Persistent and reparative neurogenesis in the juvenile masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis> telencephalon after mechanical injury

A superficially located periventricular proliferative area with PCNA-immunopositive (PCNA+) cells, which corresponds to the pallial periventricular zone (PVZ) of other fish species, including its dorsal, lateral, and medial compartments, is discovered in the telencephalon of the juvenile masu salmon Oncorhynchus masou. The PCNA+ cells are also identified in the parenchyma of the masu salmon intact brain, and their maximum concentration is observed in the medial zone. After a mechanical injury, the zones of induced neurogenesis—neurogenic niches and sites of secondary neurogenesis surrounded by radial glial fibers—appear in the masu salmon telencephalon. The PVZ of the juvenile masu salmon pallium contains clusters of undifferentiated HuCD-immunopositive (HuCD+) neurons. A change in the HuCD+ cell topography is observed in the mechanically injured masu salmon telencephalon, namely, neurogenic niches in the lateral zone and an increase in the cell distribution density and cell migration patterns in the medial zone. A high level of persistent neurogenesis is characteristic of the juvenile masu salmon brain.     

Neurochemical Organization and Connections of the Cerebral Preglomerular Complex of the Masu Salmon

Using immunohistochemical labeling of the cells containing neuronal NO synthase (nNOS), tyrosine hydroxylase (TH), GABA, and parvalbumin (PA), as well as histochemical marking of choline acetyltransferase-containing neurons, we examined the neurochemical organization of the glomerular nuclei and preglomerular complex in the brain of the masu salmon (Oncorhynchus masou). Injections of the carbocyanine dye DiI allowed us to examine projections of neurons of the preglomerular and mammillary nuclei in the salmon brain. We showed that cholinergic, GABA-, PA-, TH-, and nNOS-immunopositive neurons belonging to different morphological types are present in the glomerular and medial preglomerular nuclei. The analysis of correlations between morphometric characteristics of the cells belonging to different neurochemical types and densitometric estimates of amounts of neurochemical agents present in these cells allowed us to hypothesize that there are close morphofunctional interrelations in cell populations possessing different neurochemical and morphometric characteristics. These interrelations of the cells belonging to different chemotypes are, probably, realized as mediatory/modulatory ones. The presence of a great number of small slightly differentiated cells in the preglomerular and glomerular nuclei allows us to suppose that the growth of the greatest sensory center of the salmon brain is provided by neuroblasts that migrate from the proliferative zones in the course of postembryonal neurogenesis. It is also hypothesized that NO, TH, and GABA are involved in paracrine control of the postnatal morphogenesis of the salmon preglomerular complex. The data obtained by hodological analysis indicate that the nuclei of the preglomerular complex obtain afferent projections from the dorsomedial and ventroventral telencephalic regions, preoptic nucleus, periventricular layer of the tectum, and posterior central thalamic nucleus. Our study demonstrated the existence of reciprocal functional connections between the preglomerular complex (most important diencephalic center for transmission of sensory information) and dorsomedial and ventral regions of the telencephalon in the masu salmon.     

GFAP and PCNA Marking in the cerebellum of masu salmon’s (<Emphasis Type="Italic">Oncorhynchus masou</Emphasis>) juvenile after mechanical injury

The objective of this work was to study proliferation processes and the role of glia and neural stem cells in the event of injurious action on cerebellum of masu salmon’s (Oncorhynchus masou) juvenile. Using the immunoperoxidase staining of the glial fibrillary acidic protein (GFAP) and proliferating cells nuclear antigen (PCNA), processes of proliferation and gliogenesis after mechanical trauma of cerebellum of cherry salmon’s (Oncorhynchus masou) juvenile were studied. After the trauma, the intensity of proliferation and migration processes varies in different zones. Proliferation processes decrease after the trauma in lateral and basal zones, and migration increases. In the dorsal zone, on the contrary, migration processes significantly decrease and proliferation increases. In the dorsal matrix zone of a cerebellum, intense cell proliferation was detected. In the dorsal, lateral, and basal zone of the molecular layer of cerebellum after traumatic damage, neurogenic niches containing PCNA and cells, as well as a heterogeneous population of PCNA-cells, were identified. At the location of neurogenic niches, fibers of radial glia and small single intensely or moderately labeled GFAP cells were discovered. As a result of damaging action, GFAP+ fibers of radial glia, which form differently directed radially oriented bundles, appeared in the dorsal matrix zone. Such structural formations have not been discovered in intact animals. We suppose that, after the trauma, structural reconstruction connected with partial spatial reorientation of the radial glia fibers and formation of specific directions for cells formed in this zone occurs in the dorsal matrix zone. As a result of the trauma, in masu salmon’s cerebellum, elements of the radial glia, including both cells possessing typical morphology and cell fragments presented as long radially oriented processes or cell body containing initial fragments of radial fibers, appeared.     

