Platelet surface expression of stromal cell-derived factor-1 receptors CXCR4 and CXCR7 is associated with clinical outcomes in patients with coronary artery disease.

BACKGROUND: Surface expression of stromal cell-derived factor-1 (SDF-1, CXCL12) on platelets is enhanced during ischemic events and plays an important role in peripheral homing of stem cells and myocardial repair mechanisms. SDF-1 effects are mediated through CXCR4 and CXCR7. Both CXCR4 and CXCR7 are surface expressed on human platelets and to a higher degree in patients with coronary artery disease (CAD) compared with healthy controls. In this study, we investigated the prognostic role of platelet CXCR4- and CXCR7 surface expression in patients with symptomatic CAD. METHODS AND RESULTS: In a cohort study, platelet surface expression of CXCR4 and CXCR7 was measured by using flow cytometry in 284 patients with symptomatic CAD at the time of percutaneous coronary intervention (PCI). The primary combined end point was defined as all-cause death and/or myocardial infarction (MI) during 12-month follow-up. Secondary end points were defined as the single events of all-cause death and MI. We found significant differences of CXCR4 values in patients who developed a combined end point compared with event-free patients (mean MFIAUTHOR: Please define MFI at first use. 3.17 vs. 3.44, 95% confidence interval [CI] 0.09-0.45) and in patients who subsequently died (mean MFI 3.10 vs. 3.42, 95% CI 0.09-0.56). In multivariate Cox regression analysis, lower platelet CXCR4 levels were independently and significantly associated with all-cause mortality (hazard ratio 0.24, 95% CI 0.07-0.87) and the primary combined end point of all-cause death and/or MI (hazard ratio 0.30, 95% CI 0.13-0.72). CONCLUSION: These findings highlight a potential prognostic value of platelet expression CXCR4 on clinical outcomes in patients with CAD.

All functional aquaporin-4 isoforms are expressed in the rat cochlea and contribute to the formation of orthogonal arrays of particles.

The water channel aquaporin-4 (AQP4) is expressed in the cochlea and is essential for normal hearing. Unlike other AQPs, multiple isoforms of AQP4 have been reported in diverse tissues, three of which, M1, M23, and Mz, function as water channels. In addition, these protein isoforms are found in higher order complexes. Morphologically these higher order complexes correspond to orthogonal arrays of particles (OAPs) that are found in cell membranes by freeze fracture analysis. Using RT-PCR, quantitative PCR and blue-native PAGE immunoblots we identified all functional AQP4 isoforms -M1, M23, and Mz- and the formation of higher-order complexes in the organ of Corti of the rat. Complementary freeze-fracture studies revealed OAPs distributed in the lateral and basal membrane domains of the cochlear duct supporting cells, specifically Hensen's cells and outer sulcus cells. The unique inter- and intracellular heterogeneity in size, density and shape of OAPs suggests exceptional physiological requirements for the maintenance of water homeostasis during auditory sensory transduction in the cochlea.

Long-term engraftment of systemically transplanted, gene-modified bone marrow-derived cells in the adult mouse retina.

AIMS: To provide evidence for a novel route of gene administration to normal and diseased retinas, we performed systemic transplantation of genetically engineered bone marrow-derived cells (BMDCs) to wild-type mice and to mutant mice with retinal degeneration. METHODS: Lethally irradiated recipient mice-C57BL/6 (wild-type), SCA7 (spinocerebellar ataxia type 7) and FVB/N (rd1 mutant)-were transplanted intravenously with 5x106 BMDCs, which were transduced with a retroviral vector to express the enhanced green fluorescent protein (GFP). Chimeras were killed at 1, 3, 8, 11, 12 and 15 months (wild-type) or at 8 and 12 months (mutants) after transplantation. Eyes were enucleated, and the retinas were analysed using immunohistochemistry. RESULTS: In wild-type retinas, BMDCs preferentially engrafted in the inner and outer plexiform layers, the ganglion cell layer and the optic nerve. No BMDCs were found in the photoreceptor layer. BMDCs were more common in the degenerating retinas of the mutant mice. The majority of BMDCs in the retina were identified as microglia based on morphology and immunophenotype. Approximately 8-16% of all CD11b(+) cells in the retina expressed GFP. None of the BMDCs expressed neuronal cell markers. GFP-expressing BMDCs were found to persist for more than 1 year after transplantation. CONCLUSIONS: We demonstrate that gene-modified BMDCs show long-term engraftment and stable expression of GFP from a retrovirus in both wild-type and mutant mouse retinas. Thus, BMDCs may be used as vehicles for gene delivery to the retina.

