Pallial Mantle Research Articles

Characterization of the Abracl-Expressing Cell Populations in the Embryonic Mammalian Telencephalon.

Abracl (ABRA C-terminal-like protein) is a small, non-typical winged-helix protein that shares similarity with the C-terminal domain of the protein ABRA (Actin-Binding Rho-Activating protein). The role of Abracl in the cell remains elusive, although in cancer cells, it has been implicated in proliferation, migration and actin dynamics. Our previous study showed that Abracl mRNA was expressed in the dividing cells of the subpallial subventricular zone (SVZ), in the developing cortical plate (CP), and in the diencephalic SVZ; however, the molecular identities of the Abracl-expressing cell populations were not defined in that work. In this study, we use double immunofluorescence to characterize the expression of Abracl on sections of embryonic murine (E11.5-E18.5) and feline (E30/31-E33/34) telencephalon; to this end, we use a battery of well-known molecular markers of cycling (Ki67, Ascl1, Dlx2) or post-mitotic (Tubb3, Gad65/67, Lhx6 and Tbr1) cells. Our experiments show that Abracl protein has, compared to the mRNA, a broader expression domain, including, apart from proliferating cells of the subpallial and diencephalic SVZ, post-mitotic cells occupying the subpallial and pallial mantle (including the CP), as well as subpallial-derived migrating interneurons. Interestingly, in late embryonic developmental stages, Abracl was also transiently detected in major telencephalic fiber tracts.

Hichin, a chitin binding protein is essential for the self-assembly of organic frameworks and calcium carbonate during shell formation

Shell biomineralization is a process where inorganic minerals accumulate upon a chitinous scaffold under the control of multifunctional matrix proteins. In this study, we cloned a novel matrix protein gene from the mantle of Hyriopsis cumingii. The predicted protein, hichin, contains a chitin-binding domain and exhibited the highest expressional level in mantle tissue, with positive signals mainly detected in dorsal epithelial cells of the pallial mantle according to in situ hybridization, indicating its possible involvement in shell nacreous layer biomineralization. RNA interference showed that hichin suppression induced disordered self-assembly of the insoluble framework in the nacreous layer, and that the newly formed calcium carbonate crystals could not bind to organic frameworks. Furthermore, hichin was primarily responsible for building the framework during initial nacre deposition in pearl formation. Moreover, the chitin-binding domain of hichin also provided crystal morphology regulation in vitro crystallization assay. These results indicated that hichin is involved in the self-assembly of organic frameworks and morphological regulation in shell nacreous layer.

A shell regeneration assay to identify biomineralization candidate genes in mytilid mussels

Biomineralization processes in bivalve molluscs are still poorly understood. Here we provide an analysis of specifically expressed sequences from a mantle transcriptome of the blue mussel, Mytilus edulis. We then developed a novel, integrative shell injury assay to test, whether biomineralization candidate genes highly expressed in marginal and pallial mantle could be induced in central mantle tissue underlying the damaged shell areas. This experimental approach makes it possible to identify gene products that control the chemical micro-environment during calcification as well as organic matrix components. This is unlike existing methodological approaches that work retroactively to characterize calcification relevant molecules and are just able to examine organic matrix components that are present in completed shells. In our assay an orthogonal array of nine 1mm holes was drilled into the left valve, and mussels were suspended in net cages for 20, 29 and 36days to regenerate. Structural observations using stereo-microscopy, SEM and Raman spectroscopy revealed organic sheet synthesis (day 20) as the first step of shell-repair followed by the deposition of calcite crystals (days 20 and 29) and aragonite tablets (day 36). The regeneration period was characterized by time-dependent shifts in gene expression in left central mantle tissue underlying the injured shell, (i) increased expression of two tyrosinase isoforms (TYR3: 29-fold and TYR6: 5-fold) at day 20 with a decline thereafter, (ii) an increase in expression of a gene encoding a nacrein-like protein (max. 100-fold) on day 29. The expression of an acidic Asp-Ser-rich protein was enhanced during the entire regeneration process. This proof-of-principle study demonstrates that genes that are specifically expressed in pallial and marginal mantle tissue can be induced (4 out of 10 genes) in central mantle following experimental injury of the overlying shell. Our findings suggest that regeneration assays can be used systematically to better characterize gene products that are essential for distinct phases of the shell formation process, particularly those that are not incorporated into the organic shell matrix.

