Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a ... more Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a ''cone mosaic.'' Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish (Danio rerio) have four cone subtypes arranged into precise rows in the adult retina.
A cDNA full length encoding a putative ultraviolet (UV)-sensitive visual pigment of goldfish was ... more A cDNA full length encoding a putative ultraviolet (UV)-sensitive visual pigment of goldfish was isolated. The deduced amino acid sequence shows 64% identity to those of human blue and chicken violet, and less identity (40-49%) to those of other vertebrate visual pigment. The mRNA is localized in the miniature short single cone cells, which are known to have a sensitivity maximum in the near UV-region.
Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neur... more Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neurogenesis allows a vigorous regenerative response to damage, and fish can regenerate retinal neurons, including photoreceptors, and restore functional vision following photic, chemical, or mechanical destruction of the retina. Müller glial cells in fish function as radial-glial-like neural stem cells. During adult growth, Müller glial nuclei undergo sporadic, asymmetric, self-renewing mitotic divisions in the inner nuclear layer to generate a rod progenitor that migrates along the radial fiber of the Müller glia into the outer nuclear layer, proliferates, and differentiates exclusively into rod photoreceptors. When retinal neurons are destroyed, Müller glia in the immediate vicinity of the damage partially and transiently dedifferentiate, re-express retinal progenitor and stem cell markers, re-enter the cell cycle, undergo interkinetic nuclear migration (characteristic of neuroepithelial ...
Methods in molecular biology (Clifton, N.J.), 2012
Microarray-based gene profiling has become an important technique to measure changes in gene expr... more Microarray-based gene profiling has become an important technique to measure changes in gene expression on a genome-wide scale. Recently, cell-specific microarrays have been reported to study changes in gene expression of a particular cell type in several model organisms. Here, we describe a protocol to prepare RNA samples for microarray analysis of isolated Müller glia-derived retinal stem cells from light-damaged adult zebrafish expressing a fluorescent marker in Müller cells using enzymatic retinal dissociation followed by fluorescence-activated cell sorting (FACS).
The persistence in adult teleost fish of retinal stem cells that exhibit all of the features of t... more The persistence in adult teleost fish of retinal stem cells that exhibit all of the features of true 'adult stem cells'--self-renewal, multipotency, and the capacity to respond to injury by mitotic activation with the ability to regenerate differentiated tissues--has been known for several decades. However, the specialized cellular and molecular characteristics of these adult retinal stem cells and the microenvironmental niches that support their maintenance in the differentiated retina and regulate their activity during growth and regeneration have not yet been elucidated. Our data show that the zebrafish retina has two kinds of specialized niches that sustain retinal stem cells: 1) a neuroepithelial germinal zone at the interface between neural retina and ciliary epithelium, called the ciliary marginal zone (CMZ), a continuous annulus around the retinal circumference, and 2) the microenvironment around some Müller glia in the differentiated retina. In the uninjured retina,...
To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated... more To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated cones after laser ablation of photoreceptors in adult goldfish retina. Using a technique developed previously in this laboratory, photoreceptors in the retina of adult goldfish were ablated with an argon laser. The mitotic marker, bromodeoxyuridine, was used to label proliferating and regenerated cells, which were identified with cell-specific markers. Cells proliferating locally within lesion included microglia, Müller glia, and retinal progenitors in the inner nuclear layer (INL). The nuclei of both Müller glia and associated retinal progenitors migrated from the inner to the outer nuclear layer. The proliferating retinal progenitors, which express Notch-3 and N-cadherin, regenerated cone photoreceptors and then rod photoreceptors. Previous work has demonstrated that photoreceptors in the goldfish retina regenerate selectively after laser ablation, but the source of regenerated cones ...
