W. Ted Allison

Mutation of the bone morphogenetic protein GDF3 causes ocular and skeletal anomalies

Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.

Degeneration and regeneration of ultraviolet cone photoreceptors during development in rainbow trout

Ultraviolet‐sensitive (UVS) cones disappear from the retina of salmonid fishes during a metamorphosis that prepares them for deeper/marine waters. UVS cones subsequently reappear in the retina near sexual maturation and the return migration to natal streams. Cellular mechanisms of this UVS cone ontogeny were investigated using electroretinograms, in situ hybridization, and immunohistochemistry against opsins during and after thyroid hormone (TH) treatments of rainbow trout (Oncorhynchus mykiss). Increasing TH levels led to UVS cone degeneration. Labeling demonstrated that UVS cone degeneration occurs via programmed cell death and caspase inhibitors can inhibit this death. After the cessation of TH treatment, UVS cones regenerated in the retina. Bromodeoxyuridine (BrdU) was applied after the termination of TH treatment and was detected in the nuclei of cells expressing UVS opsin. BrdU was found in UVS cones but not other cone types. The most parsimonious explanation for the data is that UVS cones degenerated and UVS cones were regenerated from intrinsic retinal progenitor cells. Regenerating UVS cones were functionally integrated such that they were able to elicit electrical responses from second‐order neurons. This is the first report of cones regenerating during natural development. Both the death and regeneration of cones in retinae represent novel mechanisms for tuning visual systems to new visual tasks or environments. J. Comp. Neurol. 499:702–715, 2006.

Ontogeny of cone photoreceptor mosaics in zebrafish.

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. Here we describe larval zebrafish cone patterns and investigate a previously unrecognized transition between larval and adult cone mosaic patterns. Cone positions were determined in transgenic zebrafish expressing green fluorescent protein (GFP) in their UV‐sensitive cones, by the use of multiplex in situ hybridization labelling of various cone opsins. We developed a “mosaic metric” statistical tool to measure local cone order. We found that ratios of the various cone subtypes in larval and adult zebrafish were statistically different. The cone photoreceptors in larvae form a regular heterotypic mosaic array; i.e., the position of any one cone spectral subtype relative to the other cone subtypes is statistically different from random. However, the cone spectral subtypes in larval zebrafish are not arranged in continuous rows as in the adult. We used cell birth dating to show that the larval cone mosaic pattern remains as a distinct region within the adult retina and does not reorganize into the adult row pattern. In addition, the abundance of cone subtypes relative to other subtypes is different in this larval remnant compared with that of larvae or canonical adult zebrafish retina. These observations provide baseline data for understanding the development of cone mosaics via comparative analysis of larval and adult cone development in a model species. J. Comp. Neurol. 518:4182–4195, 2010.

Ontogeny of ultraviolet-sensitive cones in the retina of rainbow trout (Oncorhynchus mykiss)

In order to facilitate emerging models of retinal development, we developed electroretinogram and in situ hybridization protocols to examine the ontogeny of photoreceptors in the retina of a land‐locked salmonid, the rainbow trout (Oncorhynchus mykiss). We cloned cDNA fragments corresponding to the rod opsin and each of the four cone opsin gene families, which we utilized to produce riboprobes. We established the specificity of the in situ hybridization protocol by examining subcellular signal localization and through double‐labeling experiments. We confirm the assumption that the accessory corner cones in the square mosaic are the ultraviolet wavelength‐sensitive (UVS) cone photoreceptor (i.e., they express an SWS1 opsin) and observed UVS cones throughout the retina of small trout. Larger fish have a decrease in sensitivity to short wavelength light stimuli and the distribution of UVS cones in the mature retina is limited to the dorsal‐temporal quadrant. These larger fish also possess differentiated UVS cones in the peripheral germinal zone (PGZ), including within areas peripheral to mature retina lacking UVS cones. These data are consistent with the loss of putative UVS cones from the PGZ of a migratory salmonid of another genus, and thus the disappearance of UVS cones appears to be general to the Family Salmonidae, regardless of life history strategy. The generation, differentiation, and subsequent loss of UVS cones in the smolt PGZ is a dramatic example of the supposition that the mechanisms of PGZ development recapitulate the retinal embryogenesis of that species. J. Comp. Neurol. 461:294–306, 2003.

