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Among animals, diploblasts contain two germ layers, endoderm and ectoderm, while triploblasts have a distinct third germ layer called the mesoderm. Spiralians are a group of triploblast animals that have highly conserved development: they share the distinctive spiralian cleavage pattern as well as a unique source of mesoderm, the ectomesoderm. This population of mesoderm is distinct from endomesoderm and is considered a hallmark of spiralian development, but the regulatory network that drives its development is unknown. Here we identified ectomesoderm-specific genes in the mollusc Tritia (aka Ilyanassa) obsoleta through differential gene expression analyses comparing control and ectomesoderm-ablated embryos, followed by in situ hybridization of identified transcripts. We identified a Tritia serpin gene (ToSerpin1) that appears to be specifically expressed in the ectomesoderm of the posterior and head. Ablation of the 3a and 3b cells, which make most of the ectomesoderm, abolishes ToSerpin1 expression, consistent with its expression in these cells. Morpholino knockdown of ToSerpin1 causes ectomesoderm defects, most prominently in the muscle system of the larval head. This is the first gene identified that is specifically implicated in spiralian ectomesoderm development.

Spiralia is a large, ancient and diverse clade of animals, with a conserved early developmental program but diverse larval and adult morphologies. One trait shared by many spiralians is the presence of ciliary bands used for locomotion and feeding. To learn more about spiralian-specific traits we have examined the expression of 20 genes with protein motifs that are strongly conserved within the Spiralia, but not detectable outside of it. Here, we show that two of these are specifically expressed in the main ciliary band of the mollusc Tritia (also known as Ilyanassa). Their expression patterns in representative species from five more spiralian phyla—the annelids, nemerteans, phoronids, brachiopods and rotifers—show that at least one of these, lophotrochin, has a conserved and specific role in particular ciliated structures, most consistently in ciliary bands. These results highlight the potential importance of lineage-specific genes or protein motifs for understanding traits shared across ancient lineages., Spiralians have ciliary bands, used for locomotion and feeding, but defining molecular features of these structures are unknown. Here, the authors report a gene, Lophotrochin, that contains a protein domain only found in spiralians, and specifically expressed in diverse ciliary bands across the group, which provides a molecular signature for these structures.

Knowledge of an animal’s home range is a crucial component in making informed management decisions. However, many home range studies are limited by study area size, and therefore may underestimate the size of the home range. In many cases, individuals have been shown to travel outside of the study area and utilize a larger area than estimated by the study design. In this study, data collected by multiple research groups studying bottlenose dolphins on the east coast of Florida were combined to determine how home range estimates increased with increasing study area size. Home range analyses utilized photo-identification data collected from 6 study areas throughout the St Johns River (SJR; Jacksonville, FL, USA) and adjacent waterways, extending a total of 253 km to the southern end of Mosquito Lagoon in the Indian River Lagoon Estuarine System. Univariate kernel density estimates (KDEs) were computed for individuals with 10 or more sightings (n = 20). Kernels were calculated for the primary study area (SJR) first, then additional kernels were calculated by combining the SJR and the next adjacent waterway; this continued in an additive fashion until all study areas were included. The 95% and 50% KDEs calculated for the SJR alone ranged from 21 to 35 km and 4 to 19 km, respectively. The 95% and 50% KDEs calculated for all combined study areas ranged from 116 to 217 km and 9 to 70 km, respectively. This study illustrates the degree to which home range may be underestimated by the use of limited study areas and demonstrates the benefits of conducting collaborative science.

Caudal homeobox genes are found across animals, typically linked to two other homeobox genes in what has been called the ParaHox cluster. These genes have been proposed to pattern the anterior-posterior axis of the endoderm ancestrally, but the expression of Caudal in extant groups is varied and often occurs in other germ layers. Here we examine the role of Caudal in the embryo of the mollusc Tritia (Ilyanassa) obsoleta. ToCaudal expression is initially broad, then becomes progressively restricted and is finally only in the developing hindgut (a.k.a. intestine). Knockdown of ToCaudal using morpholino oligonucleotides specifically blocks hindgut development, indicating that despite its initially broad expression, the functional role of ToCaudal is in hindgut patterning. This is the first functional characterization of Caudal in an animal with spiralian development, which is an ancient mode of embryogenesis that arose early in bilaterian animal evolution. These results are consistent with the hypothesis that the ancestral role of the ParaHox genes was anterior-posterior patterning of the endoderm.