To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specif... more To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specifically in photoreceptors and pinealocytes, regulate photoreceptor gene expression, are necessary for normal photoreceptor differentiation, and when mutated cause a variety of photoreceptor degenerations. A zebrafish retinal cDNA library was screened with a human CRX cDNA probe. Radiation hybrid mapping, Northern blot analysis, in situ hybridization, and transient transfection studies were performed using standard methods. Based on amino acid sequence comparisons, zebrafish crx shows 50% identity with human CRX, and 85% identity in the homeodomain. A phylogenetic analysis indicates that zebrafish crx is most closely related to the mammalian Crx proteins, and more distantly related to the Otx proteins. Zebrafish crx maps between 49.6 and 54.5 cm from the top of linkage group LG05C, a map position consistent with the location of the mouse and human CRX genes. Northern blot analysis and in s...
Results and problems in cell differentiation, 2000
... Or filter your current search. Raymond PA, Find all citations by this author (default). Or fi... more ... Or filter your current search. Raymond PA, Find all citations by this author (default). Or filter your current search. Hitchcock PF. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor 48109-0616, USA. ...
To examine expression of the zebrafish ultraviolet cone opsin pigment in goldfish and zebrafish r... more To examine expression of the zebrafish ultraviolet cone opsin pigment in goldfish and zebrafish retinas. Digoxigenin-labeled cRNA probes were prepared by run-off transcription from plasmids containing cDNAs for zebrafish ultraviolet opsin, goldfish ultraviolet cone opsin, and goldfish rod opsin. Probes were hybridized to cryosections of retina and visualized with immunocytochemistry. The zebrafish ultraviolet opsin probe hybridized selectively to rod photoreceptors, but not to ultraviolet cones or any other cone type, in both zebrafish and goldfish retinas, and the pattern of expression was identical to that of the goldfish rod opsin probe. The goldfish ultraviolet opsin, in contrast, hybridized to ultraviolet cone photoreceptors in both goldfish and zebrafish. The cDNA previously identified by Robinson et al as zebrafish ultraviolet opsin is not a cone opsin but is likely to be a rod opsin.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1983
The growth and morphology of the optic tectum of adult goldfish were studied with light and elect... more The growth and morphology of the optic tectum of adult goldfish were studied with light and electron microscopy and with thymidine radioautography. The tectum is roughly hemispheric in shape, with a smaller radius of curvature rostrally than caudally. A narrow region containing proliferating cells (the germinal zone) is found along two-thirds of the rim of the tectal hemisphere but is absent rostrally, adjacent to the tectal region which receives input from the rostral visual field. New cells generated in the germinal zone are added to the tectum appositionally in crescent-shaped increments; these was no evidence of migration of new cells into the rostral region which lacks a germinal zone. Some of the new cells added to the adult tectum were shown to be neurons on the basis of cytological and ultrastructural features. Counts of tectal neurons likewise demonstrated that new cells were added with growth of the tectum; large goldfish (25 cm long) had 27% more tectal neurons than did s...
The retinas of 63 goldfish were examined after varying durations of exposure to one of three envi... more The retinas of 63 goldfish were examined after varying durations of exposure to one of three environmental lighting conditions beginning before hatching: constant light (340 lux), cyclic light (12 hr 320 lux, 12 hr dark) and constant dark. Up to 8 months, no effects of constant light or dark on photoreceptor numbers or structure were apparent. Densities of rod and cone nuclei were normal and all retinal layers appeared normal by light microscopy. Exposure to constant light for 12 months or longer resulted in a reduction in rod density by 37%. Cone numbers were unaffected by constant light, even with exposures of 3 yr, and rod and cone outer segments were normal in length at 11-20 months under all environmental conditions. Due to poor survival, only one animal was available for quantitative examination from the group reared in constant dark 12 months or longer. Photoreceptor size and number in this retina were similar to those in the constant light condition. The results suggest that...
The ability to accurately distinguish remaining or recurrent high-grade astrocytoma from necrosis... more The ability to accurately distinguish remaining or recurrent high-grade astrocytoma from necrosis or edema following treatment is essential to optimal patient management. Thallium 201 planar gamma-camera imaging has been shown to be helpful in detecting recurrent high-grade astrocytoma; however, due to tissue heterogeneity adjacent to and within tumor, the cellular specificity and quantification of 201Tl uptake are largely unknown. In order to determine which tissues are responsible for the radioisotope uptake, microautoradiographic techniques were used to examine multiple tissue sections from five patients with high-grade astrocytoma. Each patient received 5 mCi of 201Tl i.v. 1 h prior to tumor removal. Additionally, all patients received computerized tomographic and 201Tl planar gamma-camera scans prior to surgery. Following surgery, the excised tissue specimens were tentatively classified by gross pathological examination and then immediately processed for dry mount autoradiograp...
