Deviating from the Norm: Peculiarities of Aplysia cf. californica Early Cleavage Compared to Traditional Spiralian Models.

Spiralia represents one of the main clades of bilaterally symmetrical metazoans (Bilateria). This group is of particular interest due to the remarkable conservation of its early developmental pattern despite of the high diversity of larval and adult body plans. Variations during embryogenesis are considered powerful tools to determine ancestral and derived characters under a phylogenetic framework. By direct observation of embryos cultured in vitro, we analyzed the early cleavage of the euopisthobranchs Aplysia cf. californica. We used tubulin immunocytochemistry to stain mitotic spindles during early cleavages, and followed each division with the aid of an autofluorescent compound inside yolk platelets, which differed from the characteristic pink-brownish pigment of the vegetal cytoplasm in zygotes and early embryos. We found that this species exhibits an unequal cleavage characterized by ooplasmic segregation, oblique inclination of mitotic spindles, and differences in size and positioning of the asters in relation to the cellular cortex. Furthermore, we detected asynchrony in cleavage timing between the two large macromeres C and D, which increases the number of cleavage rounds required to reach a particular cell stage in comparison to other spiralians. Here, we report the presence of a transient and previously undescribed U-shaped embryo in this species. The present detailed description of A. californica early development deviates considerably from stereotypical patterns described in other spiralians. Our observations demonstrate that early spiralian development can be more plastic than previously thought.

Single-cell semiconductor sequencing.

RNA-seq or transcriptome analysis of individual cells and small-cell populations is essential for virtually any biomedical field. It is especially critical for developmental, aging, and cancer biology as well as neuroscience where the enormous heterogeneity of cells present a significant methodological and conceptual challenge. Here we present two methods that allow for fast and cost-efficient transcriptome sequencing from ultra-small amounts of tissue or even from individual cells using semiconductor sequencing technology (Ion Torrent, Life Technologies). The first method is a reduced representation sequencing which maximizes capture of RNAs and preserves transcripts' directionality. The second, a template-switch protocol, is designed for small mammalian neurons. Both protocols, from cell/tissue isolation to final sequence data, take up to 4 days. The efficiency of these protocols has been validated with single hippocampal neurons and various invertebrate tissues including individually identified neurons within a simpler memory-forming circuit of Aplysia californica and early (1-, 2-, 4-, 8-cells) embryonic and developmental stages from basal metazoans.

Developmental transcriptome of Aplysia californica.

Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages-many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization-a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms.

Development of embryonic cells containing serotonin, catecholamines, and FMRFamide-related peptides in Aplysia californica.

This study demonstrates the presence of a relatively extensive but previously unrecognized nervous system in embryonic stages of the opisthobranch mollusc Aplysia californica. During the trochophore stage, two pairs of cells were observed to be reactive to antibodies raised against the neuropeptides FMRFamide and EFLRIamide. These cells were located in the posterior region of the embryo, and their anterior projections terminated under the apical tuft. As the embryos developed into veliger stages, serotonin-like immunoreactive (LIR) cells appeared in the apical organ and were later observed to innervate the velum. Also, aldehyde-induced fluorescence indicative of catecholamines was present in cells in the foot, oral, and possibly apical regions during late embryonic veliger stages. Just before the embryo hatches as a free-swimming veliger, additional FMRFamide-LIR and catecholamine-containing cells appeared in regions that correspond to the ganglia of what will become the adult central nervous system (CNS). Neurons and connectives that will contribute to the adult CNS appear to develop along the pathways that are pioneered by the earliest posterior FMRFamide-LIR cells. These observations are consistent with the hypothesis that, besides their presumed roles in the control of embryonic behaviors, some elements may also guide the development of the CNS. Embryonic nervous systems that develop prior to and outside of the adult CNS have also been reported in pulmonate and prosobranch species of molluscs. Therefore, the demonstration of early developing neurons and their transmitter phenotypes in A. californica presents new opportunities for a better understanding of the ontogeny and phylogeny of both behavioral and neuronal function in this important model species.

Developmental emergence of different forms of neuromodulation in Aplysia sensory neurons.