Species-dependent migration of fish hatching gland cells that commonly express astacin-like proteases in common

Two constituent proteases of the hatching enzyme of the medaka ( Oryzias latipes ), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above-mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish ( Brachydanio rerio ) and masu salmon ( Oncorynchus masou ) embryos. Using the amplified fragments as probes, two full-length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.     

Gaseous transmitters in the brain of the masu salmon, <Emphasis Type="Italic">Oncorhynchus masou</Emphasis> (Salmoniformes,Salmonidae)

Distribution of nitroxidergic and H₂S-producing neurons in the brain of the masu salmon Oncorhynchus masou was studied by methods of histochemical labeling of NADPH-diaphorase and by immunohistochemical labeling of the neuronal nitric oxide synthase and cystathionine β-synthase (CBS). The established distribution of CBS and nNOS/NADPH-d of neurons and fibers in the masu salmon telencephalon, optic tectum, and cerebellum allows suggesting that the NO- and H₂S-producing systems represent individual, non-overlapping neuronal complexes performing specialized functions in the activity of local neuronal networks. In the medullar part, the nNOS-ir and NADPH-d-positive neurons were detected in the composition of viscerosensory (V, VII, and IX–X) and visceromotor (III, IV, and VI) nuclei of craniocerebral nerves, octavolateral afferent complex, reticulospinal neurons, and medial reticular formation. CBS in the masu salmon medulla was revealed in neurons of the nerve X nucleus, reticulospinal neurons, and ventrolateral reticular formation. Distribution of NO-ergic and H₂S-producing neurons in the masu salmon medullar nuclei indicates that NO in masu salmon is the predominant neuromodulator of the medullar viscerosensory systems, while H₂S seems to modulate only the descending motor systems. The results of the performed study allow suggesting that NO in the masu salmon medulla periventricular area can act as a regulator of postnatal ontogenesis.     

Comparison of the spectral sensitivity of three species of juvenile salmonids

The spectral sensitivity of chum Oncorhynchus keta , pink Oncorhynchus gorbuscha and masu Oncorhynchus masou masou salmon was measured by the optomotor reaction index in monochromatic light of 400, 440, 480, 520, 560, 600 and 620 nm using an interference filter. The reaction rate of chum salmon was highest at 520 nm but the rates of pink and masu salmon were highest at 560 nm. In addition, a high reaction rate at 400 nm was also observed in masu salmon, suggesting that masu salmon are sensitive to ultraviolet light.     

Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: evolution of neurohypophysial hormone precursors.

We have cloned and determined the nucleotide sequences of cDNAs encoding precursors of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), from the hypothalamus of masu salmon, Oncorhynchus masou. The deduced amino acid sequences of masu salmon VT and IT precursors (proVT-I and proIT-I) are highly homologous to those of chum salmon proVT-I and proIT-I, respectively. The VT and IT precursors are composed of a signal peptide, hormone and neurophysin (NP), the middle portion of which is highly conserved among vertebrates. Both the NPs extend about 30 amino acids at the C-terminal. The extended C-terminals have a leucin-rich segment in the carboxyl-terminal, as copeptin of vasopressin precursor. Southern bot analysis showed the presence of two types of proVT genes (proVT-I and proVT-II) and proIT genes (proIT-I and proIT-II) in an individual masu salmon, as in a chum salmon. Southern blot analysis with proVT probes further suggested that at least two different types of proVT-I genes exist in a single masu salmon. Northern blot analysis indicated that proVT-I and proIT-I genes are expressed in the hypothalamus, whereas proVT-II and proIT-II genes are not expressed. Evolutionary distance between proVT-I and proIT-I genes was statistically estimated based on synonymous nucleotide substitution in the coding region of the cDNAs. The magnitude of distance between masu salmon proVT-I and proIT-I genes suggested that the highly conserved central portion of NPs resulted from a gene conversion event. Between masu salmon and chum salmon, evolutionary distance for proVT-I genes is about 6-fold larger than that for proIT-I genes.     

Pax‐6 is expressed early in the differentiation of a corneal epithelial model system

Pax‐6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1‐(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax‐6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax‐6‐positive cells were also K3 keratin‐positive, suggesting that Pax‐6 is expressed in differentiating cells. Pax‐6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT‐qPCR. The raise in Pax‐6 expression preceded for 9 h the increase in LDH‐H and LDH‐M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full‐length mRNAs encoding for the two major Pax‐6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax‐6 mRNA was 2‐ to 2.5‐fold more abundant than Pax‐6(5a) mRNA. The ectopic expression of Pax‐6 or Pax‐6(5a) decreased proliferative ability leading to the formation of abortive, non‐proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax‐6. Collectively, results show that Pax‐6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax‐6 as the main driver of the differentiation of corneal epithelial cells. J. Cell. Physiol. 220: 348–356, 2009. © 2009 Wiley‐Liss, Inc.     