The photoneuroendocrine system translates environmental light conditions into the circadian produ... more The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction 1 . In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes 1 . In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus-the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ 1 . The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles 2 . Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish.
Ab&m%--New rods are continually generated and inserted across the entire differentiated retina in... more Ab&m%--New rods are continually generated and inserted across the entire differentiated retina in juvenile and adult goldfish; no other retinal cells share this characteristic. How does the preferential addition of rods affect visual function? To examine the relation between continued rod addition and visual sensitivity, we measured absolute threshold in fish of different sires. Twenty-nine fish were trained in a classical conditioning paradigm, and psychomet~c functions were obtained for each of them for detection of a 532 nm light 5 set in duration, 140 deg in angular subtense, presented while the fish was fully dark adapted. We found that absolute threshold (expressed in terms of retinal photon density) was lower in larger fish, but by a very small amount: on average, large fish (15.4 f 0.5 cm standard body length) were 1.45 times more sensitive than small fish (4.3 f 0.3 cm). Morphometric analysis showed that the planimetric density of rods in goldfish retina increases at a similar rate between small and large fish, while the density of retinal ganglion cells declines between small and large fish (by a factor of 3.8). The ratio of rods to ganglion cells (a possible indicator of neural convergence) increased, but by a factor that is too large to reconcile with the psychophysical results (5.3 x). The results suggest that absolute visual threshold in the goldfish is closely related to the density of rods in the retina.
Retinal development in teleosts can broadly be divided into three epochs. The first is the specif... more Retinal development in teleosts can broadly be divided into three epochs. The first is the specification of cellular domains in the larval forebrain that give rise to the retinal primordia and undergo early morphogenetic movements. The second is the neurogenic events within the retina proper-proliferation, cell fate determination, and pattern formation-that establish neuronal identities and form retinal laminae and cellular mosaics. The third, which is unique to teleosts and occurs in the functioning eye, is stretching of the retina and persistent neurogenesis that allows the growth of the retina to keep pace with the growth of the eye and other tissues. The first two events are rapid, complete by about 3 days postfertilization in the zebrafish embryo. The third is life-long and accounts for the bulk of retinal growth and the vast majority of adult retinal neurons. In addition, but clearly related to the retina's developmental history, lesions that kill retinal neurons elicit robust neuronal regeneration that originates from cells intrinsic to the retina. This paper reviews recent studies of retinal development in teleosts, focusing on those that shed light on the genetic and molecular regulation of retinal specification and morphogenesis in the embryo, retinal neurogenesis in larvae and adults, and injury-induced neuronal regeneration.
The retina displays numerous processes that follow a circadian rhythm. These processes are coordi... more The retina displays numerous processes that follow a circadian rhythm. These processes are coordinated through the direct action of light on photoreceptive molecules and, in the absence of light, through autocrine/paracrine actions of extracellular neuromodulators. We previously described the expression of the genes encoding the secreted heparin-binding growth factors, midkine-a (mdka) and midkine-b (mdkb), in the retina of the zebrafish. Here, we provide evidence that the expression of mdka and mdkb follows a daily rhythm, which is independent of the presence or absence of light, and we propose that the expression of mdka is regulated by the circadian clock. Both qualitative and quantitative measures show that for mdka, the levels of mRNA and protein decrease during the night and increase during the subjective day. Qualitative measures show that the expression of mdkb increases during the second half of the subjective night and decreases during the second half of the subjective day. Within horizontal cells, the two midkine paralogs show asynchronous circadian regulation. Though intensely studied in the contexts of physiology and disease, this is the first study to provide evidence for the circadian regulation of midkines in the vertebrate nervous system.