The capacity for neuromodulation and biophysical plasticity is a defining feature of most mature neuronal cell types. In several cases, modulation at the level of the individual neuron has been causally linked to changes in the functional output of a neuronal circuit and subsequent adaptive changes in the organism's behavioral responses. Understanding how such capacity for neuromodulation develops therefore may provide insights into the mechanisms both of neuronal development and learning and memory. We have examined the development of multiple forms of neuromodulation triggered by a common neurotransmitter, serotonin, in the pleural sensory neurons of Aplysia californica. We have found that multiple signaling cascades within a single neuron develop sequentially, with some being expressed only very late in development. In addition, our data suggest a model in which, within a single neuromodulatory pathway, the elements of the signaling cascade are developmentally expressed in a "retrograde" manner with the ionic channel that is modulated appearing early in development, functional elements in the second messenger cascade appearing later, and finally, coupling of the second messenger cascade to the serotonin receptor appearing quite late. These studies provide the characterization of the development of neuromodulation at the level of an identified cell type and offer insights into the potential roles of neuromodulatory processes in development and adult plasticity.

Ontogeny of serotonergic neurons in Aplysia californica.

Although the identity, projection patterns, and functions of serotonergic neurons in juvenile and adult Aplysia are relatively well understood, little is known about the development of these cells. We have used light and electron microscopic immunocytochemistry to investigate the genesis, differentiation, identity, and fate of the serotonergic cells in the embryonic, larval, and metamorphic stages of the life cycle of Aplysia. The results indicate that the first serotonergic cells emerge at midembryogenesis and that a total of five cells makes up the entire serotonergic system by hatching. These cells are part of a newly discovered ganglion in Aplysia, called the apical ganglion. This serotonergic system of five cells remains essentially intact throughout larval development. The apical ganglion, together with its serotonergic cells, is resorbed at metamorphosis. A distinct set of serotonergic cells, which begins to emerge by the end of the larval period, is rapidly elaborated during the metamorphic and early juvenile periods to form the adult serotonergic system. These results support the view that the larval and adult forms of the Aplysia nervous system consist of entirely distinct sets of serotonergic cells, each adapted to the stage-specific morphological and behavioral characteristics of the animal.

Projection patterns and target tissues of the serotonergic cells in larval Aplysia californica.

Although the functions of serotonin in adult Aplysia have been the focus of numerous investigations, our understanding of the roles played by this neurotransmitter during development is very incomplete. In the previous study (Marois and Carew [1997a] J. Comp. Neurol. 386:477-490), we showed that identified serotonergic cells are present very early during the ontogeny of Aplysia. In order to gain insight into the possible functions that these serotonergic cells may exert, we have used immuno-electron microscopy in this study to examine the projection patterns and target tissues of the serotonergic cells during the larval development of Aplysia. The results indicate that the larval serotonergic cells have numerous and precise connections to non-neuronal and neuronal target tissues: Serotonergic cells innervate the ciliated cells of the velum, numerous muscle systems, possibly visceral organs, and several cells in the central nervous system. Repeated observations of one serotonergic contact onto an undifferentiated neuron in the abdominal ganglion over a short developmental time span suggest that the serotonergic input may trigger axonogenesis in the postsynaptic cell. Apart from this possibility, we suggest that the innervation patterns of the larval serotonergic cells essentially fulfill the same primary function attributed to the adult serotonergic cells, that of modulating ongoing physiological and behavioral activity.

A mechanism of adaptation to hypergravity in the statocyst of Aplysia californica.