Evidence for a columnar organization of cones, Müller cells, and neurons in the retina of a cichlid fish.

In the retina of many lower vertebrates, the arrangement of cells, in particular of cone photoreceptors, is highly regular. The data presented in this report show that in the retina of a cichlid fish (Astatotilapia burtoni) the regular arrangement is not restricted to cone photoreceptors and their synaptic terminals but can be found in elements of the inner retina as well. A variety of immunocytochemical and other markers was used in combination with confocal microscopy on whole-mount preparations and tangential sections. Nearest neighbor analysis was performed and density recovery profiles as auto- and cross-correlograms were generated. Cells displaying a regular arrangement of their synaptic processes in matching radial register to each other were identified for each major retinal neuronal cell type except ganglion cells (i.e. photoreceptors, horizontal cells, bipolar cells, and amacrine cells). The precise location of some of the corresponding cell bodies was not as regular but still non-random, however there was no spatial cross-correlation between cell bodies of different types. The radial processes of Müller glial cells displayed a distribution correlating to the arrangement of photoreceptors and neurons. Thus, for one Müller glial cell I found two PKC-positive cone bipolar cells, a spatially corresponding grid of parvalbumin-positive amacrine cell processes, one H1 horizontal cell, and two pairs of double cones. There was no evidence among ganglion cells matching this pattern, possibly due to the lack of suitable markers. Although many other cell types do not follow this matching regular mosaic arrangement, a basic columnar building block can be postulated for the retina at least in cichlid fish. This suggests a functional radial unit from photoreceptors to the inner plexiform layer.

Distribution and colocalization of markers for proliferation, invasion, motility and neoangiogenesis in benign melanocytic naevi and malignant melanomas.

BACKGROUND: Melanomas are heterogeneous tumours, and differentiation from other melanocytic lesions may cause problems. It may be possible that the distribution and/or colocalization pattern of different markers in the lesions can enable a more accurate diagnosis of melanocytic tumours. OBJECTIVES: To test this hypothesis, melanocytic naevi, primary melanomas and metastases were investigated. METHODS: The distribution and colocalization of markers for proliferation, invasion, angiogenesis and motility of the tumour cells were investigated using antibodies directed against actin, cathepsin B (CatB), transforming growth factor-beta, vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen/Ki-67 and basic fibroblast growth factor (FGF-2). In addition, melanoma markers (HMB-45 and Melan-A) and proteins unrelated to melanoma progression [epidermal growth factor (EGF) and cathepsin H] were investigated. RESULTS: Malignant melanomas tended to express more markers of malignancy compared with melanocytic naevi, and the differences were statistically significant for EGF and actin immunoreactivity: melanocytic naevi displayed clear EGF labelling more often (60% vs. 5%) and melanomas showed more intense actin labelling (70% vs. 0%). HMB-45+ cells to a large extent also stained with antibodies to CatB but not to EGF or actin; EGF-, FGF-2- and VEGF-immunoreactive cells were predominantly HMB-45-. Similar combinations were observed in melanocytic naevi and in melanomas. CONCLUSIONS: Labelling with EGF may improve the differential diagnosis of melanocytic neoplasias. However, we did not detect a clear-cut increase of markers of malignancy in melanoma. Cells expressing multiple malignancy markers were also found in some melanocytic naevi; this may confirm the dormant potential of melanocytic naevi for melanoma development.

Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4-2 and by serum- and glucocorticoid-dependent kinase 1.

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.

Cerebral localization and regulation of the cell volume-sensitive serum- and glucocorticoid-dependent kinase SGK1.

The serum- and glucocorticoid-dependent kinase SGK1 is regulated by alterations of cell volume, whereby cell shrinkage increases and cell swelling decreases the transcription, expression and activity of SGK1. The kinase is expressed in all human tissues studied including the brain. The present study was performed to localize the sites of SGK1 transcription in the brain, to elucidate the influence of the hydration status on SGK1 transcription and to explore the functional significance of altered SGK1 expression. Northern blot analysis of human brain showed SGK1 to be expressed in all cerebral structures examined: amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus and thalamus. In situ hybridization and immunohistochemistry in the rat revealed increased expression of SGK1 in neurons of the hippocampal area CA3 after dehydration, compared with similar slices from brains of euvolaemic rats. Additionally, several oligodendrocytes, a few microglial cells, but no astrocytes, were positive for SGK1. The abundance of SGK1 mRNA in the temporal lobe, including hippocampus, was increased by dehydration and SGK1 transcription in neuroblastoma cells was stimulated by an increase of extracellular osmolarity. Co-expression studies in Xenopus laevis oocytes revealed that SGK1 markedly increased the activity of the neuronal K+ channel Kv1.3. As activation of K+ channels modifies excitation of neuronal cells, SGK1 may participate in the regulation of neuronal excitability.