Neuronal progenitors in the mammalian brain derive from radial glia or specialized astrocytes. In... more Neuronal progenitors in the mammalian brain derive from radial glia or specialized astrocytes. In developing neural retina, radial glia-like Müller cells are generated late in neurogenesis and are not considered to be neuronal progenitors, but they do proliferate after injury and can express neuronal markers, suggesting a latent neurogenic capacity. To examine the neurogenic capacity of retinal glial cells, we used lineage tracing in transgenic zebrafish with a glial-specific promoter (gfap, for glial fibrillary acid protein) driving green fluorescent protein in differentiated Müller glia. We found that all Müller glia in the zebrafish retina express low levels of the multipotent progenitor marker Pax6 (paired box gene 6), and they proliferate at a low frequency in the intact, uninjured retina. Müller glia-derived progenitors express Crx (cone rod homeobox) and are late retinal progenitors that generate the rod photoreceptor lineage in the postembryonic retina. These Müller glia-derived progenitors also remain competent to produce earlier neuronal lineages, in that they respond to loss of cone photoreceptors by specifically regenerating the missing neurons. We conclude that zebrafish Müller glia function as multipotent retinal stem cells that generate retinal neurons by homeostatic and regenerative developmental mechanisms.
Cone photoreceptors in teleost fish are organized in precise, crystalline arrays in the epithelia... more Cone photoreceptors in teleost fish are organized in precise, crystalline arrays in the epithelial plane of the retina. In zebrafish, four distinct morphological/spectral cone types occupy specific, invariant positions within a regular lattice. The cone lattice is aligned orthogonal and parallel to circumference of the retinal hemisphere: it emerges as cones generated in a germinal zone at the retinal periphery are incorporated as single-cell columns into the cone lattice. Genetic disruption of the transcription factor Tbx2b eliminates most of the cone subtype maximally sensitive to ultraviolet (UV) wavelengths and also perturbs the long-range organization of the cone lattice. In the tbx2b mutant, the other three cone types (red, green, and blue cones) are specified in the correct proportion, differentiate normally, and acquire normal, planar polarized adhesive interactions mediated by Crumbs 2a and Crumbs 2b. Quantitative image analysis of cell adjacency revealed that the cones in the tbx2b mutant primarily have two nearest neighbors and align in single-cell-wide column fragments that are separated by rod photoreceptors. Some UV cones differentiate at the dorsal retinal margin in the tbx2b mutant, although they are severely dysmorphic and are eventually eliminated. Incorporating loss of UV cones during formation of cone columns at the margin into our previously published mathematical model of zebrafish cone mosaic formation (which uses bidirectional interactions between planar cell polarity proteins and anisotropic mechanical stresses in the plane of the retinal epithelium to generate regular columns of cones parallel to the margin) reproduces many features of the pattern disruptions seen in the tbx2b mutant.
The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary... more The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. Retinal injury stimulates Müller glia to dedifferentiate, re-enter the cell cycle, and generate multipotent retinal progenitors similar to those in the CMZ to replace missing neurons. The specific signals that maintain these two distinct populations of endogenous retinal stem cells are not understood. We used genetic and pharmacological manipulation of the β-catenin/Wnt signaling pathway to show that it is required to maintain proliferation in the CMZ and that hyperstimulation of β-catenin/Wnt signaling inhibits normal retinal differentiation and expands the population of proliferative retinal progenitors. To test whether similar effects occur during regeneration, we developed a method for making rapid, selective photoreceptor ablations in larval zebrafish with intense light. We found that dephosphorylated β-catenin accumulates in Müller glia as they re-enter the cell cycle following injury, but not in Müller glia that remain quiescent. Activation of Wnt signaling is required for regenerative proliferation, and hyperstimulation results in loss of Müller glia from the INL as all proliferative cells move into the ONL. β-catenin/Wnt signaling is thus required for the maintenance of retinal progenitors during both initial development and lesion-induced regeneration, and is sufficient to prevent differentiation of those progenitors and maintain them in a proliferative state. This suggests that the β-catenin/Wnt cascade is part of the shared molecular circuitry that maintains retinal stem cells for both homeostatic growth and epimorphic regeneration.
Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a ... more Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a ''cone mosaic.'' Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish (Danio rerio) have four cone subtypes arranged into precise rows in the adult retina.
A cDNA full length encoding a putative ultraviolet (UV)-sensitive visual pigment of goldfish was ... more A cDNA full length encoding a putative ultraviolet (UV)-sensitive visual pigment of goldfish was isolated. The deduced amino acid sequence shows 64% identity to those of human blue and chicken violet, and less identity (40-49%) to those of other vertebrate visual pigment. The mRNA is localized in the miniature short single cone cells, which are known to have a sensitivity maximum in the near UV-region.
Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neur... more Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neurogenesis allows a vigorous regenerative response to damage, and fish can regenerate retinal neurons, including photoreceptors, and restore functional vision following photic, chemical, or mechanical destruction of the retina. Müller glial cells in fish function as radial-glial-like neural stem cells. During adult growth, Müller glial nuclei undergo sporadic, asymmetric, self-renewing mitotic divisions in the inner nuclear layer to generate a rod progenitor that migrates along the radial fiber of the Müller glia into the outer nuclear layer, proliferates, and differentiates exclusively into rod photoreceptors. When retinal neurons are destroyed, Müller glia in the immediate vicinity of the damage partially and transiently dedifferentiate, re-express retinal progenitor and stem cell markers, re-enter the cell cycle, undergo interkinetic nuclear migration (characteristic of neuroepithelial ...
Methods in molecular biology (Clifton, N.J.), 2012
Microarray-based gene profiling has become an important technique to measure changes in gene expr... more Microarray-based gene profiling has become an important technique to measure changes in gene expression on a genome-wide scale. Recently, cell-specific microarrays have been reported to study changes in gene expression of a particular cell type in several model organisms. Here, we describe a protocol to prepare RNA samples for microarray analysis of isolated Müller glia-derived retinal stem cells from light-damaged adult zebrafish expressing a fluorescent marker in Müller cells using enzymatic retinal dissociation followed by fluorescence-activated cell sorting (FACS).
The persistence in adult teleost fish of retinal stem cells that exhibit all of the features of t... more The persistence in adult teleost fish of retinal stem cells that exhibit all of the features of true 'adult stem cells'--self-renewal, multipotency, and the capacity to respond to injury by mitotic activation with the ability to regenerate differentiated tissues--has been known for several decades. However, the specialized cellular and molecular characteristics of these adult retinal stem cells and the microenvironmental niches that support their maintenance in the differentiated retina and regulate their activity during growth and regeneration have not yet been elucidated. Our data show that the zebrafish retina has two kinds of specialized niches that sustain retinal stem cells: 1) a neuroepithelial germinal zone at the interface between neural retina and ciliary epithelium, called the ciliary marginal zone (CMZ), a continuous annulus around the retinal circumference, and 2) the microenvironment around some Müller glia in the differentiated retina. In the uninjured retina,...
To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated... more To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated cones after laser ablation of photoreceptors in adult goldfish retina. Using a technique developed previously in this laboratory, photoreceptors in the retina of adult goldfish were ablated with an argon laser. The mitotic marker, bromodeoxyuridine, was used to label proliferating and regenerated cells, which were identified with cell-specific markers. Cells proliferating locally within lesion included microglia, Müller glia, and retinal progenitors in the inner nuclear layer (INL). The nuclei of both Müller glia and associated retinal progenitors migrated from the inner to the outer nuclear layer. The proliferating retinal progenitors, which express Notch-3 and N-cadherin, regenerated cone photoreceptors and then rod photoreceptors. Previous work has demonstrated that photoreceptors in the goldfish retina regenerate selectively after laser ablation, but the source of regenerated cones ...
