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Nax is a brain [Na+] sensor expressed in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT) in the brain. We previously demonstrated that Nax signals are involved in the control of water intake behavior through the Nax/TRPV4 pathway. Nax gene knockout mice showed significantly attenuated water intake after an intracerebroventricular (ICV) injection of a hypertonic NaCl solution; however, the induction of a certain amount of water intake still remained, suggesting that another unknown [Na+]-dependent pathway besides the Nax/TRPV4 pathway contributes to water intake. In the present study, we screened for novel [Na+] sensors involved in water intake control and identified SLC9A4 (also called sodium (Na+)/hydrogen (H+) exchanger 4 (NHE4)). SLC9A4 is expressed in angiotensin II (Ang II) receptor type 1a (AT1a)-positive neurons in the OVLT. Sodium-imaging experiments using cultured cells transfected with slc9a4 revealed that SLC9A4 was activated by increases in extracellular [Na+] ([Na+]o), but not osmolality. Moreover, the firing activity of SLC9A4-positive neurons was enhanced by increases in [Na+]o and Ang II. slc9a4 knockdown in the OVLT reduced water intake induced by increases in [Na+], but not osmolality, in the cerebrospinal fluid. ICV injection experiments of a specific inhibitor suggested that the increase in extracellular [H+] caused by SLC9A4 activation next stimulates acid-sensing channel 1a (AS1C1a) to induce water intake. Our results thus indicate that SLC9A4 in the OVLT functions as a [Na+] sensor for the control of water intake and that the SLC9A4 signal is independent of the Nax/TRPV4 pathway.

Nax is a [Na+] sensor expressed in specific glial cells in the sensory circumventricular organs (sCVOs) in the brain. We recently demonstrated that Nax signals are involved in the control of not only salt intake but also water intake behavior. Our pharmacological experiments suggested that Nax signals led to activation of neurons bearing TRPV4 by using epoxyeicosatrienoic acids (EETs) as gliotransmitters to stimulate water intake. In the present study, we performed selective lesions of individual sCVOs in wild-type (WT) mice and the site-directed rescue of Nax expression in Nax-gene knockout (Nax-KO) mice. These experiments revealed that the Nax channel in the organum vasculosum laminae terminalis (OVLT) functions as a [Na+] sensor for the control of water intake behavior. Direct measurements of 5,6-EET and 8,9-EET in the OVLT demonstrated that EET levels were indeed increased two-fold by water deprivation for two days in WT, but not Nax-KO mice, indicating that EETs were Nax-dependently produced in the OVLT in response to increases in [Na+] in body fluids. More importantly, intracerebroventricular injection of 5,6-EET at the same level was effective to induce water intake. Double staining revealed that Nax-positive cells also expressed Cyp2c44, a cytochrome P450 epoxygenase, to generate EETs. Collectively, these results indicate that Nax-positive glial cells produce EETs to activate TRPV4-positive neurons which may stimulate water intake, in response to increases in [Na+] of body fluids.

Congenital nystagmus, involuntary oscillating small eye movements, is commonly thought to originate from aberrant interactions between brainstem nuclei and foveal cortical pathways. Here we investigated whether nystagmus associated with congenital stationary nightblindness (CSNB) can result from primary deficits in the retina. We found that CSNB patients as well as an animal model (nob mice), both of which lack functional nyctalopin protein (NYX, nyx) in ON bipolar cells (ON-BC) at their synapse with photoreceptors, showed oscillating eye movements at a frequency of 4-7Hz. nob ON direction selective ganglion cells (ON-DSGC), which detect global motion and project to the accessory optic system (AOS), oscillated with the same frequency as their eyes. In the dark, individual ganglion cells (GC) oscillated asynchronously, but their oscillations became synchronized by light stimulation. Likewise, both patient and nob mice oscillating eye movements were only present in the light. Retinal pharmacological manipulations that blocked nob ON-DSGC oscillations also eliminated their oscillating eye movements, and retinal pharmacological manipulations that reduced oscillation frequency of nob ON-DSGCs also reduced oscillation frequency of their eye movements. We conclude that, in nob mice, oscillations of retinal ON-DSGCs cause nystagmus with properties similar to those associated with CSNB in humans. These results show that the nob mouse is the first animal model for a form of congenital nystagmus paving the way for development of therapeutic strategies.

