CRISPR/Cas9‐mediated genome modification in the mollusc,Crepidula fornicata

The discovery and application of the CRISPR/Cas9 genome editing method has greatly enhanced the ease with which transgenic manipulation can occur. We applied this technology to the mollusc, Crepidula fornicata, and have successfully created transgenic embryos expressing mCherry fused to endogenous β‐catenin. Specific integration of the fluorescent reporter was achieved by homologous recombination with a β‐catenin‐specific donor DNA containing the mCherry coding sequence. This fluorescent gene knock‐in strategy permits in vivo observations of β‐catenin expression during embryonic development and represents the first demonstration of CRISPR/Cas9‐mediated transgenesis in the Lophotrochozoa superphylum. The CRISPR/Cas9 method is a powerful and economical tool for genome modification and presents an option for analysis of gene expression in not only major model systems, but also in those more diverse species that may not have been amenable to the classic methods of transgenesis. This approach will allow one to generate transgenic lines of snails for future studies. genesis 53:237–244, 2015. © 2014 Wiley Periodicals, Inc.     

MAPK activation and the specification of the D quadrant in the gastropod mollusc, Crepidula fornicata

Embryos of the gastropod snail Crepidula fornicata exhibit a typical spiral cleavage pattern. Although a small polar lobe is formed at the first and second cleavage divisions, the embryo of C. fornicata exhibits a mode of development similar to that of equal-cleaving spiralians in which the D quadrant is conditionally specified by inductive interactions involving the derivatives of the first quartet micromeres. This study demonstrates that mitogen activated protein kinases, MAPK, are initially activated in the progeny of the first quartet micromeres, just prior to the birth of the third quartet (e.g., late during the 16-cell and subsequently during the 20-cell stages). Afterwards, MAPK is activated in 3D just prior to the 24-cell stage, transiently in 4d and finally in a subset of animal micromeres immediately following those stages. This pattern of MAPK activation differs from that reported for other spiralians. Using an inhibitor of MAPK kinase (MEK), we demonstrated that activated MAPK is required for the specification of the 3D macromere, during the late 16-cell through early 24-cell stages. This corresponds to the interval when the progeny of the first quartet micromeres specify the D quadrant macromere. Activated MAPK is not required in 3D later during the 24-cell stage or in the embryonic organizer, 4d, for its normal activity. Likewise, activated MAPK is not required in the animal micromeres during subsequent stages of development. Additional experiments suggest that the polar lobe, though not required for normal development, may play a role in restricting the activation of MAPK and biasing the specification of the 3D macromere.     

Cell specification and the role of the polar lobe in the gastropod mollusc Crepidula fornicata

A small polar lobe forms at the first and second cleavage divisions in the gastropod mollusc Crepidula fornicata. These lobes normally fuse with the blastomeres that give rise to the D quadrant at the two- and four-cell stages (cells ultimately generating the 4d mesentoblast and D quadrant organizer). Significantly, removal of the small polar lobe had no noticeable effect on subsequent development of the veliger larva. The behavior of the polar lobe and characteristic early cell shape changes involving protrusion of the 3D macromere at the 24-cell suggest that the D quadrant is specified prior to the sixth cleavage division. On the other hand, blastomere deletion experiments indicate that the D quadrant is not determined until the time of formation of the 4d blastomere (mesentoblast). In fact, embryos can undergo regulation to form normal-appearing larvae if the prospective D blastomere or 3D macromere is removed. Removal of the 4d mesentoblast leads to highly disorganized, radial development. Removal of the first quartet micromeres at the 8-cell stage also leads to the development of radialized larvae. These findings indicate that the embryos of C. fornicata follow the mode of development exhibited by equal-cleaving spiralians, which involves conditional specification of the D quadrant organizer via inductive interactions, presumably from the first quartet micromeres.     

Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia)

 Recent reports indicate that neuronal elements develop in early larval stages of some Gastropoda from the Pulmonata and Opisthobranchia prior to the appearance of any ganglia of the future adult central nervous system (CNS). The present study describes similar early neuronal elements in Crepidula fornicata. A posterior FMRFamide-like immunoreactive (LIR) cell with anteriorly projected fibers was observed in the trochophore stage. Additional FMRFamide-LIR and serotonin-LIR cells and fibers were found in the apical organ in the trochophore and early veliger stages. FMRFamide-LIR and serotonin-LIR projections to the velum and foot were also detected at this time. As the veliger developed, peripheral FMRFamide-LIR and later catecholaminergic cells were located in the foot region. Also during this stage, catecholaminergic cells and processes were observed near the mouth. In addition, this study tentatively identified the first serotonin- and FMRFamide-LIR cells and fibers within the developing ganglia of the adult CNS, which appeared in close proximity to the earlier developing elements. These observations are consistent with the hypothesis that, in addition to its presumed role in the control of larval behaviors, the larval nervous system guides the development of the adult CNS. Larvae from the class Bivalvia and other invertebrate phyla also have neuronal elements marked by the presence of FMRFamide, serotonin, and catecholamines, and, therefore, this study may provide additional insights into phylogenetic relationships of the Gastropoda with other representatives of the Mollusca and different invertebrate phyla. Accepted: 10 February 1999     