To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specif... more To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specifically in photoreceptors and pinealocytes, regulate photoreceptor gene expression, are necessary for normal photoreceptor differentiation, and when mutated cause a variety of photoreceptor degenerations. A zebrafish retinal cDNA library was screened with a human CRX cDNA probe. Radiation hybrid mapping, Northern blot analysis, in situ hybridization, and transient transfection studies were performed using standard methods. Based on amino acid sequence comparisons, zebrafish crx shows 50% identity with human CRX, and 85% identity in the homeodomain. A phylogenetic analysis indicates that zebrafish crx is most closely related to the mammalian Crx proteins, and more distantly related to the Otx proteins. Zebrafish crx maps between 49.6 and 54.5 cm from the top of linkage group LG05C, a map position consistent with the location of the mouse and human CRX genes. Northern blot analysis and in s...
Results and problems in cell differentiation, 2000
... Or filter your current search. Raymond PA, Find all citations by this author (default). Or fi... more ... Or filter your current search. Raymond PA, Find all citations by this author (default). Or filter your current search. Hitchcock PF. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor 48109-0616, USA. ...
To examine expression of the zebrafish ultraviolet cone opsin pigment in goldfish and zebrafish r... more To examine expression of the zebrafish ultraviolet cone opsin pigment in goldfish and zebrafish retinas. Digoxigenin-labeled cRNA probes were prepared by run-off transcription from plasmids containing cDNAs for zebrafish ultraviolet opsin, goldfish ultraviolet cone opsin, and goldfish rod opsin. Probes were hybridized to cryosections of retina and visualized with immunocytochemistry. The zebrafish ultraviolet opsin probe hybridized selectively to rod photoreceptors, but not to ultraviolet cones or any other cone type, in both zebrafish and goldfish retinas, and the pattern of expression was identical to that of the goldfish rod opsin probe. The goldfish ultraviolet opsin, in contrast, hybridized to ultraviolet cone photoreceptors in both goldfish and zebrafish. The cDNA previously identified by Robinson et al as zebrafish ultraviolet opsin is not a cone opsin but is likely to be a rod opsin.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1983
The growth and morphology of the optic tectum of adult goldfish were studied with light and elect... more The growth and morphology of the optic tectum of adult goldfish were studied with light and electron microscopy and with thymidine radioautography. The tectum is roughly hemispheric in shape, with a smaller radius of curvature rostrally than caudally. A narrow region containing proliferating cells (the germinal zone) is found along two-thirds of the rim of the tectal hemisphere but is absent rostrally, adjacent to the tectal region which receives input from the rostral visual field. New cells generated in the germinal zone are added to the tectum appositionally in crescent-shaped increments; these was no evidence of migration of new cells into the rostral region which lacks a germinal zone. Some of the new cells added to the adult tectum were shown to be neurons on the basis of cytological and ultrastructural features. Counts of tectal neurons likewise demonstrated that new cells were added with growth of the tectum; large goldfish (25 cm long) had 27% more tectal neurons than did s...
The retinas of 63 goldfish were examined after varying durations of exposure to one of three envi... more The retinas of 63 goldfish were examined after varying durations of exposure to one of three environmental lighting conditions beginning before hatching: constant light (340 lux), cyclic light (12 hr 320 lux, 12 hr dark) and constant dark. Up to 8 months, no effects of constant light or dark on photoreceptor numbers or structure were apparent. Densities of rod and cone nuclei were normal and all retinal layers appeared normal by light microscopy. Exposure to constant light for 12 months or longer resulted in a reduction in rod density by 37%. Cone numbers were unaffected by constant light, even with exposures of 3 yr, and rod and cone outer segments were normal in length at 11-20 months under all environmental conditions. Due to poor survival, only one animal was available for quantitative examination from the group reared in constant dark 12 months or longer. Photoreceptor size and number in this retina were similar to those in the constant light condition. The results suggest that...
The ability to accurately distinguish remaining or recurrent high-grade astrocytoma from necrosis... more The ability to accurately distinguish remaining or recurrent high-grade astrocytoma from necrosis or edema following treatment is essential to optimal patient management. Thallium 201 planar gamma-camera imaging has been shown to be helpful in detecting recurrent high-grade astrocytoma; however, due to tissue heterogeneity adjacent to and within tumor, the cellular specificity and quantification of 201Tl uptake are largely unknown. In order to determine which tissues are responsible for the radioisotope uptake, microautoradiographic techniques were used to examine multiple tissue sections from five patients with high-grade astrocytoma. Each patient received 5 mCi of 201Tl i.v. 1 h prior to tumor removal. Additionally, all patients received computerized tomographic and 201Tl planar gamma-camera scans prior to surgery. Following surgery, the excised tissue specimens were tentatively classified by gross pathological examination and then immediately processed for dry mount autoradiograp...