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Congenital nystagmus, involuntary oscillating small eye movements, is commonly thought to originate from aberrant interactions between brainstem nuclei and foveal cortical pathways. Here, we investigated whether nystagmus associated with congenital stationary night blindness (CSNB) results from primary deficits in the retina. We found that CSNB patients as well as an animal model (nob mice), both of which lacked functional nyctalopin protein (NYX, nyx) in ON bipolar cells (BCs) at their synapse with photoreceptors, showed oscillating eye movements at a frequency of 4–7 Hz. nob ON direction-selective ganglion cells (DSGCs), which detect global motion and project to the accessory optic system (AOS), oscillated with the same frequency as their eyes. In the dark, individual ganglion cells (GCs) oscillated asynchronously, but their oscillations became synchronized by light stimulation. Likewise, both patient and nob mice oscillating eye movements were only present in the light when contrast was present. Retinal pharmacological and genetic manipulations that blocked nob GC oscillations also eliminated their oscillating eye movements, and retinal pharmacological manipulations that reduced the oscillation frequency of nob GCs also reduced the oscillation frequency of their eye movements. We conclude that, in nob mice, synchronized oscillations of retinal GCs, most likely the ON-DCGCs, cause nystagmus with properties similar to those associated with CSNB in humans. These results show that the nob mouse is the first animal model for a form of congenital nystagmus, paving the way for development of therapeutic strategies., Nystagmus (involuntary eye movements) in cases of congenital stationary night blindness arises from light-induced synchronization of oscillating retinal ganglion cells, revealing that some forms of nystagmus can have a retinal origin, rather than originating from aberrant interactions between brainstem nuclei and foveal cortical pathways, as commonly thought.

Increases in sodium concentrations ([Na+]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on increased [Na+] is translated to SNA have not yet been elucidated. We herein reveal that sympathetic activation leading to BP increases is not induced by mandatory high salt intakes or the intraperitoneal/intracerebroventricular infusions of hypertonic NaCl solutions in Nax-knockout mice in contrast to wild-type mice. We identify Nax channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT) as the sensors detecting increases in [Na+] in body fluids and show that OVLT neurons projecting to the paraventricular nucleus (PVN) are activated via acid-sensing ion channel 1a (ASIC1a) by H+ ions exported from Nax-positive glial cells. The present results provide an insight into the neurogenic mechanisms responsible for salt-induced BP elevations.

Growth cones at the tip of growing axons are key cellular structures that detect guidance cues and mediate axonal growth. An increasing number of studies have suggested that the dynamic regulation of microtubules in the growth cone plays an essential role in growth cone steering. The dynamic properties of microtubules are considered to be regulated by variegated cellular factors but, in particular, through microtubule-interacting proteins. Here, we examined the functional role of adenomatous polyposis coli-like molecule 2 (APC2) in the development of axonal projections by using the chick retinotectal topographic projection system. APC2 is preferentially expressed in the nervous system from early developmental stages through to adulthood. Immunohistochemical analysis revealed that APC2 is distributed along microtubules in growth cones as well as axon shafts of retinal axons. Overexpression of APC2 in cultured cells induced the stabilization of microtubules, whereas the knockdown ofAPC2in chick retinas with specific short hairpin RNA reduced the stability of microtubules in retinal axons.APC2 knockdown retinal axons showed abnormal growth attributable to a reduced response to ephrin-A2in vitro. Furthermore, they showed drastic alterations in retinotectal projections without making clear target zones in the tectumin vivo. These results suggest that APC2 plays a critical role in the development of the nervous system through the regulation of microtubule stability.