It has been proposed that animals have a pattern of developmental evolution resembling an hourglass because the most conserved development stage—often called the phylotypic stage—is always in midembryonic development. Although the topic has been debated for decades, recent studies using molecular data such as RNA-seq gene expression data sets have largely supported the existence of periods of relative evolutionary conservation in middevelopment, consistent with the phylotypic stage and the hourglass concepts. However, so far this approach has only been applied to a limited number of taxa across the tree of life. Here, using established phylotranscriptomic approaches, we found a surprising reverse hourglass pattern in two molluscs and a polychaete annelid, representatives of the Spiralia, an understudied group that contains a large fraction of metazoan body plan diversity. These results suggest that spiralians have a divergent midembryonic stage, with more conserved early and late development, which is the inverse of the pattern seen in almost all other organisms where these phylotranscriptomic approaches have been reported. We discuss our findings in light of proposed reasons for the phylotypic stage and hourglass model in other systems.

Gastropod shell morphologies are famously diverse but generally share a common geometry, the logarithmic coil. Variations on this morphology have been modeled mathematically and computationally but the developmental biology of shell morphogenesis remains poorly understood. Here we characterize the organization and growth patterns of the shell-secreting epithelium of the larval shell of the basket whelk Tritia (also known as Ilyanassa). Despite the larval shell’s relative simplicity, we find a surprisingly complex organization of the shell margin in terms of rows and zones of cells. We examined cell division patterns with EdU incorporation assays and found two growth zones within the shell margin. In the more anterior aperture growth zone, we find that inferred division angles are biased to lie parallel to the shell edge, and these divisions occur more on the margin’s left side. In the more posterior mantle epithelium growth zone, inferred divisions are significantly biased to the right, relative to the anterior–posterior axis. These growth zones, and the left–right asymmetries in cleavage patterns they display, can explain the major modes of shell morphogenesis at the level of cellular behavior. In a gastropod with a different coiling geometry, Planorbella sp., we find similar shell margin organization and growth zones as Tritia, but different left–right asymmetries than we observed in the helically coiled shell of Tritia. These results indicate that differential growth patterns in the mantle edge epithelium contribute to shell shape in gastropod shells and identify cellular mechanisms that may vary to generate shell diversity in evolution.

In some animal groups, the secondary embryonic axis is patterned by a small group of cells, often called an organizer, that signals to other cells to establish the correct pattern of cell fates. The Dpp/BMP2-4 pathway plays a central role in secondary axis patterning in many animals [1-11], but it has not been examined during early axial patterning in spiralian embryogenesis. This is a deeply conserved mode of development found in mollusks, annelids, nemerteans, entoprocts, and some marine platyhelminth groups (reviewed in [12, 13]). In the spiralian embryo of the mollusk Ilyanassa, we find that the Dpp ortholog (IoDpp) is expressed most strongly on the dorsal side, in cells of the embryonic organizer and its neighbors. Phospho-smad staining indicates that the pathway is active in all lineages during organizer signaling, but activation is strongest on the dorsal side. Knockdown of IoDpp by morpholino oligos prevents the development of all structures that require organizer signaling and ventralizes the embryo. Ectopic activation of the pathway can induce eyes and external shell, which require organizer signaling. These results indicate that Dpp/BMP2-4 signaling is a key part of the spiralian organizer and suggest similarity with other metazoan organizers. However, the fact that IoDpp/BMP2-4 is inducing, rather than repressing, the neuroectoderm is a surprising difference that may be conserved among spiralians. These results connect the spiralian organizer to this general aspect of secondary axis patterning but highlight the significant variation across animals in effects of the pathway on particular cell types and tissues.

A critical component of maintaining biodiversity in fragmented habitats is maintaining connectivity among the usable fragments. Least cost path (LCP) analysis is a tool that can be used for predicting the ability of an organism to move from one habitat patch to another, based on geographical features of the landscape and life history traits of the organism. While this analysis has been utilized for terrestrial habitats, it is rarely applied to aquatic environments. Aquatic hypoxic conditions occur when dissolved oxygen falls below 2 mg/L. These conditions can create barriers in the water column that can either force fish to leave a habitat, or avoid that habitat altogether. Using the lower St. Johns River (LSJR) estuary in Florida, USA, as a study system, the ability of an adult silver perch, Bairdiella chrysoura, to escape a large-scale hypoxic event was modeled using a multicriteria LCP approach. Criteria-specific cost grids were constructed based upon current speed, risk of predation, and whether oxyge...