The photoneuroendocrine system translates environmental light conditions into the circadian produ... more The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction 1 . In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes 1 . In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus-the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ 1 . The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles 2 . Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish.
Ab&m%--New rods are continually generated and inserted across the entire differentiated retina in... more Ab&m%--New rods are continually generated and inserted across the entire differentiated retina in juvenile and adult goldfish; no other retinal cells share this characteristic. How does the preferential addition of rods affect visual function? To examine the relation between continued rod addition and visual sensitivity, we measured absolute threshold in fish of different sires. Twenty-nine fish were trained in a classical conditioning paradigm, and psychomet~c functions were obtained for each of them for detection of a 532 nm light 5 set in duration, 140 deg in angular subtense, presented while the fish was fully dark adapted. We found that absolute threshold (expressed in terms of retinal photon density) was lower in larger fish, but by a very small amount: on average, large fish (15.4 f 0.5 cm standard body length) were 1.45 times more sensitive than small fish (4.3 f 0.3 cm). Morphometric analysis showed that the planimetric density of rods in goldfish retina increases at a similar rate between small and large fish, while the density of retinal ganglion cells declines between small and large fish (by a factor of 3.8). The ratio of rods to ganglion cells (a possible indicator of neural convergence) increased, but by a factor that is too large to reconcile with the psychophysical results (5.3 x). The results suggest that absolute visual threshold in the goldfish is closely related to the density of rods in the retina.
Retinal development in teleosts can broadly be divided into three epochs. The first is the specif... more Retinal development in teleosts can broadly be divided into three epochs. The first is the specification of cellular domains in the larval forebrain that give rise to the retinal primordia and undergo early morphogenetic movements. The second is the neurogenic events within the retina proper-proliferation, cell fate determination, and pattern formation-that establish neuronal identities and form retinal laminae and cellular mosaics. The third, which is unique to teleosts and occurs in the functioning eye, is stretching of the retina and persistent neurogenesis that allows the growth of the retina to keep pace with the growth of the eye and other tissues. The first two events are rapid, complete by about 3 days postfertilization in the zebrafish embryo. The third is life-long and accounts for the bulk of retinal growth and the vast majority of adult retinal neurons. In addition, but clearly related to the retina's developmental history, lesions that kill retinal neurons elicit robust neuronal regeneration that originates from cells intrinsic to the retina. This paper reviews recent studies of retinal development in teleosts, focusing on those that shed light on the genetic and molecular regulation of retinal specification and morphogenesis in the embryo, retinal neurogenesis in larvae and adults, and injury-induced neuronal regeneration.
The retina displays numerous processes that follow a circadian rhythm. These processes are coordi... more The retina displays numerous processes that follow a circadian rhythm. These processes are coordinated through the direct action of light on photoreceptive molecules and, in the absence of light, through autocrine/paracrine actions of extracellular neuromodulators. We previously described the expression of the genes encoding the secreted heparin-binding growth factors, midkine-a (mdka) and midkine-b (mdkb), in the retina of the zebrafish. Here, we provide evidence that the expression of mdka and mdkb follows a daily rhythm, which is independent of the presence or absence of light, and we propose that the expression of mdka is regulated by the circadian clock. Both qualitative and quantitative measures show that for mdka, the levels of mRNA and protein decrease during the night and increase during the subjective day. Qualitative measures show that the expression of mdkb increases during the second half of the subjective night and decreases during the second half of the subjective day. Within horizontal cells, the two midkine paralogs show asynchronous circadian regulation. Though intensely studied in the contexts of physiology and disease, this is the first study to provide evidence for the circadian regulation of midkines in the vertebrate nervous system.