Owing to its external position in the embryo, the chick eye has been used as a readily accessible model for studying the molecular mechanisms behind the patterning of the central nervous system. Although methods of genetic analysis have not been established as in the mouse, the chick is convenient for analyzing the functions of genes by in ovo electroporation of retroviral vectors. In this review, we describe the retroviral vector-mediated transfer of genes into the chick optic vesicle by in ovo electroporation. A rapid, efficient, and sustained expression of transgenes is achieved by this approach.

Water-intake behavior is under the control of brain systems that sense body fluid conditions at sensory circumventricular organs (sCVOs); however, the underlying mechanisms have not yet been elucidated in detail. Nax is a sodium (Na+) level sensor in the brain, and the transient receptor potential vanilloid (TRPV) channels TRPV1 and TRPV4 have been proposed to function as osmosensors. We herein investigated voluntary water intake immediately induced after an intracerebroventricular administration of a hypertonic NaCl solution in TRPV1-, TRPV4-, Na x-, and their double-gene knockout (KO) mice. The induction of water intake by TRPV1-KO mice was normal, whereas intake by TRPV4-KO and Na x-KO mice was significantly less than that by WT mice. Water intake by Na x /TRPV4-double KO mice was similar to that by the respective single KO mice. When TRPV4 activity was blocked with a specific antagonist HC-067047, water intake by WT mice was significantly reduced, whereas intake by TRPV4-KO and Na x-KO mice was not. Similar results were obtained with the administration of miconazole, which inhibits the biosynthesis of epoxyeicosatrienoic acids (EETs), endogenous agonists for TRPV4, from arachidonic acid (AA). Intracerebroventricular injection of hypertonic NaCl with AA or 5,6-EET restored water intake by Na x-KO mice to the wild-type level but not that by TRPV4-KO mice. These results suggest that the Na+ signal generated in Nax-positive glial cells leads to the activation of TRPV4-positive neurons in sCVOs to stimulate water intake by using EETs as gliotransmitters. Intracerebroventricular injection of equiosmolar hypertonic sorbitol solution induced small but significant water intake equally in all the genotypes, suggesting the presence of an unknown osmosensor in the brain.

It has been long believed that the anteroposterior (A-P) and dorsoventral (D-V) axes in the developing retina are determined independently and also that the retinotectal projection along the two axes is controlled independently. However, we recently demonstrated that misexpression ofVentroptin,a bone morphogenic protein (BMP) antagonist, in the developing chick retina alters the retinotectal projection not only along the D-V (or mediolateral) axis but also along the A-P axis. Moreover, the dorsal-high expression ofBMP4is relieved by the dorsotemporal-high expression ofBMP2at embryonic day 5 (E5) in the retina, during whichVentroptincontinuously counteracts the two BMPs keeping on the countergradient expression pattern, respectively. Here, we show that the topographic molecules so far reported to have a gradient only along the D-V axis and ephrin-A2 so far only along the A-P axis are both controlled by the BMP signal, and that they are expressed in a gradient manner along the tilted axis from E6 on in the developing chick retina: the expression patterns of these oblique-gradient molecules are all changed, whenBMP2expression is manipulated in the developing retina. Furthermore, in bothBMP2knockdown embryos andephrin-A2-misexpressed embryos, the retinotectal projection is altered along the two orthogonal axes. The expressional switching fromBMP4toBMP2thus appears to play a key role in the retinal patterning and topographic retinotectal projection by tilting the D-V axis toward the posterior side during retinal development. Our results also indicate thatBMP2expression is essential for the maintenance of regional specificity along the revised D-V axis.

Eph receptors are activated by the autophosphorylation of tyrosine residues upon the binding of their ligands, the ephrins; however, the protein tyrosine phosphatases (PTPs) responsible for the negative regulation of Eph receptors have not been elucidated. Here, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that efficiently dephosphorylates both EphA and EphB receptors as substrates. Biochemical analyses revealed that Ptpro dephosphorylates a phosphotyrosine residue conserved in the juxtamembrane region, which is required for the activation and signal transmission of Eph receptors. Ptpro thus seems to moderate the amount of maximal activation of Eph receptors. Using the chick retinotectal projection system, we show that Ptpro controls the sensitivity of retinal axons to ephrins and thereby has a crucial role in the establishment of topographic projections. Our findings explain the molecular mechanism that determines the threshold of the response of Eph receptors to ephrins in vivo.