The gravity-sensing organ of Aplysia californica consists of bilaterally paired statocysts containing statoconia, which are granules composed of calcium carbonate crystals in an organic matrix. In early embryonic development, Aplysia contain a single granule called a statolith, and as the animal matures, statoconia production takes place. The objective of this study was to determine the effect of hypergravity on statoconia production and homeostasis and explore a possible physiologic mechanism for regulating this process. Embryonic Aplysia were exposed to normogravity or 3 x g or 5.7 x g and each day samples were analyzed for changes in statocyst, statolith, and body dimensions until they hatched. In addition, early metamorphosed Aplysia (developmental stages 7-10) were exposed to hypergravity (2 x g) for 3 weeks, and statoconia number and statocyst and statoconia volumes were determined. We also determined the effects of hypergravity on statoconia production and homeostasis in statocysts isolated from developmental stage 10 Aplysia. Since prior studies demonstrated that urease was important in the regulation of statocyst pH and statoconia formation, we also evaluated the effect of hypergravity on urease activity. The results show that hypergravity decreased statolith and body diameter in embryonic Aplysia in a magnitude-dependent fashion. In early metamorphosed Aplysia, hypergravity decreased statoconia number and volume. Similarly, there was an inhibition of statoconia production and a decrease in statoconia volume in isolated statocysts exposed to hypergravity in culture. Urease activity in statocysts decreased after exposure to hypergravity and was correlated with the decrease in statoconia production observed. In short, there was a decrease in statoconia production with exposure to hypergravity both in vivo and in vitro and a decrease in urease activity. It is concluded that exposure to hypergravity downregulates urease activity, resulting in a significant decrease in the formation of statoconia.

Early FMRFamide-like immunoreactive cells in gastropod neurogenesis.

The somata of the first FMRF amide-like immunoreactive (Fa-LIR) cells appear early during neurogenesis and are located posteriorly within the embryo. By the time that Aplysia californica hatches into a free-living veliger, it already contains 3 intensely immunoreactive cells with fibres which project anteriorly to cross the cerebral commissure and invade the developing pedal ganglia. Fa-LIR elements are also present by 30-33% of embryonic development in Lymnaea stagnalis, which hatches as a fully metamorphosed juvenile. The cells in L. stagnalis, are similarly located posteriorly in the embryo and have fibres which project to the cerebral and pedal ganglia. These findings suggest that such Fa-LTR cells are among the very first to develop within the gastropod nervous system and that they may be play important roles in subsequent neuronal development.

Effects of hypergravity on statocyst development in embryonic Aplysia californica.

Aplysia californica is a marine gastropod mollusc with bilaterally paired statocysts as gravity-receptor organs. Data from three experiments in which embryonic Aplysia californica were exposed to 2 x g are discussed. The experimental groups were exposed to excess gravity until hatching (9-12 day), whereas control groups were maintained at normal gravity. Body diameter was measured before exposure to 2 x g. Statocyst, statolith and body diameter were each determined for samples of 20 embryos from each group on successive days. Exposure to excess gravity led to an increase in body size. Statocyst size was not affected by exposure to 2 x g. Statolith size decreased with treatment as indicated by smaller statolith-to-body ratios observed in the 2 x g group in all three experiments. Mean statolith diameter was significantly smaller for the 2 x g group in Experiment 1 but not in Experiments 2 and 3. Defective statocysts, characterized by very small or no statoliths, were found in the 2 x g group in Experiments 1 and 2.

Development of the statocyst in Aplysia californica. I. Observations on statoconial development.

The gravity receptor organs of gastropod molluscs, such as Aplysia californica, are bilateral paired statocysts, which contain dense statoconia within a fluid-filled cyst. Gravitational forces on the statoconia are sensed through their interaction with ciliated mechanoreceptor cells in the wall of the cyst. Larval Aplysia contain a single statolith within each statocyst; when the animals grow to a critical size, they begin producing multiple statoconia, a process that continues throughout life. The number of statoconia is highly correlated with animal weight but poorly correlated with age, indicating that stone production is related to total metabolism. The single statolith has an amorphous internal structure whereas the multiple statoconia have calcification deposited on concentric layers of membrane or matrix protein. The statolith appears to be produced within the cyst lumen but the multiple statoconia are produced within supporting cells between the receptor cells. Large adult animals have statoconia larger than those in early post-metamorphic animals which have just started producing multiple stones. The maximum statocyst diameter at which the receptor-cell cilia can suspend the statolith in the center of the cyst lumen is 45 microns; production of multiple stones begins when the cyst reaches this size. The mechanisms by which statoconia production is initiated and controlled are discussed.