Ultrastructure of melanocytes from retina and choroid of the Pacific salmon

The ultrastructure of the retina and choroid cells in three species of the Pacific salmon, Oncohrynchus gorbuscha, O. keta and O. masou, was studied. The structure of retina pigment cells is similar in all the three species, only a small difference was found in the percentage of lengthened and rounded forms of melanosomes. Melanocytes of the masu salmon differ also in the structure of their nuclei. The pigment cells of choroid differ from those of retina by a more extended form of melanosomes and by the presence of less rounded melanosomes. In the chum salmon retina we found electron lucent "contact vesicles", whose assignment is open to discussion. In retina pigment cells of the masu salmon smolts ready for migration, the activity of Golgi appararus rises, mitochondria and mature melanosomes increase in number. The choroid pigment cells are slightly swollen, their processes more often and deeper penetrate into the walls of vessels, down to the endothelium. Results of the experiment with the application of an artificial magnetic field (AMF) have shown that the retina and choroid pigment cells in the masu salmon fry react to the field of a certain direction. The phenomenon of magnetosensitivity of pigment cells is discussed in addition to their possible involvement in magnetoreception.     

Pax6 regulates regional development and neuronal migration in the cerebral cortex

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.     

Regularities of the life history strategy adoption in masu salmon Oncorhynchus masou from the Kol River (Western Kamchatka) in regard to the processes of growth and sexual maturation

Growth, age structure, sexual maturation, and peculiarities of the life history strategy adoption were studied for the population of masu salmon Oncorhynchus masou inhabiting the Kol River, Western Kamchatka. The growth rate and gametogenesis depended on the peculiarities of masu salmon generations in certain years and were the limiting factors for the juvenile differentiation and the direction of its ontogenetic development. The adoption of the resident or anadromous life history strategy in masu salmon is the epigenetic process, and the bifurcation during the life span of a single specimen is observed only once in a life, but twice in the generation, at the age of 1+ or 2+. The diversity of the life history strategy patterns was less at the northern boundary of the geographical range of masu salmon compared to the area of ecological optimum (the basin of the Sea of Japan). In the Kol River basin, nearby the northern boundary of its geographical range, the reproductive success and formation of the life history strategy diversity in the population depended strictly on the temperature regime of the water bodies, where the spawning and the freshwater period of life cycle of masu salmon take place. In Kamchatka, all the breeders were monocyclic, i.e. anadromous and resident precocious males spawned only once in a life.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Hierarchical genetic structure of native masu salmon populations in Hokkaido,Japan

Identification of the spatial extent of genetic structuring that may be influenced by evolutionary, ecological and historical factors is critical for effective conservation or management strategies. Masu salmon Oncorhynchus masou is commonly distributed in Far East, however, many local populations have been under threats of decline due to habitat destruction, overexploitation, and genetic introgression. To reveal the spatial genetic structure of native masu salmon populations in Hokkaido, masu salmon samples were collected from 16 rivers in which there was no official record of artificial releases of any masu salmon stock and were analyzed using 15 microsatellite loci. A Bayesian assignment test revealed that masu salmon populations were divided into two genetically distinct groups: the northeastern and southwestern groups. For within-group genetic structure, all populations, except for geographically proximate populations, were significantly different from each other. AMOVA revealed that genetic variation at among-group level based on groups identified assignment test was greater than that of groups based on geographic locations. There was no significant IBD for the 16 populations. However, the Mantel test revealed significant IBD for the northeastern group, but did not for the southwestern group. This study suggested that native masu salmon populations in Hokkaido exhibit a hierarchical genetic structure that is largely a result of their precise homing behavior. The results of this study also highlight the importance of defining populations by using genetic data rather than by using predefined populations based on geographic locations for the correct determination of genetic structure.     

Application of a bioenergetics model to estimate the influence of habitat degradation by check dams and potential recovery of masu salmon populations

Using a bioenergetics model, we examined how check dams negatively effect masu salmon (Oncorhynchus masou) populations by causing habitat loss in upstream areas and habitat degradation in downstream areas. The potential recovery of masu salmon populations in the upstream area was estimated based on the expected biomass and potential recovery area. We also determined if and how fish carrying capacity is affected by degradation of substrate conditions (armoring and compaction) in the downstream area. Recovery of upstream areas was considered to be effective in enhancing and conserving masu salmon populations. We demonstrated that the dam-induced altered substrate conditions and habitat degradation in the downstream area resulted in a considerable reduction of drifting prey. Simulation analysis revealed that a 40 % increase in the abundance of masu salmon juveniles in the downstream area could be expected if substrate conditions were restored. We concluded that both improvement of migration barriers and restoring the sediment regime would be important in enhancing and conserving wild masu salmon populations.