To understand the molecular basis of topographic retinotectal projection, an overall view of the asymmetrically expressed molecules in the developing retina is needed. We performed a large-scale screening using restriction landmark cDNA scanning (RLCS) in the embryonic day 8 (E8) chick retina. RLCS is a cDNA display system, in which a large number of cDNA species are displayed as two-dimensional spots with intensities reflecting their expression levels as mRNA. We searched for spots that gave different signal intensities between the nasal and temporal retinas or between the dorsal and ventral retinas, and detected about 200 spots that were preferential on one side in the retina. The asymmetric expression of each gene was verified by Northern blotting and in situ hybridization. By subsequent analyses using molecular cloning, DNA sequencing, and database searching, 33 asymmetric molecules along the nasotemporal (N-T) axis and 20 along the dorsoventral (D-V) axis were identified. These included transcription factors, secretory factors, transmembrane proteins, and intracellular proteins with various putative functions. Their expression profiles revealed by in situ hybridization are highly diverse and individual. Moreover, many of them begin to be expressed in the retina from the early developmental stages, suggesting that they are implicated in the establishment and maintenance of regional specificity in the developing retina. The molecular repertoire revealed by this work will provide candidates for future studies to elucidate the molecular mechanisms of topographic retinotectal map formation.

In the developing retina, a retinoic acid (RA) gradient along the dorso-ventral axis is believed to be a prerequisite for the establishment of dorso-ventral asymmetry. This RA gradient is thought to result from the asymmetrical distribution of RA-generating aldehyde dehydrogenases along the dorso-ventral axis. Here, we identified a novel aldehyde dehydrogenase specifically expressed in the chick ventral retina, using restriction landmark cDNA scanning (RLCS). Since this molecule showed enzymatic activity to produce RA from retinaldehyde, we designated it retinaldehyde dehydrogenase 3 (RALDH-3). Structural similarity suggested that RALDH-3 is the orthologue of human aldehyde dehydrogenase 6. We also isolated RALDH-1 which is expressed in the chick dorsal retina and implicated in RA formation. Raldh-3 was preferentially expressed first in the surface ectoderm overlying the ventral portion of the prospective eye region and then in the ventral retina, earlier than Raldh-1 in chick and mouse embryos. High level expression of Raldh-3 was also observed in the nasal region. In addition, we found that Pax6 mutants are devoid of Raldh-3 expression. These results suggested that Raldh-3 is the key enzyme in the formation of an RA gradient along the dorso-ventral axis during the early eye development, and also in the development of the olfactory system.

We previously identified SPARC-related protein-containing immunoglobulin domains 1 (SPIG1, also known as Follistatin-like protein 4) as one of the dorsal-retina-specific molecules expressed in the developing chick retina. We here demonstrated that the knockdown ofSPIG1in the retinal ganglion cells (RGCs) of developing chick embryos induced the robust ectopic branching of dorsal RGC axons and failed to form a tight terminal zone at the proper position on the tectum. The knockdown ofSPIG1in RGCs also led to enhanced axon branchingin vitro. However, this was canceled by the addition of a neutralizing antibody against brain-derived neurotrophic factor (BDNF) to the culture medium. SPIG1 and BDNF were colocalized in vesicle-like structures in cells. SPIG1 bound with the proform of BDNF (proBDNF) but very weakly with mature BDNFin vitro. The expression and secretion of mature BDNF were significantly decreased when SPIG1 was exogenously expressed with BDNF in HEK293T or PC12 cells. The amount of mature BDNF proteins as well as the tyrosine phosphorylation level of the BDNF receptor, tropomyosin-related kinase B (TrkB), in the hippocampus were significantly higher inSPIG1-knockout mice than in wild-type mice. Here the spine density of CA1 pyramidal neurons was consistently increased. Together, these results suggest that SPIG1 negatively regulated BDNF maturation by binding to proBDNF, thereby suppressing axonal branching and spine formation.

