A dual involvement of Protocadherin 18a in stromal cell development guides the formation of a functional hematopoietic niche.

Hematopoietic stem and progenitor cells emerge from the aorta and migrate to the caudal hematopoietic tissue (CHT) of zebrafish larvae, the hematopoietic equivalent of the mammalian fetal liver, for their proliferation and differentiation. We previously reported that somite-derived stromal cells were a key component of the CHT niche. Here, we found that the cell adhesion protein Protocadherin 18a (Pcdh18a) is expressed in the stromal cell progenitors (SCPs) emigrating from somites toward the future CHT. Deletion of most of the Pcdh18a intracellular domain caused a decrease in the number of SCPs, the directionality of their migration, and the cell-contact mediated repulsion that normally occurs between migrating SCPs. These defects were followed by abnormal morphogenesis of the venous plexus that forms the CHT framework, and the inability of the CHT to function as a niche for hematopoietic stem and progenitor cells. Finally, we found that the extracellular domain of Pcdh18a mediates trans heterophilic adhesion of stromal cells to endothelial cells in vivo and thereby the reticular versus perivascular fate of SCPs. Thus, Pcdh18a expression in SCPs is essential for the proper development of the hematopoietic niche.

Trim33 is essential for macrophage and neutrophil mobilization to developmental or inflammatory cues.

Macrophages infiltrate and establish in developing organs from an early stage, often before these have become vascularized. Similarly, leukocytes, in general, can quickly migrate through tissues to any site of wounding. This unique capacity is rooted in their characteristic amoeboid motility, the genetic basis of which is poorly understood. Trim33 (also known as Tif1-γ), a nuclear protein that associates with specific DNA-binding transcription factors to modulate gene expression, has been found to be mainly involved in hematopoiesis and gene regulation mediated by TGF-ß. Here, we have discovered that in Trim33-deficient zebrafish embryos, primitive macrophages are unable to colonize the central nervous system to become microglia. Moreover, both macrophages and neutrophils of Trim33-deficient embryos display a reduced basal mobility within interstitial tissues, and a profound lack of a response to inflammatory recruitment signals, including local bacterial infections. Correlatively, Trim33-deficient mouse bone marrow-derived macrophages display a strongly reduced three-dimensional amoeboid mobility in fibrous collagen gels. The transcriptional regulator Trim33 is thus revealed as being essential for the navigation of macrophages and neutrophils towards developmental or inflammatory cues within vertebrate tissues.

Alcam-a and Pdgfr-α are essential for the development of sclerotome-derived stromal cells that support hematopoiesis.

Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fates. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the mammalian fetal liver, arise from the ventral part of caudal somites. We have now found that this ventral domain is the sclerotome, and that two markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-α, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that trunk somites are similarly dependent on Alcam and Pdgfr-α to produce mesenchymal cells that foster HSPC emergence from the aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis.

A short-term memory trace persists for days in the mouse hippocampus.

Active recall of short-term memory (STM) is known to last for a few hours, but whether STM has long-term functions is unknown. Here we show that STM can be optogenetically retrieved at a time point during which natural recall is not possible, uncovering the long-term existence of an STM engram. Moreover, re-training within 3 days led to natural long-term recall, indicating facilitated consolidation. Inhibiting offline CA1 activity during non-rapid eye movement (NREM) sleep, N-methyl-D-aspartate receptor (NMDAR) activity, or protein synthesis after first exposure to the STM-forming event impaired the future re-exposure-facilitated consolidation, which highlights a role of protein synthesis, NMDAR and NREM sleep in the long-term storage of an STM trace. These results provide evidence that STM is not completely lost within hours and demonstrates a possible two-step STM consolidation, first long-term storage as a behaviorally inactive engram, then transformation into an active state by recurrence within 3 days.

A cortical cell ensemble in the posterior parietal cortex controls past experience-dependent memory updating.

When processing current sensory inputs, animals refer to related past experiences. Current information is then incorporated into the related neural network to update previously stored memories. However, the neuronal mechanism underlying the impact of memories of prior experiences on current learning is not well understood. Here, we found that a cellular ensemble in the posterior parietal cortex (PPC) that is activated during past experience mediates an interaction between past and current information to update memory through a PPC-anterior cingulate cortex circuit in mice. Moreover, optogenetic silencing of the PPC ensemble immediately after retrieval dissociated the interaction without affecting individual memories stored in the hippocampus and amygdala. Thus, a specific subpopulation of PPC cells represents past information and instructs downstream brain regions to update previous memories.