Pronounced infracuticular endocytosis in mammalian outer hair cells.

Endocytosis in cochlear hair cells was investigated by staining with the vital fluorescent dye FM 1-43, that partitions reversibly into membranes and is trapped in vesicles during endocytosis. The temporal development and spatial distribution of FM 1-43 induced fluorescence was investigated using confocal laser-scanning microscopy. FM 1-43 rapidly and intensely stained cochlear hair cells, leaving the supporting cells unstained. For short application (0.2-30 s), only the infracuticular region of outer hair cells (OHCs) was labeled, whereas for long application (30-60 s), the OHCs were also labeled in the infranuclear zone and along a central strand extending from the infracuticular zone down to the nucleus, as well as along the entire cell membrane. Except for the cell membrane, the infracuticular zone, directly below the cuticular plate, showed the most rapid and intense staining, and in most cases staining was spherically shaped with a diameter of 3-7 microm. Localization and size of this infracuticular staining coincided with Hensen's body, a specialized variant of the endoplasmic reticulum. In contrast to the OHCs, apical fluorescence of inner hair cells presented a homogeneous distribution. When OHCs were incubated in FM 1-43 for longer than 1 min, many points of contact between the central strand, the infracuticular zone and the lateral cell membrane were observed. Since Hensen's bodies are a specialty of OHCs and the fluorescent staining pattern of these cells was unique, it is proposed that Hensen's body is involved in the turnover of OHC-specific proteins, such as those involved in the molecular machinery of the motor action of the plasma membrane.

Inhibitory control of synaptic activity in goldfish Mb bipolar cell terminals visualized by FM1-43.

To investigate the physiology and plasticity of mixed rod-cone ON-bipolar cells (Mb) in the goldfish retina, we established a slice preparation which allows us to optically monitor the synaptic activity of bipolar cell axon terminals. We used the styryl dye FM1-43 which is incorporated into active axon terminals due to synaptic vesicle cycling and thus reflects synaptic activity. Different activity states of the axon terminals were revealed when slices prepared from light-adapted retinae were incubated in the presence of FM1-43 under various conditions. Depolarizing high K+ Ringer (50 mM) and the gamma-butyric acid (GABA) antagonist bicuculline (100 microM) resulted in more than two-fold increase in the number of stained terminals compared to slices stained in normal Ringer. In contrast, GABA treatment (0.5 mM) reduced the frequency of stained terminals. Thus, in light-adapted retinal slices the synaptic activity of Mb axon terminals can be modulated towards higher and lower activity states. The fact that the GABA antagonist bicuculline had similar effects as stimulatory high K+ Ringer suggests that inhibitory control is an important component in the regulation of synaptic activity and transmitter release in Mb terminals.

The embryogenesis of rod photoreceptors in the teleost fish retina, Haplochromis burtoni.

Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer (ONL) produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors.

Müller (glial) cells in the teleost retina: consequences of continuous growth.

In the continuously enlarging eye of teleost fishes retinal growth is achieved by the generation of new cells and by stretching the existing tissue. As a consequence of stretching, the density for most neurons decreases except for rod photoreceptors, which are produced by stem cells in the outer nuclear layer (ONL). We investigated retinal Müller glial cells with immunocytochemical markers against vimentin and glutamine synthetase in animals of various sizes. In addition, we used Western immunoblot analysis to investigate the changes in the glia-specific enzyme glutamine synthetase in the enlarging retina. We found that in the cichlid fish Haplochromis burtoni the density of Müller cells decreases from about 14 cells/mm2 to 4 cells/mm2 with increasing body size. Since it is known that the density of rod photoreceptors remains constant, it follows that the neuron to Müller cell ratio increases in the growing eye. In our estimates, this ratio ranges from around 54:1 in small fish to more than 67:1 in larger animals. Quantified Western blots revealed that the amount of glutamine synthetase per retinal area does not change in the growing eye, which means that the amount of this enzyme in each Müller cell must increase. Staining isolated cells and retinal sections from small and large fish with an antibody against glutamine synthetase showed stronger immunoreactivity in larger animals, especially in the areas of the photoreceptor cell bodies and outer limiting membrane and a more extensive branching of Müller cell processes. Thus, Müller cells in growing fish appear to compensate for the increasing metabolic challenge and are able to maintain their function.

Distribution of mitochondria within Müller cells--II. Post-natal development of the rabbit retinal periphery in vivo and in vitro: dependence on oxygen supply.

