Hox gene mutations result in transformation of segment identities. Despite the fact that variation in segment number/character was common in the diversification of arthropods, few examples of Hox gene gains/losses have been correlated with morphological evolution. Furthermore, a full appreciation of the variation in the genomic arrangement of Hox genes in extant arthropods has not been recognized, as genome sequences from each major arthropod clade have not been reported until recently. Initial genomic analysis of the chelicerate Tetranychus urticae suggested that loss of Hox genes and Hox gene clustering might be more common than previously assumed. To further characterize the genomic evolution of arthropod Hox genes, we compared the genomic arrangement and general characteristics of Hox genes from representative taxa from each arthropod subphylum. RESULTS: In agreement with others, we find arthropods generally contain ten Hox genes arranged in a common orientation in the genome, with an increasing number of sampled species missing either Hox3 or abdominal-A orthologs. The genomic clustering of Hox genes in species we surveyed varies significantly, ranging from 0.3 to 13.6 Mb. In all species sampled, arthropod Hox genes are dispersed in the genome relative to the vertebrate Mus musculus. Differences in Hox cluster size arise from variation in the number of intervening genes, intergenic spacing, and the size of introns and UTRs. In the arthropods surveyed, Hox gene duplications are rare and four microRNAs are, in general, conserved in similar genomic positions relative to the Hox genes. CONCLUSIONS: The tightly clustered Hox complexes found in the vertebrates are not evident within arthropods, and differential patterns of Hox gene dispersion are found throughout the arthropods. The comparative genomic data continue to support an ancestral arthropod Hox cluster of ten genes with a shared orientation, with four Hox gene-associated miRNAs, although the degree of dispersion between genes in an ancestral cluster remains uncertain. Hox3 and abdominal-A orthologs have been lost in multiple, independent lineages, and current data support a model in which inversions of the Abdominal-B locus that result in the loss of abdominal-A correlate with reduced trunk segmentation.

Evidence for the plasticity of arthropod signal transduction pathways.

Metazoans are known to contain a limited, yet highly conserved, set of signal transduction pathways that instruct early developmental patterning mechanisms. Genomic surveys that have compared gene conservation in signal transduction pathways between various insects and Drosophila support the conclusion that these pathways are conserved in evolution. However, the degree to which individual components of signal transduction pathways vary among more divergent arthropods is not known. Here, we report our results of a survey of the genome of the two-spotted spider mite Tetranychus urticae, using a set of 294 Drosophila orthologs of genes that function in signal transduction. We find a third of all genes surveyed absent from the spider mite genome. We also identify several novel duplications that have not been previously reported for a chelicerate. In comparison with previous insect surveys, Tetranychus contains a decrease in overall gene conservation, as well as an unusual ratio of ligands to receptors and other modifiers. These findings suggest that gene loss and duplication among components of signal transduction pathways are common among arthropods and suggest that signal transduction pathways in arthropods are more evolutionarily labile than previously hypothesized.

Ilyanassa Notch signaling implicated in dynamic signaling between all three germ layers.

Two cells (3D and 4d) in the mud snail Ilyanassa obsoleta function to induce proper cell fate. In this study, we provide support for the hypothesis that Notch signaling in Ilyanassa obsoleta functions in inductive signaling at multiple developmental stages. The expression patterns of Notch, Delta and Suppressor of Hairless (SuH) are consistent with a function for Notch signaling in endoderm formation, the function of 3D/4d and the sublineages of 4d. Veligers treated with DAPT show a range of defects that include a loss of endodermal structures, and varying degrees of loss of targets of 4d inductive signaling. Veligers that result from injection of Ilyanassa Delta siRNAi in general mimic the defects observed in the DAPT treated larvae. The most severe DAPT phenotypes mimic early ablations of 4d. However, the early specification of 4d itself appears normal and MAPK activation in both 3D/4d and the micromeres, which are known to activate MAPK as a result of 3D/4d induction, are normal in DAPT treated larvae. Treating larvae at successively later timepoints with DAPT suggests that Notch/Delta signaling is not only required during early 4d inductive signaling, but during subsequent stages of cell fate determination as well. Based on our results, combined with previous reports implicating the endoderm in maintaining induced fate specification in Ilyanassa, we propose a speculative model that Notch signaling is required to specify endoderm fates and 4d sublineages, as well as to maintain cell fates induced by 4d.

Analysis of ciliary band formation in the mollusc Ilyanassa obsoleta.

Two primary ciliary bands, the prototroch and metatroch, are required for locomotion and in the feeding larvae of many spiralians. The metatroch has been reported to have different cellular origins in the molluscs Crepidula fornicata and Ilyanassa obsoleta, as well as in the annelid Polygordius lacteus, consistent with multiple independent origins of the spiralian metatroch. Here, we describe in further detail the cell lineage of the ciliary bands in the gastropod mollusc I. obsoleta using intracellular lineage tracing and the expression of an acetylated tubulin antigen that serves as a marker for ciliated cells. We find that the I. obsoleta metatroch is formed primarily by third quartet derivatives as well as a small number of second quartet derivatives. These results differ from the described metatrochal lineage in the mollusc C. fornicata that derives solely from the second quartet or the metatrochal lineage in the annelid P. lacteus that derives solely from the third quartet. The present study adds to a growing body of literature concerning the evolution of the metatroch and the plasticity of cell fates in homologous micromeres in spiralian embryos.