Hippocampus as a sorter and reverberatory integrator of sensory inputs.

The hippocampus must be capable of sorting and integrating multiple sensory inputs separately but simultaneously. However, it remains to be elucidated how the hippocampus executes these processes simultaneously during learning. Here we found that synchrony between conditioned stimulus (CS)-, unconditioned stimulus (US)- and future retrieval-responsible cells occurs in the CA1 during the reverberatory phase that emerges after sensory inputs have ceased, but not during CS and US inputs. Mutant mice lacking N-methyl-D-aspartate receptors (NRs) in CA3 showed a cued-fear memory impairment and a decrease in synchronized reverberatory activities between CS- and US-responsive CA1 cells. Optogenetic CA3 silencing at the reverberatory phase during learning impaired cued-fear memory. Thus, the hippocampus uses reverberatory activity to link CS and US inputs, and avoid crosstalk during sensory inputs.

Identification of CEACAM6 as an intermediate filament-associated protein expressed in Sertoli cells of rat testis.

Ceacam6 (carcinoembryonic antigen-related cell adhesion molecule 6 gene) has recently been isolated by differential display followed by RT-PCR and DNA sequence analyses. Ceacam6 is a member of an immunoglobulin superfamily and encodes a protein of 266 amino acid residues possessing one immunoglobulin (Ig)-like domain. RT-PCR analysis showed that Ceacam6 was dominantly expressed in rat testis and its expression level prominently increased after 6 wk of postnatal development in testis. Immunohistochemical analyses using the anti-CEACAM6 antibody revealed that CEACAM6 colocalized with intermediate filaments (vimentin) in Sertoli cells and interstitial cells. The association between CEACAM6 and vimentin was observed throughout postnatal development in rat testis. Transfection experiments performed in COS-7 cells suggested that overexpression of CEACAM6 brought about aggregation of vimentin filament around nuclei with which CEACAM6 colocalized and that the N-terminus region of CEACAM6, including the Ig-like domain, seemed to be required for association with vimentin filaments. Interaction between CEACAM6 and vimentin in rat testis and transfected COS-7 cells was confirmed by immunoprecipitation. Our observations strongly suggested that CEACAM6 might be a novel intermediate filament-associated protein involved in regulation of vimentin architecture in Sertoli cells.

Characterization and subcellular localization of Tektin 3 in rat spermatozoa.

Mammalian sperm flagella have filament-forming Tektin proteins (Tektin 1-5) reported to be involved in the stability and structural complexity of flagella. Male mice null for Tektin3 produce spermatozoa with reduced forward progression and increased flagellar structural bending defects. The subcellular localization of Tektin3 (TEKT3) in spermatozoa, however, has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT3 in flagella of rat spermatozoa, we performed extraction studies followed by immunoblot analysis, immunofluorescence microscopy, and immunogold electron microscopy. Extraction of sperm flagella from the cauda epididymis resulted in complete removal of axonemal tubulins, while TEKT3 was resistant to extraction with the same S-EDTA (1% SDS, 75 mM NaCl, 24 mM EDTA, pH 7.6) solution, suggesting that TEKT3 might be present in the peri-axonemal component and not directly associated with axonemal tubulins. Resistance to S-EDTA extraction might be due to disulfide bond formation during epididymal maturation since concentrations of DTT greater than 5 mM drastically promoted release of TEKT3 from flagella. Immunofluorescence microscopy and pre-embedding immunoelectron microscopy revealed that TEKT3 was predominantly associated with the surface of mitochondria and outer dense fibers in the middle piece. In addition, TEKT3 was found to be present at the equatorial segment region of the acrosome membrane in sperm heads. TEKT3 might not only work as a flagellar constituent required for flagellar stability and sperm motility but also may be involved in acrosome-related events, such as the acrosome reaction or sperm-egg fusion.

Immunolocalization of spetex-1 at the connecting piece in spermatozoa of the musk shrew (Suncus murinus).