Spiralian embryogenesis is found in a number of animal phyla, but the molecular mechanisms that pattern these embryos remain poorly understood. A hallmark of spiralian development is the production of tiers of cells, called quartets, that share distinct developmental potentials. Many RNAs have been discovered that are segregated into particular quartets, raising the possibility that such RNAs could be involved in establishing quartet-specific developmental potentials. In the spiralian embryo of the mollusc Ilyanassa, the IoTis11 RNA is segregated into the second and third quartets, then decays in nearly all lineages except for the ventral-anterior cells of the third quartet, 3a and 3b. Previously published fate-mapping studies, extended here, show that 3a and 3b make bilaterally symmetrical contributions to the esophagus, head ectoderm, and larval musculature. Deletion of either 3a or 3b has only mild effects on development, but ablating both cells impairs development of the esophagus and several other organs. Knockdown of IoTis11 with a translation-blocking morpholino oligonucleotide causes a very similar set of phenotypes as ablation of 3a and 3b, showing that translation of this transcript is required for normal development of 3a and 3b. These results show that a segregated RNA is necessary for the cells that inherit it in a spiralian embryo. Given that RNAs are asymmetrically segregated in nearly all the early cleavages in this embryo, these results suggest that the embryo is extensively patterned by segregated factors. Our experiments also uncovered two previously unappreciated non-autonomous events during Ilyanassa development. First, we found that the embryo can regulate to develop normal esophagus after deletion of either 3a or 3b. Second, we found that the 3a or 3b lineages are required for normal development of the digestive glands, which arise from the fourth order macromeres.

The snail Ilyanassa obsoleta is a useful model for a variety of investigations in the fields of developmental biology, cell biology, larval ecology, ecotoxicology, parasitology, and chemical ecology. To enhance such studies, we have carried out two cDNA sequencing projects to characterize the mRNA transcripts that are present during development of this embryo. These efforts have generated 480 megabases of new sequence, which have been assembled into transcript contigs and represent thousands of newly identified Ilyanassa genes. We identified the orthologs of 182 transcription factors in these data, focusing on families that are likely to be sequence-specific transcriptional regulators. To demonstrate the utility of identifying and examining such transcripts, we describe the expression pattern during organogenesis for IoOnecut, an Ilyanassa ortholog of the HNF6/onecut family of transcription factors.

Summary During animal development, blast cell lineages are generated by repeated divisions of a mother cell into a series of daughter cells, often with a specific series of distinct fates. Nanos is a translational regulator that is involved in germline development in diverse animals [1–4] and also involved in somatic patterning in insects [5, 6]. Recently, Nanos was found to be required for maintenance of stem cell divisions in the Drosophila germline [7, 8]. We have found that in the mollusk Ilyanassa , Nanos messenger RNA and protein are specifically localized in the mesendodermal blast cell lineage derived from the strongly conserved 4d cell. Nanos activity is required for differentiation of multiple tissues that are derived from the 4d cell, showing that IoNanos is required for somatic development in this embryo. At the cellular level, we show that IoNanos activity is required for the highly stereotyped cleavage pattern of the 4d lineage, the proliferative capacity of the blast cells, and the marked asymmetry of the blast cell divisions. These results suggest that IoNanos is involved in regulating blast cell behaviors in the 4d lineage.