Visual pigment composition in zebrafish: Evidence for a rhodopsin-porphyropsin interchange system

Numerous reports have concluded that zebrafish (Danio rerio) possesses A1-based visual pigments in their rod and cone photoreceptors. In the present study, we investigated the possibility that zebrafish have a paired visual pigment system. We measured the spectral absorption characteristics of photoreceptors from zebrafish maintained in different temperature regimes and those treated with exogenous thyroid hormone using CCD-based microspectrophotometry. Rods from fish housed at 15 degrees C and 28 degrees C were not significantly different, having lambda max values of 503 +/- 5 nm (n = 106) and 504 +/- 6 nm (n = 88), respectively. Thyroid hormone treatment (held at 28 degrees C), however, significantly shifted the lambda max of rods from 503 +/- 5 nm (n = 194) to 527 +/- 8 nm (n = 212). Cone photoreceptors in fish housed at 28 degrees C (without thyroid hormone treatment) had lambda max values of 361 +/- 3 nm (n = 2) for ultraviolet-, 411 +/- 5 nm (n = 18) for short-, 482 +/- 6 nm (n = 9) for medium-, and 565 +/- 10 nm (n = 14) for long-wavelength sensitive cones. Thyroid hormone treatment of fish held at 28 degrees C significantly shifted the lambda max of long-wavelength sensitive cones to 613 +/- 11 nm (n = 20), substantially beyond that of the lambda max of the longest possible A1-based visual pigment (approximately 580 nm). Thyroid hormone treatment produced smaller shifts of lambda max in other cone types and increased the half-band width. All shifts in photoreceptor lambda max values resulting from thyroid hormone treatment matched predictions for an A1- to A2-based visual pigment system. We therefore conclude that zebrafish possess a rhodopsin-porphyropsin interchange system that functions to spectrally tune rod and cone photoreceptors. We believe that these observations should be carefully considered during analysis of zebrafish spectral sensitivity.

Investigating regeneration and functional integration of CNS neurons: lessons from zebrafish genetics and other fish species.

Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in the zebrafish spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput) indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.

Spatio-temporal characterization of retinal opsin gene expression during thyroid hormone-induced and natural development of rainbow trout

The abundance and spatial distribution of retinal cone photoreceptors change during thyroid hormone (TH)-induced and natural development of rainbow trout (Oncorhynchus mykiss). These changes are thought to allow the fish to adapt to different photic environments throughout its life history. To date, the ontogeny of rainbow trout cone photoreceptors has been examined using physiological and morphological approaches. In this study, we extended these observations by measuring opsin gene expression in retinal quadrants during natural and TH-induced development. Gene expression during natural development was investigated in retinae from fish at both parr and smolt stages. The role of TH in modulating opsin gene expression was determined in TH-treated parr and control fish sampled after two, nine, and 22 days of treatment. Total RNA was isolated from each retinal quadrant and steady-state opsin mRNA levels were measured using reverse transcriptase real-time quantitative polymerase chain reaction (QPCR) analysis. Expression of ultraviolet-sensitive opsin (SWS1), rod opsin (RH1), middle wavelength-sensitive opsin (RH2), and long wavelength-sensitive opsin (LWS) transcripts vary spatially in the parr retina. Smolts, compared to parr, had downregulated SWS1 expression in all quadrants, lower LWS expression dorsally, higher RH1 expression nasally, and higher RH2 expression dorsally. In TH-treated parr, SWS1 opsin expression was downregulated in the nasal quadrants by two days. SWS1 displayed the greatest degree of downregulation in all quadrants after nine days of treatment, with an increase in short wavelength-sensitive (SWS2) and RH2 opsin mRNA expression in the temporal quadrants. This study reveals that opsin genes display spatially significant differences within rainbow trout retina in their level of mRNA expression, and that regulation of opsin expression is a dynamic process that is influenced by TH. This is particularly evident for SWS1 gene expression in parr following TH-induced and natural development.