Spetex-1, which has been isolated by differential display and rat cDNA library screening as a haploid spermatid-specific gene, encodes a protein with two coiled-coil motifs that locates at both the segmented column in the connecting piece and outer dense fibers-affiliated satellite fibrils in rat sperm flagella. Orthologs of Spetex-1 are identified in many animal species, including human, chimpanzee, macaque, cow, dog, African clawed frog, green spotted puffer, and zebrafish. In this study, we used RT-PCR in combination with 5' and 3' RACE (Rapid Amplification of cDNA End) technique to isolate Spetex-1 ortholog of the musk shrew (Suneus murinus), which yielded a full-length Suncus Spetex-1 gene containing an open reading frame of 1,908 base pairs encoding a protein of 636 amino acids with the predicted molecular mass of 72,348 Da. Suncus Spetex-1 has two coiled-coil motifs at 118-184 and 242-276 amino acid residues, which is a characteristic shared by mammalian Spetex-1 proteins. To examine the subcellular localization of Spetex-1 in Suncus spermatozoa, we produced the anti-Suncus Spetex-1 antibody and carried out immunocytochemistry. In spite of that the primary structure of Suncus Spetex-1 is basically similar to that of rat and mouse Spetex-1, confocal laser scanning microscopy and immunoelectron microscopy revealed that Spetex-1 was restricted to the segmented column and capitulum in the connecting piece of Suncus spermatozoa and was not detected in other parts of flagella, suggesting a diversity of Spetex-1 localization in mammalian spermatozoa.

Characterization of Spetex-1, a new component of satellite fibrils associated with outer dense fibers in the middle piece of rodent sperm flagella.

Spetex-1, which has been isolated by differential display as a haploid spermatid-specific gene, encodes a protein with two coiled-coil motifs located in the middle piece of flagella in rodent spermatozoa. The middle piece of flagella is composed of axoneme and peri-axonemal elements including outer dense fibers (ODFs) and satellite fibrils. Pre-embedding immunoelectron microscopy clearly demonstrated that Spetex-1 is located at satellite fibrils associated with ODFs in the middle piece of flagella of rat spermatozoa. Extraction of Spetex-1 from spermatozoa by SDS or urea required dithiothreitol, suggesting crosslinking by disulfide bond is involved in the assembly of satellite fibrils containing Spetex-1. We identified putative Spetex-1 orthologs in many animal species, and both cysteine residues and coiled-coil motifs were well conserved in mammalian orthologs of Spetex-1. When Spetex-1 was co-transfected into COS-7 cells with myc-tagged Tektin4, another filamentous protein associated with ODFs, the two molecules were co-localized in various sizes of aggregates in the cells. These data suggested that Spetex-1, a new component of satellite fibrils, might be involved in the structural stability of the sperm flagellar middle piece and functions in co-operation with Tektin4.

Subcellular localization of Tektin2 in rat sperm flagellum.

Tektins are evolutionarily conserved filament-forming proteins localized in flagella and cilia, and have been reported to be involved in the stability and structural complexity of axonemal microtubules. Five mammalian Tektins (Tektin1-5) have been reported. Of these, Tektin2 (TEKT2) has been found to be required for normal flagellum structure and function. Tekt2-null sperm display flagellum bending and reduced motility, probably due to disruption of the dynein inner arm. However, the subcellular localization of TEKT2 in spermatozoa has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT2 in flagella of rat spermatozoa, we performed confocal laser scanning microscopy, extraction of flagella followed by immunoblot analysis, and immunogold electron microscopy. Extraction of sperm flagella by SDS-EDTA resulted in complete extraction of axonemal tubulins, while TEKT2 was only partially released from flagella, suggesting that TEKT2 might be present in the peri-axonemal component, not directly associated with axonemal tubulins. Confocal laser scanning microscopy and pre-embedding immunoelectron microscopy revealed that TEKT2 is associated with the surface of outer dense fibers (ODFs). TEKT2 may function as an ODF-affiliated molecule required for flagellum stability and sperm motility.

Pcdhß deficiency affects hippocampal CA1 ensemble activity and contextual fear discrimination.