Decoupling elongation and segmentation: notch involvement in anostracan crustacean segmentation.

Repeated body segments are a key feature of arthropods. The formation of body segments occurs via distinct developmental pathways within different arthropod clades. Although some species form their segments simultaneously without any accompanying measurable growth, most arthropods add segments sequentially from the posterior of the growing embryo or larva. The use of Notch signaling is increasingly emerging as a common feature of sequential segmentation throughout the Bilateria, as inferred from both the expression of proteins required for Notch signaling and the genetic or pharmacological disruption of Notch signaling. In this study, we demonstrate that blocking Notch signaling by blocking γ-secretase activity causes a specific, repeatable effect on segmentation in two different anostracan crustaceans, Artemia franciscana and Thamnocephalus platyurus. We observe that segmentation posterior to the third or fourth trunk segment is arrested. Despite this marked effect on segment addition, other aspects of segmentation are unaffected. In the segments that develop, segment size and boundaries between segments appear normal, engrailed stripes are normal in size and alignment, and overall growth is unaffected. By demonstrating Notch involvement in crustacean segmentation, our findings expand the evidence that Notch plays a crucial role in sequential segmentation in arthropods. At the same time, our observations contribute to an emerging picture that loss-of-function Notch phenotypes differ significantly between arthropods suggesting variability in the role of Notch in the regulation of sequential segmentation. This variability in the function of Notch in arthropod segmentation confounds inferences of homology with vertebrates and lophotrochozoans.

Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid.

Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.

The snail Ilyanassa: a reemerging model for studies in development.

Ilyanassa obsoleta is a marine gastropod that is a long-standing and very useful model for studies of embryonic development. It is especially important as a model for the spiralian development program, a distinctive mode of early development shared by a large group of animal phyla, but poorly understood. Ilyanassa adults are readily obtainable and easy to keep in the laboratory, and they produce large numbers of embryos throughout most of the year. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. In this article, we present an overview of aspects of its biology and use as a model organism.

Obtaining Ilyanassa snail embryos.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa adults are readily obtainable and easy to keep in the laboratory. Although the normal spawning season for Ilyanassa is during early summer, they can produce high-quality embryos nearly year-round in the laboratory. Snails collected in the late fall, winter, or spring can be induced to deposit zygotes before the natural spawning season by warming them to room temperature, and snails collected before the natural spawning season can be made to postpone zygote deposition until needed (up to at least 6 mo) by maintaining them in tanks in a cold room at 4 degrees C-8 degrees C. This protocol describes how to induce embryo production in Ilyanassa snails, collect the embryos, and rear them to the stage required for study.

Induction of larval metamorphosis in the snail Ilyanassa.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa adults are readily obtainable and easy to keep in the laboratory, and they can produce high-quality embryos nearly year-round in the laboratory. After hatching from capsules, larval Ilyanassa can be maintained in culture, feeding on single-celled algae. The larvae will become competent to undergo metamorphosis after approximately 3 wk in culture. Metamorphosis can be induced artificially by treating with either the neurotransmitter serotonin or the nitric oxide synthase inhibitor 7-nitroindazole. Both of these reagents have been shown to induce metamorphosis in >75% of larvae within 48 h. This protocol describes the induction of metamorphosis in snail larvae.

Pressure injection of Ilyanassa snail embryos.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques, as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Intracellular microinjection is an important tool, especially for lineage tracing and perturbations of specific genes by knockdown approaches and synthetic mRNA injections. Two methods for the introduction of lineage tracers into particular cells are routine in Ilyanassa. Iontophoresis of charged molecules, such as fluorophore-dextran conjugates can be accomplished using a simply built current generator. Injection of an oil-based solution containing the fluorescent probe 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) is also straightforward. However, injection of oil-based solutions and iontophoresis have not been useful for delivering water-soluble reagents to perturb gene function, and pressure injection of aqueous solutions has been more challenging. This protocol describes a recently optimized procedure for the pressure injection of aqueous solutions into Ilyanassa embryos and zygotes with high rates of survival and normal development. The key parameters seem to be the injection needles, injection media, and the stage of injected embryos.

Fixation of Ilyanassa snail embryos and larvae.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques, as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa embryos are particularly well suited for RNA and protein localization studies because of the relatively large cells and favorable properties for imaging. This protocol describes how to fix and store Ilyanassa embryos and larvae for use in whole-mount in situ hybridization and immunohistochemical studies.

Isolation of genomic DNA from Ilyanassa snail larvae.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa is host to several species of parasitic trematode worms, so care must be taken to avoid contamination of Ilyanassa genomic DNA with that of the parasites. The easiest way to avoid this contamination is to isolate DNA from veliger larvae, which are not parasitized. This also avoids other problems that can be encountered when isolating DNA from adult mollusc tissues, such as the presence of large amounts of polysaccharides. This protocol describes the isolation of genomic DNA from Ilyanassa larvae.

Isolating protein from Ilyanassa snail embryos.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. This protocol describes the procedure for extracting protein from Ilyanassa embryos for use in techniques such as Western blotting or two-dimensional (2D) gel electrophoresis.