Contrasting patterns of genome‐wide polymorphism in the native and invasive range of the marine mollusc Crepidula fornicata

Selection processes are believed to be an important evolutionary driver behind the successful establishment of nonindigenous species, for instance through adaptation for invasiveness (e.g. dispersal mechanisms and reproductive allocation). However, evidence supporting this assumption is still scarce. Genome scans have often identified loci with atypical patterns of genetic differentiation (i.e. outliers) indicative of selection processes. Using microsatellite‐ and AFLP‐based genome scans, we looked for evidence of selection following the introduction of the mollusc Crepidula fornicata. Native to the northwestern Atlantic, this gastropod has become an emblematic invader since its introduction during the 19th and 20th centuries in the northeastern Atlantic and northeastern Pacific. We examined 683 individuals from seven native and 15 introduced populations spanning the latitudinal introduction and native ranges of the species. Our results confirmed the previously documented high genetic diversity in native and introduced populations with little genetic structure between the two ranges, a pattern typical of marine invaders. Analysing 344 loci, no outliers were detected between the introduced and native populations or in the introduced range. The genomic sampling may have been insufficient to reveal selection especially if it acts on traits determined by a few genes. Eight outliers were, however, identified within the native range, underlining a genetic singularity congruent with a well‐known biogeographical break along the Florida. Our results call into question the relevance of AFLP genome scans in detecting adaptation on the timescale of biological invasions: genome scans often reveal long‐term adaptation involving numerous genes throughout the genome but seem less effective in detecting recent adaptation from pre‐existing variation on polygenic traits. This study advocates other methods to detect selection effects during biological invasions—for example on phenotypic traits, although genome scans may remain useful for elucidating introduction histories.     

Unexpected collective larval dispersal but little support for sweepstakes reproductive success in the highly dispersive brooding mollusc Crepidula fornicata

In many marine invertebrates, long‐distance dispersal is achieved during an extended pelagic larval phase. Although such dispersal should result in high gene flow over broad spatial scales, fine‐scale genetic structure has often been reported, a pattern attributed to interfamilial variance in reproductive success and limited homogenization during dispersal. To examine this hypothesis, the genetic diversity of dispersing larvae must be compared with the postdispersal stages, that is benthic recruits and adults. Such data remain, however, scarce due to the difficulty to sample and analyse larvae of minute size. Here, we carried out such an investigation using the marine gastropod Crepidula fornicata. Field sampling of three to four larval pools was conducted over the reproductive season and repeated over 3 years. The genetic composition of larval pools, obtained with 16 microsatellite loci, was compared with that of recruits and adults sampled from the same site and years. In contrast to samples of juveniles and adults, large genetic temporal variations between larval pools produced at different times of the same reproductive season were observed. In addition, full‐ and half‐sibs were detected in early larvae and postdispersal juveniles, pointing to correlated dispersal paths between several pairs of individuals. Inbred larvae were also identified. Such collective larval dispersal was unexpected given the long larval duration of the study species. Our results suggest that each larval pool is produced by a small effective number of reproducers but that, over a reproductive season, the whole larval pool is produced by large numbers of reproducers across space and time.     

Nitric oxide inhibits metamorphosis in larvae of Crepidula fornicata, the slippershell snail

This paper concerns the role of nitric oxide (NO) in controlling metamorphosis in the marine gastropod Crepidula fornicata. Metamorphosis was stimulated by the nitric oxide synthase (NOS) inhibitors AGH (aminoguanidine hemisulfate) and SMIS (S-methylisothiourea sulfate) at concentrations of about 100-1000 micromol l(-1) and 50-200 micromol l(-1), respectively. Metamorphosis was not, however, induced by the NOS inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) at even the highest concentration tested, 500 micromol l(-1). Moreover, pre-incubation with l-NAME at 20 and 80 micromol l(-1) did not increase the sensitivity of competent larvae to excess K(+), a potent inducer of metamorphosis in this species; we suggest that either l-NAME is ineffective in suppressing NO production in larvae of C. fornicata, or that it works only on the constitutive isoform of the enzyme. In contrast, metamorphosis was potentiated by the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) in response to a natural metamorphic inducer derived from conspecific adults. Because NO typically stimulates cGMP production through the activation of soluble guanylate cyclase, this result supports the hypothesis that NO acts as an endogenous inhibitor of metamorphosis in C. fornicata. The expression of NOS, shown by immunohistochemical techniques, was detected in the apical ganglion of young larvae but not in older larvae, further supporting the hypothesis that metamorphosis in C. fornicata is made possible by declines in the endogenous concentration of NO during development.     