T lymphocytes recirculate continually through the T cell areas of peripheral lymph nodes. During each passage, the T cells survey the surface of large dendritic cells (DCs), also known as interdigitating cells. However, these DCs have been difficult to release from the lymph node. By emphasizing the use of calcium-free media, as shown by Vremec et al. (Vremec, D., M. Zorbas, R. Scollay, D.J. Saunders, C.F. Ardavin, L. Wu, and K. Shortman. 1992. J. Exp. Med. 176:47–58.), we have been able to release and enrich DCs from the T cell areas. The DCs express the CD11c leukocyte integrin, the DEC-205 multilectin receptor for antigen presentation, the intracellular granule antigens which are recognized by monoclonal antibodies M342, 2A1, and MIDC-8, very high levels of MHC I and MHC II, and abundant accessory molecules such as CD40, CD54, and CD86. When examined with the Y-Ae monoclonal which recognizes complexes formed between I-Ab and a peptide derived from I-Eα, the T cell area DCs expressed the highest levels. The enriched DCs also stimulated a T-T hybridoma specific for this MHC II–peptide complex, and the hybridoma underwent apoptosis. Therefore DCs within the T cell areas can be isolated. Because they present very high levels of self peptides, these DCs should be considered in the regulation of self reactivity in the periphery.

Body-fluid homeostasis is essential to life, and the concentration of Na + ([Na + ]) and osmolality in plasma and the cerebrospinal fluid (CSF) are continuously monitored in the brain. To maintain a physiological level of Na/osmolality in body fluids, the control of Na and water intake and excretion are of prime importance. Two independent sensing systems for [Na + ] and osmolality in circumventricular organs (CVOs) have long been postulated to be involved in the monitoring of body-fluid conditions. In the past decade, several molecules were reported as promising candidates for these sensors – Na x for the [Na + ] sensor and transient receptor potential (TRP) channels for the osmosensor. This review presents a summary of developments in these areas over recent years.

We investigated the effect of type I IFNs (IFN-alpha and IFN-beta) on IFN-gamma-induced nitric oxide (NO) production by murine peritoneal macrophages. It was found that exogenous and also endogenous type I IFNs suppressed IFN-gamma-induced NO production, cytosolic inducible NO synthase (iNOS) activity, and iNOS mRNA accumulation in macrophages. Furthermore, we show here that type I IFNs prevent the NO-mediated deterioration of mitochondrial respiratory activity in macrophages. These results seem to indicate a possible protective role of type I IFNs against the NO-mediated immunosuppressive and/or cytotoxic effect of macrophages.

The present study was performed to investigate the effect of beta- endorphin on macrophage colony-stimulating factor (M-CSF)-induced differentiation of macrophages from bone marrow cells in a semisolid culture system. beta-endorphin increased the number of macrophage colonies when bone marrow cells were cultured in the presence of M-CSF plus lipopolysaccharide (LPS). This was not the case with LPS- unresponsive C3H/HeJ mouse bone marrow cells. alpha-endorphin and gamma- endorphin were as effective as beta-endorphin in enhancing the colony formation. Exogenous interleukin-1 (IL-1), but neither IL-6 nor tumor necrosis factor (TNF), collaborated with beta-endorphin even in the absence of LPS, suggesting that IL-1 is a primary mediator of the effect of LPS. Indeed, anti-IL-1 antibody abolished the collaborative effect of beta-endorphin with LPS. Moreover, IL-1 was effective even for C3H/HeJ mouse bone marrow cells. Naloxone, an antagonist of endorphins for opioid-receptors, completely abrogated the effect of beta-endorphin. In a single-cell culture system, the collaboration between beta-endorphin and IL-1 was revealed by the increase in number and size of macrophage colonies, but collaboration between beta- endorphin and LPS did not occur. These results indicate that, in mixed cell culture, beta-endorphin acts in concert with paracrinal IL-1 induced by LPS to enhance M-CSF-dependent macrophage differentiation from immature precursor cells.