PUBLISHED, (213) Attention-deficit hyperactivity disorder (ADHD) is a heritable childhood onset disorder that is marked by variability at multiple levels including clinical presentation, cognitive profile, and response to stimulant medications. It has been suggested that this variability may reflect etiological differences, particularly, at the level of underlying genetics. This study examined whether an attentional phenotype-spatial attentional bias could serve as a marker of symptom severity, genetic risk, and stimulant response in ADHD. A total of 96 children and adolescents with ADHD were assessed on the Landmark Task, which is a sensitive measure of spatial attentional bias. All children were genotyped for polymorphisms (3' untranslated (UTR) and intron 8 variable number of tandem repeats (VNTRs)) of the dopamine transporter gene (DAT1). Spatial attentional bias correlated with ADHD symptom levels and varied according to DAT1 genotype. Children who were homozygous for the 10-repeat allele of the DAT1 3'-UTR VNTR displayed a rightward attentional bias and had higher symptom levels compared to those with the low-risk genotype. A total of 26 of these children who were medication naive performed the Landmark Task at baseline and then again after 6 weeks of stimulant medication. Left-sided inattention (rightward bias) at baseline was associated with an enhanced response to stimulants at 6 weeks. Moreover, changes in spatial bias with stimulant medications, varied as a function of DAT1 genotype. This study suggests an attentional phenotype that relates to symptom severity and genetic risk for ADHD, and may have utility in predicting stimulant response in ADHD., Attention-deficit hyperactivity disorder (ADHD) is a heritable childhood onset disorder that is marked by variability at multiple levels including clinical presentation, cognitive profile, and response to stimulant medications. It has been suggested that this variability may reflect etiological differences, particularly, at the level of underlying genetics. This study examined whether an attentional phenotype-spatial attentional bias could serve as a marker of symptom severity, genetic risk, and stimulant response in ADHD. A total of 96 children and adolescents with ADHD were assessed on the Landmark Task, which is a sensitive measure of spatial attentional bias. All children were genotyped for polymorphisms (3' untranslated (UTR) and intron 8 variable number of tandem repeats (VNTRs)) of the dopamine transporter gene (DAT1). Spatial attentional bias correlated with ADHD symptom levels and varied according to DAT1 genotype. Children who were homozygous for the 10-repeat allele of the DAT1 3'-UTR VNTR displayed a rightward attentional bias and had higher symptom levels compared to those with the low-risk genotype. A total of 26 of these children who were medication naive performed the Landmark Task at baseline and then again after 6 weeks of stimulant medication. Left-sided inattention (rightward bias) at baseline was associated with an enhanced response to stimulants at 6 weeks. Moreover, changes in spatial bias with stimulant medications, varied as a function of DAT1 genotype. This study suggests an attentional phenotype that relates to symptom severity and genetic risk for ADHD, and may have utility in predicting stimulant response in ADHD., This work was supported by grants from the Irish Health Research Board, Science Foundation Ireland, the Irish Higher Education Authority's Programme for Research in Third-Level Institutions. MAB is supported by an Australian National Health and Medical Research Council Howard Florey Centenary Fellowship. KAJ is supported by the Health Research Board of Ireland.

Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3-4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four-cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24-cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups.

Of the North American gomphotheres, the status of Tetralophodon, primarily known from the Old World, is among the most controversial. This controversy stems from the incompleteness of the North American specimens. Along with elongation of the M/m3s and a tendency towards double trefoiling on the cheek teeth, Tetralophodon is diagnosed by complete tetralophodonty (i.e., four lophs on D/d 4-M/m2). However, of those progressive North American gomphothere specimens that have plausibly been referred to Tetralophodon only the M/m2s are known to be tetralophodont, with the condition of the M/m1s and P/p4s unknown. Thus, it has proven impossible to determine whether these purported Tetralophodon species represent true Tetralophodon immigrants, or one or more parallel radiations from North American Gomphotherium stock. The discovery of an unusual gomphothere in the Clarendonian Kepler Quarry fauna of Nebraska has possible bearing on North American Tetralophodon. This gomphothere resembles North American T...

According to Joseph Roach, ‘surrogation’ is a key mechanism for the reproduction of collective social memory within the ‘circum-Atlantic world’, a term he uses in Cities of the Dead (1996) to describe the oceanic system connecting the Americas, Africa and Europe that emerged in the sixteenth century and that was forged, in particular, through the trade in and enslavement of millions of African people. Surrogation consists of a process through which attempts are made to fill ‘cavities’ created by death or other forms of departure with replacements. While this idea has mainly been used to understand processes of cultural representation in the colonial circum-Atlantic world, it can also be applied to postcolonial contexts, such as the processes of nation-building in the formally decolonized Caribbean that rest on the articulation of history and memory. In Barbados, this process involved the creation in 1998 of a pantheon of ten National Heroes. First among these is Bussa, an enslaved man commemorate...

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.