Clustered protocadherins (Pcdhs), a large group of adhesion molecules, are important for axonal projections and dendritic spread, but little is known about how they influence neuronal activity. The Pcdhß cluster is strongly expressed in the hippocampus, and in vivo Ca2+ imaging in Pcdhß-deficient mice revealed altered activity of neuronal ensembles but not of individual cells in this region in freely moving animals. Specifically, Pcdhß deficiency increased the number of large-size neuronal ensembles and the proportion of cells shared between ensembles. Furthermore, Pcdhß-deficient mice exhibited reduced repetitive neuronal population activity during exploration of a novel context and were less able to discriminate contexts in a contextual fear conditioning paradigm. These results suggest that one function of Pcdhßs is to modulate neural ensemble activity in the hippocampus to promote context discrimination.

Coronin 1A depletion restores the nuclear stability and viability of Aip1/Wdr1-deficient neutrophils.

Actin dynamics is central for cells, and especially for the fast-moving leukocytes. The severing of actin filaments is mainly achieved by cofilin, assisted by Aip1/Wdr1 and coronins. We found that in Wdr1-deficient zebrafish embryos, neutrophils display F-actin cytoplasmic aggregates and a complete spatial uncoupling of phospho-myosin from F-actin. They then undergo an unprecedented gradual disorganization of their nucleus followed by eruptive cell death. Their cofilin is mostly unphosphorylated and associated with F-actin, thus likely outcompeting myosin for F-actin binding. Myosin inhibition reproduces in WT embryos the nuclear instability and eruptive death of neutrophils seen in Wdr1-deficient embryos. Strikingly, depletion of the main coronin of leukocytes, coronin 1A, fully restores the cortical location of F-actin, nuclear integrity, viability, and mobility of Wdr1-deficient neutrophils in vivo. Our study points to an essential role of actomyosin contractility in maintaining the integrity of the nucleus of neutrophils and a new twist in the interplay of cofilin, Wdr1, and coronin in regulating F-actin dynamics.

Tektin5, a new Tektin family member, is a component of the middle piece of flagella in rat spermatozoa.

Tektins are composed of a family of filament-forming proteins localized in cilia and flagella. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin2 and Tektin4, have been verified to be present in sperm flagella. A new member of the TEKTIN gene family, which was designated as rat Tektin5, was obtained by PCR technique. Rat Tektin5 cDNA consists of 1,674 bp encoding a 62.8 kDa protein of 558 amino acids. Tektin5 protein contains a Tektin domain as well as a nonapeptide signature sequence that is a prominent feature of Tektin proteins. RT-PCR analysis indicated that Tektin5 was predominantly expressed in testis and that its expression was up-regulated during testis development. Immunoblot analyses revealed that Tektin5 is present in sperm flagella but not in heads and that it is completely released from rat spermatozoa by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Confocal laser scanning microscopy revealed that Tektin5 was located in the middle piece of flagella in rat spermatozoa with no immunolabeling in the heads and the principal piece. Immunogold electron microscopy adopting pre-embedding method discovered that Tektin5 is predominantly associated with the inner side of the mitochondrial sheath. Tektin5 might work as a middle piece component requisite for flagellar stability and sperm motility.

Population structures of the water-borne plant pathogen Phytopythium helicoides reveal its possible origins and transmission modes in Japan.

The purpose of this study was to clarify the genetic diversity of Phytopythium helicoides and to understand the transmission mode of the pathogen in Japan. In total, 232 P. helicoides isolates were collected from various host plants and geographic origins, including farms and natural environments. We developed 6 novel microsatellite markers for use in the study and found 90 alleles among the 6 markers in the 232 isolates. The analysis of molecular variance suggested that P. helicoides has high variance within individuals and low fixation indices between populations. A phylogenetic analysis revealed that isolates collected from the same hosts and/or geographic origins were often grouped together. For example, several isolates from natural environments were grouped with isolates from nearby agricultural areas. On the other hand, 2 geographically distant populations collected from the same host plant had similar genotypes. Our results suggested that migration of the pathogen could be facilitated naturally via drainage systems or by human activity in the transport of agricultural materials.

Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae.