B7-2 is a recently discovered, second ligand for the CTLA-4/CD28, T cell signaling system. Using the GL-1 rat monoclonal antibody (mAb), we monitored expression of B7-2 on mouse leukocytes with an emphasis on dendritic cells. By cytofluorography, little or no B7-2 was detected on most cell types isolated from spleen, thymus, peritoneal cavity, skin, marrow, and blood. However, expression of B7-2 could be upregulated in culture. In the case of epidermal and spleen dendritic cells, which become highly immunostimulatory for T cells during a short period of culture, the upregulation of B7-2 was dramatic and did not require added stimuli. Lipopolysaccharide did not upregulate B7-2 levels on dendritic cells, in contrast to macrophages and B cells. By indirect immunolabeling, the level of staining with GL-1 mAb exceeded that seen with rat mAbs to several other surface molecules including intercellular adhesion molecule 1, B7-1, CD44, and CD45, as well as new hamster mAbs to CD40, CD48, and B7-1/CD80. Of these accessory molecules, B7-2 was a major species that increased in culture, implying a key role for B7-2 in the functional maturation of dendritic cells. B7-2 was the main (> 90%) CTLA-4 ligand on mouse dendritic cells. When we applied GL-1 to tissue sections of a dozen different organs, clear-cut staining with B7-2 antigen was found in many. B7-2 staining was noted on liver Kupffer cells, interstitial cells of heart and lung, and profiles in the submucosa of the esophagus. B7-2 staining was minimal in the kidney and in the nonlymphoid regions of the gut, and was not observed at all in the brain. In the tongue, only rare dendritic cells in the oral epithelium were B7-2+, but reactive cells were scattered about the interstitial spaces of the muscle. In all lymphoid tissues, Gl-1 strongly stained certain distinct regions that are occupied by dendritic cells and by macrophages. For dendritic cells, these include the thymic medulla, splenic periarterial sheaths, and lymph node deep cortex; for macrophages, the B7-2-rich regions included the splenic marginal zone and lymph node subcapsular cortex. Splenic B7-2+ cells were accessible to labeling with GL-1 mAb given intravenously. Dendritic cell stimulation of T cells (DNA synthesis) during the mixed leukocyte reaction was significantly (35-65%) blocked by GL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Two winged-helix transcription factors, FoxG1 (previously called chick brain factor1, CBF1) and FoxD1 (chick brain factor2, CBF2), are expressed specifically in the nasal and temporal regions of the developing chick retina, respectively. We previously demonstrated that FoxG1 controls the expression of topographic molecules including FoxD1, and determines the regional specificity of the nasal retina. FoxD1 is known to prescribe temporal specificity, however, molecular mechanisms and downstream targets have not been elucidated. Here we addressed the genetic mechanisms for establishing temporal specificity in the developing retina using an in ovo electroporation technique. Fibroblast growth factor (Fgf) and Wnt first play pivotal roles in inducing the region-specific expression of FoxG1 and FoxD1 in the optic vesicle. Misexpression of FoxD1 represses the expression of FoxG1, GH6, SOHo1, and ephrin-A5, and induces that of EphA3 in the retina. GH6 and SOHo1 repress the expression of FoxD1. In contrast to the inhibitory effect of FoxG1 on bone morphogenic protein (BMP) signaling, FoxD1 does not alter the expression of BMP4 or BMP2. Studies with chimeric mutants of FoxD1 showed that FoxD1 acts as a transcription repressor in controlling its downstream targets in the retina. Taken together with previous findings, our data suggest that FoxG1 and FoxD1 are located at the top of the gene cascade for regional specification along the nasotemporal (anteroposterior) axis in the retina, and FoxD1 determines temporal specificity.