Neuronal progenitors in the mammalian brain derive from radial glia or specialized astrocytes. In... more Neuronal progenitors in the mammalian brain derive from radial glia or specialized astrocytes. In developing neural retina, radial glia-like Müller cells are generated late in neurogenesis and are not considered to be neuronal progenitors, but they do proliferate after injury and can express neuronal markers, suggesting a latent neurogenic capacity. To examine the neurogenic capacity of retinal glial cells, we used lineage tracing in transgenic zebrafish with a glial-specific promoter (gfap, for glial fibrillary acid protein) driving green fluorescent protein in differentiated Müller glia. We found that all Müller glia in the zebrafish retina express low levels of the multipotent progenitor marker Pax6 (paired box gene 6), and they proliferate at a low frequency in the intact, uninjured retina. Müller glia-derived progenitors express Crx (cone rod homeobox) and are late retinal progenitors that generate the rod photoreceptor lineage in the postembryonic retina. These Müller glia-derived progenitors also remain competent to produce earlier neuronal lineages, in that they respond to loss of cone photoreceptors by specifically regenerating the missing neurons. We conclude that zebrafish Müller glia function as multipotent retinal stem cells that generate retinal neurons by homeostatic and regenerative developmental mechanisms.
Cone photoreceptors in teleost fish are organized in precise, crystalline arrays in the epithelia... more Cone photoreceptors in teleost fish are organized in precise, crystalline arrays in the epithelial plane of the retina. In zebrafish, four distinct morphological/spectral cone types occupy specific, invariant positions within a regular lattice. The cone lattice is aligned orthogonal and parallel to circumference of the retinal hemisphere: it emerges as cones generated in a germinal zone at the retinal periphery are incorporated as single-cell columns into the cone lattice. Genetic disruption of the transcription factor Tbx2b eliminates most of the cone subtype maximally sensitive to ultraviolet (UV) wavelengths and also perturbs the long-range organization of the cone lattice. In the tbx2b mutant, the other three cone types (red, green, and blue cones) are specified in the correct proportion, differentiate normally, and acquire normal, planar polarized adhesive interactions mediated by Crumbs 2a and Crumbs 2b. Quantitative image analysis of cell adjacency revealed that the cones in the tbx2b mutant primarily have two nearest neighbors and align in single-cell-wide column fragments that are separated by rod photoreceptors. Some UV cones differentiate at the dorsal retinal margin in the tbx2b mutant, although they are severely dysmorphic and are eventually eliminated. Incorporating loss of UV cones during formation of cone columns at the margin into our previously published mathematical model of zebrafish cone mosaic formation (which uses bidirectional interactions between planar cell polarity proteins and anisotropic mechanical stresses in the plane of the retinal epithelium to generate regular columns of cones parallel to the margin) reproduces many features of the pattern disruptions seen in the tbx2b mutant.
The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary... more The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. Retinal injury stimulates Müller glia to dedifferentiate, re-enter the cell cycle, and generate multipotent retinal progenitors similar to those in the CMZ to replace missing neurons. The specific signals that maintain these two distinct populations of endogenous retinal stem cells are not understood. We used genetic and pharmacological manipulation of the β-catenin/Wnt signaling pathway to show that it is required to maintain proliferation in the CMZ and that hyperstimulation of β-catenin/Wnt signaling inhibits normal retinal differentiation and expands the population of proliferative retinal progenitors. To test whether similar effects occur during regeneration, we developed a method for making rapid, selective photoreceptor ablations in larval zebrafish with intense light. We found that dephosphorylated β-catenin accumulates in Müller glia as they re-enter the cell cycle following injury, but not in Müller glia that remain quiescent. Activation of Wnt signaling is required for regenerative proliferation, and hyperstimulation results in loss of Müller glia from the INL as all proliferative cells move into the ONL. β-catenin/Wnt signaling is thus required for the maintenance of retinal progenitors during both initial development and lesion-induced regeneration, and is sufficient to prevent differentiation of those progenitors and maintain them in a proliferative state. This suggests that the β-catenin/Wnt cascade is part of the shared molecular circuitry that maintains retinal stem cells for both homeostatic growth and epimorphic regeneration.
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Papers by Pamela Raymond