Regeneration of Cone Photoreceptors when Cell Ablation Is Primarily Restricted to a Particular Cone Subtype

We sought to characterize the regenerated cells, if any, when photoreceptor ablation was mostly limited to a particular cone subtype. This allowed us to uniquely assess whether the remaining cells influence specification of regenerating photoreceptors. The ability to replace lost photoreceptors via stem cell therapy holds promise for treating many retinal degenerative diseases. Zebrafish are potent for modelling this because they have robust regenerative capacity emanating from endogenous stem cells, and abundant cone photoreceptors including multiple spectral subtypes similar to human fovea. We ablated the homolog of the human S-cones, the ultraviolet-sensitive (UV) cones, and tested the hypothesis that the photoreceptors regenerating in their place take on identities matching those expected from normal cone mosaic development. We created transgenic fish wherein UV cones can be ablated by addition of a prodrug. Thus photoreceptors developed normally and only the UV cones expressed nitroreductase; the latter converts the prodrug metronidazole to a cell-autonomous neurotoxin. A significant increase in proliferation of progenitor cell populations (p<0.01) was observed when cell ablation was primarily limited to UV cones. In control fish, we found that BrdU primarily incorporated into rod photoreceptors, as expected. However the majority of regenerating photoreceptors became cones when retinal cell ablation was predominantly restricted to UV cones: a 2-fold increase in the relative abundance of cones (p = 0.008) was mirrored by a 35% decrease in rods. By primarily ablating only a single photoreceptor type, we show that the subsequent regeneration is biased towards restoring the cognate photoreceptor type. We discuss the hypothesis that, after cone death, the microenvironment formed by the remaining retinal cells may be influential in determining the identity of regenerating photoreceptors, though other interpretations are plausible. Our novel animal model provides control of ablation that will assist in identifying mechanisms required to replace cone photoreceptors clinically to restore daytime vision.

Contribution of growth differentiation factor 6-dependent cell survival to early-onset retinal dystrophies

Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-β (TGF-β) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-β signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.

Amyloid Beta Precursor Protein and Prion Protein Have a Conserved Interaction Affecting Cell Adhesion and CNS Development

Genetic and biochemical mechanisms linking onset or progression of Alzheimer Disease and prion diseases have been lacking and/or controversial, and their etiologies are often considered independent. Here we document a novel, conserved and specific genetic interaction between the proteins that underlie these diseases, amyloid-β precursor protein and prion protein, APP and PRP, respectively. Knockdown of APP and/or PRNP homologs in the zebrafish (appa, appb, prp1, and prp2) produces a dose-dependent phenotype characterized by systemic morphological defects, reduced cell adhesion and CNS cell death. This genetic interaction is surprisingly exclusive in that prp1 genetically interacts with zebrafish appa, but not with appb, and the zebrafish paralog prp2 fails to interact with appa. Intriguingly, appa & appb are largely redundant in early zebrafish development yet their abilities to rescue CNS cell death are differentially contingent on prp1 abundance. Delivery of human APP or mouse Prnp mRNAs rescue the phenotypes observed in app-prp-depleted zebrafish, highlighting the conserved nature of this interaction. Immunoprecipitation revealed that human APP and PrPC proteins can have a physical interaction. Our study reports a unique in vivo interdependence between APP and PRP loss-of-function, detailing a biochemical interaction that considerably expands the hypothesized roles of PRP in Alzheimer Disease.