The occurrence and localization of mitochondria within glial (Müller) cells and neurons of the peripheral (avascular) rabbit retina was studied electron microscopically and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). Post-natal development in vivo was compared with development of organ cultures from neonatal rabbit retinae, grown over 2 weeks in vitro. The adult pattern of mitochondrial localization (restriction to the sclerad end of the cells) was observed from the beginning of enzyme expression at early post-natal stages. However, when neonatal retinal pieces were grown in vitro with their vitread surface exposed to the air, their Müller cells contained mitochondria along most of their length. When functionally developed retinae from postnatal day 14 were explanted in vitro, they retained their sclerad mitochondrial distribution for almost 24 h but thereafter the inner portions of their cytoplasm became occupied by mitochondria within a few hours. This was achieved mainly by mitochondrial migration rather than by formation of new mitochondria because it was not prevented by cycloheximide-induced inhibition of protein synthesis. These data support the following hypotheses: (1) the mitochondrial distribution in Müller cells is determined by the local cytoplasmic O2 pressure (pO2), (2) existing mitochondria move towards cytoplasmic regions of sufficient pO2 by rather rapid migration and (3) the start of this migration is delayed by almost 24 h due to the action of as yet unknown control mechanisms. In contrast, the mitochondrial content of retinal ganglion and amacrine cells in the vitread retinal layers was virtually independent of the source and level of oxygen supply.

Distribution of mitochondria within Müller cells--I. Correlation with retinal vascularization in different mammalian species.

The distribution of mitochondria within retinal glial (Müller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Müller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Müller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Müller cell processes. (3) Müller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Müller cells from the peripapillar myelinated region ('medullary rays') of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Müller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Müller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.

Cell movement and cell cycle dynamics in the retina of the adult teleost Haplochromis burtoni.

The authors analyzed the pattern of neurogenesis, the time frame of cell movement, and the cell cycle kinetics of a population of stem cells located in the outer nuclear layer in the retina of the adult teleost Haplochromis burtoni. These stem cells continue to give rise to new rod photoreceptors throughout life. The new rods move vitread after the last cell division. The authors investigated events during cell division and cell differentiation by using one marker that labels dividing cells transiently (proliferating cell nuclear antigen) along with another marker that labels dividing cells permanently (bromodeoxyuridine). The bulk of cell movement does not occur within 24 hours after S-phase labeling but is clearly underway 12 hours later, shortly after mitosis. The cell cycle length was estimated to be approximately 25 hours. The distribution of labeled cells at various times after S-phase suggests that new rods are generated by asymmetric cell division, that is, one of the daughter cells moves after mitosis and becomes postmitotic, while the other daughter cell remains in place and reenters the cell cycle. The proliferation patterns across the retina suggest that the location of areas of mitotic activity changes over time. The authors hypothesize that local extracellular factors control the rate of cell division in a given area, thereby keeping the overall rod density constant.

Development of the neonatal rabbit retina in organ culture. 1. Comparison with histogenesis in vivo, and the effect of a gliotoxin (alpha-aminoadipic acid).

Organ cultures from neonatal rabbit retinae grew well over periods of up to 2 weeks in vitro. Proliferation in vitro declined in parallel with the decline seen in vivo, although the rate of proliferation in the explants was slightly reduced. The proliferation of progenitor cells in vitro produced the same cell types produced postnatally in vivo. Postnatally generated cell clones, labeled by means of a retroviral vector, consisted mainly of rods and Müller cells. The layers of the retinae developed as in vivo; an outer plexiform layer occurred after the first 2 days in vitro. Ultrastructurally, ribbon synapses (outer and inner plexiform layer) and conventional synapses (inner plexiform layer) were observed. The photoreceptor cells grew well-developed inner segments and cilia but no mature outer segments. The cultured retinae contained a well-developed, regular lattice of Müller cells expressing vimentin as in vivo. The neuron-to-Müller cell-ratios were essentially the same as in vivo, viz. about 15 to 16 neurons, among them about 10 to 11 (rod) photoreceptor cells per Müller cell. When the glia cell-specific toxin alpha-aminoadipic acid (alpha AAA) was applied, the pattern of vimentin-positive Müller cells became irregular, or even locally missing. In such cases, the tissue became disorganized as indicated by a local disappearance of the regular layering, and development of many rosettes. It is concluded that an intact lattice of Müller cells is necessary for the migration of young neurons, and for correct formation of retinal layers.

Nonuniform distribution of cell proliferation in the adult teleost retina.