Fish otoliths are highly calcified concretions deposited in the inner ear and serve as a part of the hearing and balance systems. They consist mainly of calcium carbonate and a small amount of organic matrix. The latter component is considered to play important roles in otolith formation. Previously, we identified two major otolith matrix proteins, OMP-1 (otolith matrix protein-1) and Otolin-1, from salmonid species. To assess the function of these proteins in otolith formation, we performed antisense morpholino oligonucleotide (MO)-mediated knockdown of omp-1 and otolin-1 in zebrafish embryos. We first identified zebrafish cDNA homologs of omp-1 (zomp-1) and otolin-1 (zotolin-1). Whole-mount in situ hybridization then revealed that the expression of both zomp-1 and zotolin-1 mRNAs is restricted to the otic vesicles. zomp-1 mRNA was expressed from the 14-somite stage in the otic placode, but the zOMP-1 protein was detected only from 26-somite stage onwards. In contrast, zotolin-1 mRNA expression became clear around 72 hpf. MOs designed to inhibit zomp-1 and zotolin-1 mRNA translation, respectively, were injected into 1-2 cell stage embryos. zomp-1 MO caused a reduction in otolith size and an absence of zOtolin-1 deposition, while zotolin-1 MO caused a fusion of the two otoliths, and an increased instability of otoliths after fixation. We conclude that zOMP-1 is required for normal otolith growth and deposition of zOtolin-1 in the otolith, while zOtolin-1, a collagenous protein, is involved in the correct arrangement of the otoliths onto the sensory epithelium of the inner ear and probably in stabilization of the otolith matrix.

Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss.

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with "high activity CA" in the zebrafish, and its mRNA expression showed variation in the range 1.9-11.4 x 10(4) copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 x 10(4) copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.

Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata).

The mollusc shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are believed to play important roles in shell formation. In the present study, we extracted and purified a novel matrix protein, named Prismalin-14, from the acid-insoluble fraction of the prismatic layer of the shell of the Japanese pearl oyster (Pinctada fucata), and determined its whole amino acid sequence by a combination of amino acid sequence analysis and MS analysis of the intact protein and its enzymic digests. Prismalin-14 consisted of 105 amino acid residues, including PIYR repeats, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed inhibitory activity on calcium carbonate precipitation and calcium-binding activity in vitro. The scanning electron microscopy images revealed that Prismalin-14 affected the crystallization of calcium carbonate in vitro. A cDNA encoding Prismalin-14 was cloned and its expression was analysed. The amino acid sequence deduced from the nucleotide sequence of Prismalin-14 cDNA was identical with that determined by peptide sequencing. Northern-blot analysis showed that a Prismalin-14 mRNA was expressed only at the mantle edge. In situ hybridization demonstrated that a Prismalin-14 mRNA was expressed strongly in the inner side of the outer fold of the mantle. These results suggest that Prismalin-14 is a framework protein that plays an important role in the regulation of calcification of the prismatic layer of the shell.

NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation.

The ontogeny of haematopoietic niches in vertebrates is essentially unknown. Here we show that the stromal cells of the caudal haematopoietic tissue (CHT), the first niche where definitive haematopoietic stem/progenitor cells (HSPCs) home in zebrafish development, derive from the caudal somites through an epithelial-mesenchymal transition (EMT). The resulting stromal cell progenitors accompany the formation of the caudal vein sinusoids, the other main component of the CHT niche, and mature into reticular cells lining and interconnecting sinusoids. We characterize a zebrafish mutant defective in definitive haematopoiesis due to a deficiency in the nascent polypeptide-associated complex alpha subunit (NACA). We demonstrate that the defect resides not in HSPCs but in the CHT niche. NACA-deficient stromal cell progenitors initially develop normally together with the sinusoids, and HSPCs home to the resulting niche, but stromal cell maturation is compromised, leading to a niche that is unable to support HSPC maintenance, expansion and differentiation.

Artificial association of pre-stored information to generate a qualitatively new memory.

Memory is thought to be stored in the brain as an ensemble of cells activated during learning. Although optical stimulation of a cell ensemble triggers the retrieval of the corresponding memory, it is unclear how the association of information occurs at the cell ensemble level. Using optogenetic stimulation without any sensory input in mice, we found that an artificial association between stored, non-related contextual, and fear information was generated through the synchronous activation of distinct cell ensembles corresponding to the stored information. This artificial association shared characteristics with physiologically associated memories, such as N-methyl-D-aspartate receptor activity and protein synthesis dependence. These findings suggest that the association of information is achieved through the synchronous activity of distinct cell ensembles. This mechanism may underlie memory updating by incorporating novel information into pre-existing networks to form qualitatively new memories.