Catecholamines in larvae and juveniles of the prosobranch gastropod, Crepidula fornicata

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K+. Competent larvae contained 1.58 +/- 0.26 (S.E.M.) x 10(-2) pmol DOPA, 0.91 +/- 0.45 x 10(-2) pmol NE, and 0.290 +/- 0.087 pmol DA (mean values per microg total protein, n = 4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-DL-m-tyrosine (alpha-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K+. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1-10 microM. In juveniles induced by 10 microM DDTC, levels of both NE and DA averaged approximately 80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.     

Sex and genetic structure across age groups in populations of the European marine invasive mollusc, Crepidula fornicata L. (Gastropoda)

In long-lived species, variance in allele frequencies over time may vary according to the number of generations contributing to progeny. Here, we investigate the temporal stability of genetic diversity and structure in relation to sex and age in introduced populations of Crepidula fornicata , an exotic gastropod that successfully invaded Europe . This protandrous species has the potential to change sex from male to female according not only to age, but also to local sex ratio (social environment). This mechanism may adjust the reproduction efficiency across different cohorts and thus decrease the likelihood of genetic drift in the following generations. Based on crude demographic structure analysis in two spatially closed introduced French populations, we demonstrate that recruitment is discontinuous. Although timing of sex change is different across populations, both populations have a similar age structure characterized by distributions of males and females changing across generations. Using five microsatellite loci, we show that both populations display a temporal genetic homogeneity and a stability in genetic diversity indices across age groups examined. Our results highlight that the social control of sex change in C. fornicata has strong implications to the maintenance of high genetic diversity by enhancing breeding across several generations at each reproductive season.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 365–374.     

Neural correlates of settlement in veliger larvae of the gastropod,Crepidula fornicata

Settlement behavior of molluscan veliger larvae prior to metamorphosis requires cessation of swimming, accomplished by arrest of prototrochal cilia on the margin of the velum (the larval swimming organ). Ciliary arrest in larvae of gastropods is mediated by an action potential that occurs synchronously across the velum as a consequence of electrical coupling between the prototrochal ciliated cells. We developed a preparation for extracellular recording of such ciliary arrest spikes from intact swimming and crawling veliger larvae of the caenogastropod Crepidula fornicata, using a fine wire electrode. Ciliary arrest spike rates during bouts of substrate crawling were significantly higher than those recorded during preceding swimming periods in larvae that were competent for metamorphosis, but not in precompetent larvae. Spike rates were similar on clean polystyrene substrates, and on substrates that had been coated with a natural cue for metamorphosis (mucus from conspecific adults). We used immunohistochemical methods to localize neuromodulators that might regulate the function of velar cilia. Labeled terminals for serotonin, FMRFamide, and tyrosine hydroxylase (an enzyme for catecholamine synthesis) were located in positions consistent with modulatory effects on the prototrochal ciliated cells. Prototrochal ciliary arrest spike rates and beat frequencies were measured in isolated velar lobes from competent larvae, which were exposed to serotonin, FMRFamide, and dopamine (10^?5 mol L^?1). Serotonin abolished arrest spiking and increased beat frequency; dopamine also increased beat frequency, and FMRFamide depressed it. Competent larvae tested in a small static water column swam to the top of the column when exposed to serotonin, but occupied lower positions than controls when in the presence of dopamine and FMRFamide. The larval nervous system appears to regulate velar functions that are critical for settlement behavior, and is likely to do so by integrating different sensory modalities in an age‐dependent manner.     

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian,Styela

The regional distribution of poly(A)⁺ RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [³H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [³H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [³H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [³H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [³H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [³H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A)⁺ RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela.     

High-resolution fate map of the snail Crepidula fornicata: the origins of ciliary bands, nervous system, and muscular elements

The littorinimorph gastropod Crepidula fornicata shows a spiralian cleavage pattern and has been the subject of studies in experimental embryology, cell lineage, and the organization of the larval nervous system. To investigate the contribution of early blastomeres to the veliger larva, we used intracellular cell lineage tracers in combination with high-resolution confocal imaging. This study corroborates many features derived from other spiralian fate maps (such as the origins of the hindgut and mesoderm from the 4d mesentoblast), but also yields new findings, particularly with respect to the origins of internal structures, such as the nervous system and musculature that have never been described in detail. The ectomesoderm in C. fornicata is mainly formed by micromeres of the 3rd quartet (principally 3a and 3b), which presumably represents a plesiomorphic condition for molluscs. The larval central nervous system is mainly formed by the micromeres of the 1st and 2nd quartet, of which 1a, 1c, and 1d form the anterior apical ganglion and nerve tracks to the foot and velum, and 2b and 2d form the visceral loop and the mantle cell. Our study shows that both first and second velar ciliary bands are generated by the same cells that form the prototroch in other spiralians and apparently bear no homology to the metatroch found in annelids.     