Topographic maps are a fundamental feature of neural networks in the nervous system. Understanding the molecular mechanisms by which topographically ordered neuronal connections are established during development has long been a major challenge in developmental neurobiology. The retinotectal projection of lower vertebrates including birds has been used as a readily accessible model system. In this projection, the temporal (posterior) retina is connected to the rostral (anterior) part of the contralateral optic tectum, the nasal (anterior) retina to the caudal (posterior) tectum, and likewise the dorsal and ventral retina to the ventral (lateral) and dorsal (medial) tectum, respectively. Thus, images received by the retina are precisely projected onto the tectum in a reversed manner. For the formation of topographic maps, molecular gradients in origin and targets are essential. To search for topographic molecules in the embryonic retina, we performed a large-scale screening and successfully identified a variety of molecules with various asymmetrical expression patterns along both axes in the developing retina. Included were many novel molecules with unknown functions, together with known molecules. Through analyses of these molecules, we can now present gene cascades for the retinal patterning and for the establishment of topographic retinotectal projection. In addition, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that regulates Eph receptors. We show that Ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays crucial roles in the topographic projection.

To understand the molecular basis of topographic retinotectal projection, an overall view of the asymmetrically expressed molecules in the developing retina is needed. We performed a large-scale screening using restriction landmark cDNA scanning (RLCS) in the embryonic day 8 (E8) chick retina. RLCS is a cDNA display system, in which a large number of cDNA species are displayed as two-dimensional spots with intensities reflecting their expression levels as mRNA. We searched for spots that gave different signal intensities between the nasal and temporal retinas or between the dorsal and ventral retinas, and detected about 200 spots that were preferential on one side in the retina. The asymmetric expression of each gene was verified by Northern blotting and in situ hybridization. By subsequent analyses using molecular cloning, DNA sequencing, and database searching, 33 asymmetric molecules along the nasotemporal (N-T) axis and 20 along the dorsoventral (D-V) axis were identified. These included transcription factors, secretory factors, transmembrane proteins, and intracellular proteins with various putative functions. Their expression profiles revealed by in situ hybridization are highly diverse and individual. Moreover, many of them begin to be expressed in the retina from the early developmental stages, suggesting that they are implicated in the establishment and maintenance of regional specificity in the developing retina. The molecular repertoire revealed by this work will provide candidates for future studies to elucidate the molecular mechanisms of topographic retinotectal map formation.

In the visual system, the establishment of the anteroposterior and dorsoventral axes in the retina and tectum during development is important for topographic retinotectal projection. We identified chick Ventroptin, an antagonist of bone morphogenetic protein 4 (BMP-4), which is mainly expressed in the ventral retina, not only with a ventral high–dorsal low gradient but also with a nasal high–temporal low gradient at later stages. Misexpression of Ventroptin altered expression patterns of several topographic genes in the retina and projection of the retinal axons to the tectum along both axes. Thus, the topographic retinotectal projection appears to be specified by the double-gradient molecule Ventroptin along the two axes.

The effect of calcitonin gene-related peptide (CGRP) was investigated on cytokine-induced IL-9 production by Swiss 3T3 fibroblasts. CGRP by itself had little effect on IL-6 production, and substantially enhanced that induced by IL-1 beta or TNF alpha at concentrations of 10(-8) M or higher. The action of CGRP was mediated by a CGRP-specific receptor. In contrast, calcitonin had no effect at all. A kinetic study of IL-6 activity in the culture supernatant showed that CGRP not only accelerated but also increased IL-6 production. Enhancement by CGRP of cytokine-dependent IL-6 production was ascribed to the increased accumulation of IL-6 mRNA. IL-1 beta-induced IL-6 production was maintained by CGRP after removal of IL1 beta. The addition of CGRP to the culture following stimulation with IL-1 beta or IL-1 beta plus CGRP decelerated the decay of IL-6 mRNA. These results indicate that the augmenting effect of CGRP is mediated, at least in part, by the stabilization of IL-6-specific mRNA.