There is disagreement among ecologists as to whether ecosystem system behavior in general is the net result of all of the complex internal system interactions (bottom-driven) or if the behavior is driven by a limited number of key processes (top-driven). If ecosystems are primarily bottom-driven in nature, then it is unlikely that any two complex ecosystems will ever behaviorally converge as a simple matter of probability, or that their behaviors will ever be predictable. Conversely, evidence of ecosystem convergence would suggest that the systems are top-driven, with the corollary that their behaviors can be understood (and therefore in principle predicted) without a complete understanding of their internal workings. Research has demonstrated that body mass distributions of terrestrial animals broadly reflect ecosystem function. Thus, comparable but causally disconnected terrestrial ecosystems that demonstrate similar body mass distributions would suggest ecosystem convergence. To look for this possible convergence, I generated body mass distributions in a time series for late Miocene North American mammal faunas from the Gulf Coastal Plains, Great Plains, and Pacific coastal region, and compared them with data from the modern Serengeti savanna region. The data show that during the early late Miocene Gulf Coastal Plain faunas resembled each other but were distinctly different from that of the Serengeti, the Great Plains fauna resembled the Serengeti, while the Pacific Coast fauna showed no resemblance to any of the others. However, during the latest Miocene the Gulf Coastal Plain faunas were transformed so as to strongly resemble the Serengeti fauna. The resemblance with the Serengeti was maintained by the Great Plains faunas until at least the end of the Miocene, while the Pacific Coast faunas remained distinctly different from the others. These findings suggest that the late Miocene ecosystems of the Gulf Coastal Plain and Great Plains regions (but not the Pacific Coast region) converged with that of the Serengeti savanna fauna and thus that these ecosystems were/are top-driven rather than bottom-driven in nature.

Spiralian development is shared by several protostome phyla and characterized by regularities in early cleavage, fate map, and larva. Experimental evidence from multiple spiralian species implicates cells in the D quadrant lineage as the organizer of future axial development of the embryo. However, the mechanisms by which the D quadrant is specified differ between species with equal and unequal spiral cleavage. Equally cleaving mollusc embryos establish the D quadrant via cell–cell interactions between the micromeres and macromeres at the 24- to 36-cell stage. In unequally cleaving embryos, the D quadrant is established at the 4-cell stage via asymmetries in the first 2 cell divisions. We have begun to explore the molecular mechanisms of D quadrant patterning in spiralians. Previously, we showed that, in the unequally cleaving embryo of the mollusc Ilyanassa obsoleta, the MAPK pathway is activated and functionally required in 3D and also in the micromeres known to require a signal from 3D. Here, we examine the role of MAPK signaling in 4 spiralians with equal cleavage. In 3 equally cleaving molluscs, the chiton Chaetopleura, the limpet Tectura, and the snail Lymnaea, the MAPK pathway is activated in the 3D cell but not in the overlying micromeres. In the equally cleaving embryo of the polychaete annelid Hydroides, MAPK activation was not detected in the 3D macromere but was observed in one of its daughter cells, 4d. In addition, inhibiting Tectura MAPK activation disrupts differentiation of 3D and cells induced by it, supporting a functional role for MAPK in axis specification in equally cleaving spiralians. Thus, MAPK signaling may have a conserved role in the D quadrant organizer cell 3D in molluscs. However, there have been at least 2 evolutionary changes in the activation of the MAPK pathway during spiralian evolution. MAPK function in the Ilyanassa micromeres is a recent cooption and, since the divergence of annelids and molluscs, there has been a shift in onset of MAPK activation between 3D and 4d. We propose that this latter shift correlates with a change in the timing of specification of the secondary embryonic axis.

We have completed the first stage of a two-stage genome wide screen designed to identify chromosomal regions that may harbour susceptibility genes for bipolar affective disorder. The first stage screening sample included 509 subjects from 151 nuclear families recruited within the United Kingdom and Republic of Ireland. This sample contained 154 narrowly defined affected sibling pairs (DSM-IV BPI) and 258 broadly defined affected sibling pairs (DSM-IV BPI, SABP, BPII, BPNOS or MDD(R)), approximately two thirds of all families contained at least one other additional typed individual. All individuals were genotyped using 398 highly polymorphic microsatellite markers from Applied Biosystems's Linkage Mapping Set Version 2. The average inter-marker distance was 9.6 cM and the mean heterozygosity was 0.78. Analysis of these data using non-parametric linkage methods (MAPMAKER/SIBS) found no evidence for loci of major effect and no regions reached genome-wide significance for either suggestive or significant linkage. We identified 19 points across the genome where the MLS exceeded a value set for follow up in our second stage screen (MLSor = 0.74 (equivalent to a nominal pointwise significance of 5%) under the narrowest diagnostic model). These points were on chromosomes 2, 3, 4, 6, 7, 9, 10, 12, 17, 18X. Some of these points overlapped with previous linkage reports both within bipolar affective disorder and other psychiatric illnesses. Under the narrowest diagnostic model, the single most significant multipoint linkage was on chromosome 18 at marker D18S452 (MLS=1.54). Overall the highest MLS was 1.70 on chromosome 2 at marker D2S125, under the broadest diagnostic model.