Teleost fish continue to grow throughout life, and their eyes enlarge correspondingly. Within the eye, the retina grows by stretching existing tissue and adding new cells. Cell addition occurs in two ways: First, all cell types except rod photoreceptors are added circumferentially at the edge of the eye where the retina meets the iris; second, rod photoreceptors are generated from a population of rod progenitor cells which divide throughout the outer nuclear layer (ONL). To determine the spatial distribution of rod progenitor cells across the teleost retina, we labeled dividing cells with an antibody to proliferating cell nuclear antigen (PCNA) throughout a 24 h period. We found a significantly higher density of dividing rod precursor cells at the nasal and temporal margins than in the central retina throughout the 24 h cycle. At night, the density of dividing cells is significantly greater at the nasal pole of the eye. The difference between cell division at the center and the margin was reduced at night when the density of cell division in the central retina increased significantly. Taken together, these data suggest that the eye grows asymmetrically, with more cells added at the nasal pole. Possible developmental causes and functional consequences of the reported distribution of cell divisions in time and location are presented.

A novel, rapid flat-mounting technique for visualizing antibody labeling in the retina.

Complete analysis of retinal tissue is difficult because it consists of a thin neural tissue spread across the back of a hemispheric surface. Conventional sectioning in a plane parallel to a central axis of symmetry produces a large number of samples, each containing only a small amount of the tissue of interest. Consequently, quantitative comparison of any feature of interest typically uses a small fraction of the sections from each retina, because analysis of the entire collection of sections is too time consuming. Such a sampling process can lead to misleading or erroneous conclusions. We present a new method which allows complete analysis of the retina using a small number of samples produced by sectioning flattened retinas. This procedure is straightforward as illustrated using an antibody against proliferating cell nuclear antigen (PCNA) to locate dividing cells in the teleost fish retina. Immunocytochemical staining on flat-sectioned retinas was quantified using a computer-based image analysis system. When the cells of interest are randomly distributed, conventional sampling procedures can seriously under- or over-estimate their number. The new technique presented allows significantly more efficient examination and quantification of the entire retina as compared to conventional techniques.

New rods move before differentiating in adult teleost retina.

To understand the cellular context of neuronal differentiation in the vertebrate retina, we analyzed the behavior of a class of progenitor cells in the outer nuclear layer of the teleost retina which divide throughout the animal's life and generate only rod photoreceptors. We present evidence that these progenitors reside adjacent to the outer limiting membrane of the retina during all phases of cell cycle. After final cell division, postmitotic cells move inward, toward the vitreal boundary of the outer nuclear layer, before they give rise to new rods. This movement is remarkable because it occurs in the mature, functioning retina. We hypothesize that only one of the two progenitor daughter cells moves while the other remains adjacent to the outer limiting membrane to divide again.

Localization and expression of insulin-like growth factor in the teleost retina.

Teleost fish retinas continue to add neurons throughout life, and evidence from in vitro experiments have implicated insulin-like growth factors (IGFs) in this process. To discover whether these factors are expressed in vivo, we have examined their expression in the cichlid fish, Haplochromis burtoni. Three lines of evidence show that IGFs are present in the fish retina. An IGF-I specific antibody, sm 1.2, binds preferentially to the retinal outer plexiform layer, in areas of cone photoreceptor synaptic endings. Northern blots of mRNA hybridized with riboprobes from trout IGF-I and IGF-II genes revealed transcripts of approximately 6.5 and 4.9 kb, respectively. The IGF-I probe detected an additional transcript of 1.2 kb in liver but not in retinal mRNA. In situ hybridization with digoxigenin-labeled riboprobes revealed that the IGF gene product is localized in the cone photoreceptors. These results show that cone photoreceptors are the source of IGFs in the fish retina, consistent with the hypothesis that IGFs play a role in regulating production of new neurons in the teleost retina.

Daily rhythm of cell proliferation in the teleost retina.

To determine whether the number of cell divisions in the teleost retina exhibited a regular daily variation, we labeled dividing cells with an antibody to proliferating cell nuclear antigen. The number of dividing rod precursor cells in the outer nuclear layer of the retina were counted in retinas from the telost fish Haplochromis burtoni, sacrificed at 4-h intervals during a standard light-dark cycle and in constant darkness. These rod precursor cells exhibited a striking rhythm of cell division. The highest number of cell divisions (acrophase) was found to occur at night when it was approximately 3 times higher than during the day. The observed rhythm persisted in animals held in constant darkness. We suggest that this endogenous 24-h rhythm of rod precursor cell division may be controlled by a circadian clock. Although there are several examples of continuously proliferating cell populations which exhibit circadian or diurnal rhythms, this appears to be the first documentation of a rhythm of division in cells destined to become neurons.