Differential localization of TGF-beta 2 in mouse preimplantation and early postimplantation development

The localization of transforming growth factor type beta 2 (TGF-beta 2) has been followed during preimplantation and early postimplantation murine development using an anti-peptide antibody that specifically recognizes TGF-beta 2. The staining pattern showed that TGF-beta 2 is expressed from the four-cell stage onward and is differentially regulated as cells diverge to various lineages. High levels of staining were found in the trophectoderm of the blastocyst but no staining was observed in the inner cell mass. During postimplantation development the primitive and embryonic ectoderm also lacked detectable staining while visceral endoderm stained well. Parietal endoderm cells also showed positive staining reaction although to a lesser extent than visceral endoderm cells. These findings were confirmed in model systems of the embryo, namely, embryonal carcinoma and embryonic stem cells differentiated to to cells with either visceral or parietal endoderm characteristics. The possible regulatory role of this factor in early embryogenesis is discussed.     

Evaluating whether velar lobe size indicates food limitation among larvae of the marine gastropod Crepidula fornicata

Disproportionately large feeding structures have been used to infer food limitation in some marine invertebrate larvae, but few studies have investigated whether other factors alter larval morphology in similar ways. In this study, larvae of Crepidula fornicata were reared either at five different food concentrations of Isochrysis galbana (clone T-ISO) at a single temperature (22 degrees C) (Experiments I and II); or on three different phytoplankton species (Isochrysis galbana, Dunaliella tertiolecta, and Pavlova lutheri) at both high and low concentrations at a single temperature (22 degrees C) (Experiment III); or at high and low concentrations of Isochrysis galbana at four different temperatures between 16 and 25 degrees C (Experiment IV). Shell lengths and velar lobe dimensions were determined for individual larvae at intervals to monitor relative rates of velar and shell growth. In addition (Experiment V), fast growing and slow growing larvae in Experiment I were examined separately to determine whether velar lobes developed at similar rates (relative to shell growth) for fast and slow growing larvae within individual cultures. In general, velar lobes grew significantly larger, relative to shell length, when larvae were reared at low food concentrations (P<0.0001); for larvae of similar shell length, the velar lobes of those fed 1x10(4) cells ml(-1) were on average 17.7% larger than those of larvae fed 18x10(4) cells ml(-1) of T-ISO. In contrast, larvae fed different phytoplankton species at equivalently high food concentrations did not differ in relative velum size (P=0.2666), even though shell growth rates differed significantly for larvae raised on the different diets, indicating substantial variation in food quality. We also found that relative rates of velum and shell growth differed among fast and slow growing individuals within treatments. Temperature had no significant effect on relative rates of velar and shell growth within the 16-25 degrees C range tested (P=0.121), but may have altered the relationship between food concentration and relative velar growth. These results indicate that dramatically reduced food concentration induces disproportionate growth in the velar lobes of C. fornicata, but that interpretation of data from field-collected individuals of this species will be made difficult by the potentially confounding effects of temperature, food quality, and differences in individual growth potential. Assessments of food limitation using morphological measurements for field-collected larvae will need to be supplemented with other indicators before convincing conclusions about the extent of food limitation in C. fornicata can be drawn.     

Differential expression of two skeletal muscle beta-tropomyosin mRNAs during Xenopus laevis development

A cDNA clone for a Xenopus laevis skeletal muscle beta-tropomyosin (beta-TMad) isoform was isolated from an adult skeletal muscle cDNA library. Sequence analysis revealed that this clone corresponded to a second beta-tropomyosin mRNA distinct from the one that was previously characterized (beta-TMemb). The two skeletal beta-TM mRNAs originate from distinct genes and are differentially expressed during development. Beta-TMemb mRNA is expressed only in the somites of the early embryo while beta-TMad mRNA is expressed in pre-metamorphic tadpoles and adult skeletal muscles. We have isolated the promoter region of the beta-TMemb gene and shown that a DNA construct containing 2.9 kb of promoter region is properly expressed after injection in the embryo.     

Differential subcellular localization of particular mRNAs in hippocampal neurons in culture

In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and beta-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that is selectively distributed in dendrites and cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.