Spiralian embryogenesis is deeply conserved and seems to have been in place in the last common ancestor of the large assemblage of protostome phyla known as the Lophotrochozoa. While the blastula fate maps of several spiralian embryos have been determined, little is known about the events that link the early embryo and the larva. For all cells in the Ilyanassa blastula, we determined the clonal morphology at four time points between the blastula and veliger stages. We found that ectomesoderm comes mostly from 3a and 3b, but also from 2c and 2b. We also observed the ingression and early proliferation of 3a- and 3b-derived ectomesoderm. We found cells in the 2b clone that marked the anterior edge of the blastopore and later the mouth and cells in the 3c/3d clones that marked the posterior edges of these structures. This demonstrates directly that the mouth forms in the same location as the blastopore. In the development of the shell field, we observed dramatic cell migration events that invert the positions of the 2b and 2d clones that contribute to the shell. Using time-lapse imaging, we followed and described the cleavage pattern of the conserved endomesodermal blast cell, 4d, up to 4d + 45 h, when there were 52 cells in the clone. Our results show the growth and movement of clones derived from cells of the spiralian blastula as they transform into the trochophore-like and veliger stages. They have implications for the evolution of the shell in gastropods, the origins of mesoderm in spiralians, and the evolution of mouth formation in metazoans.

In populations that are small and asexual, mutations with slight negative effects on fitness will drift to fixation more often than in large or sexual populations in which they will be eliminated by selection. If such mutations occur in substantial numbers, the combined effects of long-term asexuality and small population size may result in substantial accumulation of mildly deleterious substitutions. Prokaryotic endosymbionts of animals that are transmitted maternally for very long periods are effectively asexual and experience smaller effective population size than their free-living relatives. The contrast between such endosymbionts and related free-living bacteria allows us to test whether a population structure imposing frequent bottlenecks and asexuality does lead to an accumulation of slightly deleterious substitutions. Here we show that several independently derived insect endosymbionts, each with a long history of maternal transmission, have accumulated destabilizing base substitutions in the highly conserved 16S rRNA. Stabilities of Domain I of this subunit are 15–25% lower in endosymbionts than in closely related free-living bacteria. By mapping destabilizing substitutions onto a reconstructed phylogeny, we show that decreased ribosomal stability has evolved separately in each endosymbiont lineage. Our phylogenetic approach allows us to demonstrate statistical significance for this pattern: becoming endosymbiotic predictably results in decreased stability of rRNA secondary structure.

The giant otter Enhydritherium terraenovae, a New World taxon thought to be related to both the Old World otter Enhydriodon and the extant sea otter Enhydra, was originally described on the basis of relatively limited material, mostly dental. However, an incomplete skeleton of E. terraenovae recovered from the early Hemphillian Moss Acres Racetrack site in northern Florida includes a skull, mandible, part of the axial skeleton, and most of both the fore- and hindlimbs, adding greatly to knowledge of this animal. On the basis of this new material, the osteology of this species is described in detail for the first time, with its diagnosis revised accordingly. This new osteological information, along with information about the nature of the Moss Acres Racetrack site, allows such paleoecological and functional aspects of E. terraenovae as habitat preference, mode of swimming, and diet to be inferred or interpreted in a detail previously impossible. E. terraenovae: 1) was a habitat generalist rather t...