The Importance of Total Genome Databases in Research on Akoya Pearl Oyster

Pearl is the most valuable form of gem made by organisms and its beauty has been considered attractive and precious by people all over the world. Pearl oyster aquaculture system, in which a graft from the mantle pallium is transplanted with a nucleus into mother pearl oysters, is currently conducted at the industrial level. However, it is unclear what molecular mechanisms are involved in this system of pearl formation, which produces brilliant and beautiful nacreous layers. Since it takes long time to look for the proteins and genes possibly participating in the molluskan shell formation, another sophisticated strategy has been expected to screen candidate genes. One breakthrough in the latter area was the development of next-generation sequencing (NGS) in place of the automated Sanger method, the first generation sequencing technique. Using NGS, expressed sequence tag (EST) libraries were constructed from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in the Akoya pearl oyster Pinctada fucata. Subsequently, the total genome sequence of the Akoya pearl oyster was analyzed. This database, together with the EST database mentioned above, is useful for identifying genes responsible for superior phenotype characters for pearl oyster aquaculture such as fast growth, disease resistance, easy domestication, and ability to produce high quality pearls. Subsequent marker-assisted breeding of the Akoya pearl oyster should establish strains with superior genotypes, producing even higher quality Akoya pearls.

Deep Sequencing of ESTs from Nacreous and Prismatic Layer Producing Tissues and a Screen for Novel Shell Formation-Related Genes in the Pearl Oyster

BackgroundDespite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates.Principal FindingsWe employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions.ConclusionsWe constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster. Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20 degrees C in 5% CO2:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2'-deoxyuridine was added to the culture at 30 microns. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2'-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.

Homology and Evolutionary Origins of the 'Neocortex'

The telencephalon of mammals is characterized by the presence of a hexalaminated structure on its external surface, with specific auditory, visual, somatosensory and motor regions. Due to its seeming unique presence in mammals, it is frequently designated as the neocortex. The evolutionary origins of the so-called neocortex have long puzzled comparative neuroanatomists, in view of the seeming absence of a neocortical-like anlage in nonmammalian amniotes. The resolution of this puzzle requires analysis of both adult and embryonic brains. Experimental neuroanatomical, physiological and behavioral methods applied to adult avian and reptilian brains have finally clarified several fundamental questions regarding the origins of 'neocortex' and have indicated that these origins can be viewed as consequent to two separate events: (1) The elaboration of constituent neuronal populations and their associated connections that are common to the telencephalae of both nonmammalian and mammalian amniotes. In mammals these populations are found within the so-called neocortex. In birds and reptiles, most of these neurons are found within the dorsal and dorsolateral ventricular ridges (DVR and DLVR). (2) In mammals, the components of the DVR and DLVR are incorporated into the thin overlying pallium to form a laminated 'neocortex'. Analysis of development in domestic chicks suggests that the DVR is one of several prosencephalic neuromeres ('Prosomeres') that contribute to the ontogeny of comparable structures in birds. Perhaps in mammals, as well, cortical development is consequent to incorporation of these several prosomeres into proliferative ependyma of the pallial mantle.

Cadmium kinetics in freshwater clams. IV. Histochemical localization of cadmium in Anodonta cygnea and Anodonta anatina, exposed to cadmium chloride.

By means of a histochemical staining method, the sulphide-silver technique, the localization of cadmium was studied in several tissues of freshwater mussels,Anodonta cygnea andAnodonta anatina, exposed to CdCl2 (25 Μg/L Cd) for periods up to 16 weeks. After three weeks of exposure, reaction products indicative of the presence of free or loosely bound Cd were observed in the pallial mantle epithelium. After six weeks, reaction products appeared in gill epithelia of both species, and in kidney epithelia ofA. anatina. After nine weeks of exposure, reaction products were present also in kidney epithelia ofA. cygnea. In most cases, reaction products were concentrated in distinct particles in the apical cell region. The initial appearance of reaction products in mantle and gill epithelia was followed by a stationary phase, in which the reaction pattern did not change significantly, despite a continued accumulation of the metal. After 16 weeks of exposure, a further increase in the amount of reaction products was noticed in midgut gland and kidney epithelia ofA. cygnea, and in nearly all epithelia ofA. anatina. In the latter species, reaction products were no longer restricted to distinct particles, but were distributed throughout the epithelial cells and the underlying connective tissue.