Unusual shovel-tusked gomphothere material from the late Clarendonian Black Butte Local Fauna (Juntura Formation, Oregon), including a lower tusk, partial upper tusk, and two mandibles including a second and a third molar, has been described and referred to Platybelodon. Platybelodon has the following diagnostic characteristics: molars strongly double-trefoiled; third molar with five lophs; lower tusks short with broad and very flat profiles lacking significant dorsoventral curvature; and lower tusks lacking obvious lamination but instead filled with small, short dentinal rods. However, the Black Butte shovel-tusked gomphothere was referred to Platybelodon despite having the following conflicting features: molars with weakly developed single trefoiling; third molar with only four lophids (the fourth little more than a talonid); broad, moderately flattened, relatively elongated lower tusk with strong dorsal curvature; and lower tusk internal structure showing lamination rather than dentinal rods. I...

Spiralian embryos are found in a large group of invertebrate phyla but are largely uncharacterized at a molecular level. These embryos are thought to be particularly reliant on autonomous cues for patterning, and thus represent potentially useful models for understanding asymmetric cell division. The series of asymmetric divisions that produce the micromere quartets are particularly important for patterning because they subdivide the animal-vegetal axis into tiers of cells with different developmental potentials. In the embryo of the snail Ilyanassa, the IoLR5 RNA is specifically segregated to the first quartet cells during the third cleavage. Here, we show that this RNA, and later the protein, are maintained in the 1q121 cells and their descendents throughout development. Some IoLR5-expressing cells become internalized and join the developing cerebral ganglia. Knockdown of IoLR5 protein results in loss of the larval eyes, which normally develop in association with these ganglia. Segregation of this RNA to the first quartet cells does not occur if centrosomal localization is bypassed. We show that the specific inheritance of the RNA by the first quartet cells is driven by a discrete RNA sequence in the 3′ UTR that is necessary and sufficient for localization and segregation, and that localization of another RNA to the first quartet is mediated by a similar element. These results demonstrate that micromere quartet identity, a hallmark of the ancient spiralian developmental program, is controlled in part by specific RNA localization motifs.

INTRODUCTIONThe 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.

INTRODUCTIONThe 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.

The shovel-tusked gomphotheres are normally portrayed scooping up water plants with their shovellike mandibular tusks. This portrayal is based on speculation about the possible functions of the lower tusks and misinterpretation of mandibular-tusk wear patterns that goes back to the 1920s and 1930s. In addition, most literature concerning shovel-tusker feeding behavior ignores the possibility that the upper tusks may have had a role in feeding, as they do in modern African elephants. Because wear patterns on tusks provide direct evidence as to how tusks were used during feeding, I examined and interpreted patterns on the lower and upper tusks of shovel-tusked gomphotheres on the basis of theoretical models of tusk wear to determine how the tusks were used in feeding.This examination led to the following conclusions. (1) The genus Amebelodon was characterized by feeding opportunism, gathering food with both its lower and upper tusks in a variety of ways. (2) There is evidence for similar flexibility within the genus Serbelodon. (3) Wear patterns on mandibular tusks of Platybelodon grangeri and Torynobelodon barnumbrowni indicate that these taxa did not practice mandibular shoveling but used these tusks to cut tough vegetation in a specialized fashion. (4) The flaplike trunks (probosces) generally attributed to shovel-tusked gomphotheres in restorations are shown almost certainly to be erroneous; morphological and functional evidence suggests that these animals had trunks similar to those of modern elephants. (5) Strong tusk sexual dimorphism is unknown in the shovel-tusked gomphotheres except for Platybelodon and should not be found in any taxa that relied on their tusks for procuring food. New restorations of both Amebelodon and Platybelodon are provided that are more consistent with the actual evidence bearing on their feeding behavior.

The spiralian developmental program is a highly conserved mode of early development that is characterized by regularities in cleavage pattern, fate map, and larval morphology. It is found in a number of animal phyla, and was likely present in the last common ancestor of the large superphylum Lophotrochozoa. Despite this key position for understanding the evolution of development in animals, and the intrinsic advantages for using spiralian embryos to study embryonic development and asymmetric cell division, very little is known about the molecular mechanisms of spiralian embryogenesis. The snail Ilyanassa has typical spiralian development, as well as a number of practical and experimental advantages that have made it a useful model for understanding spiralian embryogenesis and basic processes in metazoan development. Here, I describe the key embryological experiments that inform our understanding of spiralian development in Ilyanassa, and I review recent progress in understanding the molecular basis of patterning this embryo.

The Spiralia is a clade of protostome invertebrate phyla that share a highly conserved mode of early development. Spiralian development is characterized by regularities in the arrangement of early cleavages, the fates of the cells that are produced by these divisions, and the development of the distinctive trochophore larva. Because of the strong conservation in early development, homologies can be identified between cells in divergent taxa. Some of the most striking examples of conservation in the spiralian embryo are in the cells that generate the mesoderm. The specification of the mesodermal precursors has been well characterized by embryological approaches, and recently the molecular mechanisms of mesoderm specification are starting to be elucidated. This review examines the development of mesoderm in spiralians in a comparative context, with particular focus on the relationship between the mesendodermal cell 4d and the embryonic organizer.

In everyday life, our sensory system is bombarded with visual input and we rely upon attention to select only those inputs that are relevant to behavioural goals. Typically, humans can shift their attention from one visual field to the other with little cost to perception. In cases of 'unilateral neglect', however, there is a persistent bias of spatial attention towards the same side as the damaged cerebral hemisphere. We used a visual orienting task to examine the influence of functional polymorphisms of the dopamine transporter gene (DAT1) on individual differences in spatial attention in normally developing children. DAT1 genotype significantly influenced spatial bias. Healthy children who were homozygous for alleles that influence the expression of dopamine transporters in the brain displayed inattention for left-sided stimuli, whereas heterozygotes did not. Our data provide the first evidence in healthy individuals of a genetically mediated bias in spatial attention that is related to dopamine signalling.

PUBLISHED, Several independent studies and meta-analyses aimed at identifying genomic regions linked to bipolar disorder (BP) have failed to find clear and consistent evidence of linkage regions. Our hypothesis is that combining the original genotype data provides benefits of increased power and control over sources of heterogeneity that outweigh the difficulty and potential pitfalls of the implementation. We conducted a combined analysis using the original genotype data from 11 BP genomewide linkage scans comprising 5,179 individuals from 1,067 families. Heterogeneity among studies was minimized in our analyses by using uniform methods of analysis and a common, standardized marker map and was assessed using novel methods developed for meta-analysis of genome scans. To date, this collaboration is the largest and most comprehensive analysis of linkage samples involving a psychiatric disorder. We demonstrate that combining original genome-scan data is a powerful approach for the elucidation of linkage regions underlying complex disease. Our results establish genomewide significant linkage to BP on chromosomes 6q and 8q, which provides solid information to guide future gene-finding efforts that rely on fine-mapping and association approaches.

During development, different cell fates are generated by cell-cell interactions or by the asymmetric distribution of patterning molecules. Asymmetric inheritance is known to occur either through directed transport along actin microfilaments into one daughter cell or through capture of determinants by a region of the cortex inherited by one daughter. Here we report a third mechanism of asymmetric inheritance in a mollusc embryo. Different messenger RNAs associate with centrosomes in different cells and are subsequently distributed asymmetrically during division. The segregated mRNAs are diffusely distributed in the cytoplasm and then localize, in a microtubule-dependent manner, to the pericentriolar matrix. During division, they dissociate from the core mitotic centrosome and move by means of actin filaments to the presumptive animal daughter cell cortex. In experimental cells with two interphase centrosomes, mRNAs accumulate on the correct centrosome, indicating that differences between centrosomes control mRNA targeting. Blocking the accumulation of mRNAs on the centrosome shows that this event is required for subsequent cortical localization. These events produce a complex pattern of mRNA localization, in which different messages distinguish groups of cells with the same birth order rank and similar developmental potentials.

We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCbeta1, gamma1, and delta1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCgamma1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCgamma1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCgamma1 on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 microM. These results demonstrate the presence of PLCgamma1 in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.

INTRODUCTIONThe 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.

INTRODUCTIONThe 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°C-8°C. This protocol describes how to induce embryo production in Ilyanassa snails, collect the embryos, and rear them to the stage required for study.

INTRODUCTIONThe 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.

INTRODUCTIONThe 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 ~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.

INTRODUCTIONIlyanassa 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.

At least five animal phyla exhibit spiralian development, which is characterized by striking similarities in the geometry of the early cleavage pattern and the fate map of the blastula, along with similarities in larval morphology. Recent advances in reconstructing the phylogeny of spiralians and their relatives suggest that the common ancestor of a large clade of protostome phyla known as the Lophotrochozoa had spiralian development. In this minireview, I describe characteristics of spiralian development and some recent insights into its mechanisms and evolution.