A JAK-STAT pathway regulates wing vein formation in Drosophila.

We present evidence that the JAK-STAT signal transduction pathway regulates multiple developmental processes in Drosophila. We screened for second-site mutations that suppress the phenotype of the hyperactive hopTum-1 Jak kinase, and recovered a mutation that meiotically maps to the known chromosomal position of D-Stat, a Drosophila stat gene. This hypomorphic mutation, termed statHJ contains a nucleotide substitution in the first D-Stat intron, resulting in a reduction in the number of correctly processed transcripts. Further, the abnormally processed mRNA encodes a truncated protein that has a dominant negative effect on transcriptional activation by the wild-type cDNA in cell culture. statHJ mutants exhibit patterning defects that include the formation of ectopic wing veins, similar to those seen in mutants of the epidermal growth factor/receptor pathway. Abnormalities in embryonic and adult segmentation and in tracheal development were also observed. The hopTum-1 and statHJ mutations can partially compensate for each other genetically, and Hop overexpression can increase D-Stat transcriptional activity in vitro, indicating that the gene products interact in a common regulatory pathway.

A circadian clock regulates rod and cone input to fish retinal cone horizontal cells.

In the vertebrate retina, the light responses of post-receptor neurons depend on the ambient or background illumination. Using intracellular recording, we have found that a circadian clock regulates the light responses of dark-adapted fish cone horizontal cells. Goldfish were maintained on a 12-hr light/12-hr dark cycle. At different times of the day or night, retinas were superfused in darkness for 90 min ("prolonged darkness"), following which horizontal cells were impaled without the aid of any light flashes. In some of the experiments, fish were kept in constant darkness for 3-48 hr prior to surgery. After prolonged darkness during the night, but not during the day, the light responses of L-type cone horizontal cells resembled those of rod horizontal cells with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Following light sensitization during the night and day, the light responses of rod and cone horizontal cells were clearly different with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Under conditions of constant darkness for two full light/dark cycles, average responses of cone horizontal cells to a bright light stimulus during the subjective day were greater than during the subjective night. Prior reversal of the light/dark cycle reversed the 24-hr rhythm of cone horizontal cell responses to bright lights. In addition, following one full cycle of constant darkness, average cone horizontal cell spectral sensitivity during the subjective night closely matched that of rod horizontal cells, whereas average cone horizontal cell spectral sensitivity during the subjective day was similar to that of red (625 nm) cones. These results indicate that the effects of dark adaptation depend on the time of day and are regulated by a circadian clock so that cone input to cone horizontal cells predominates in the day and rod input predominates in the night.

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Retinal glial cell glutamate transporter is coupled to an anionic conductance.

Application of L-glutamate to retinal glial (Müller) cells results in an inwardly rectifying current due to the net influx of one positive charge per molecule of glutamate transported into the cell. However, at positive potentials an outward current can be elicited by glutamate. This outward current is eliminated by removal of external chloride ions. Substitution of external chloride with the anions thiocyanate, perchlorate, nitrate, and iodide, which are known to be more permeant at other chloride channels, results in a considerably larger glutamate-elicited outward current at positive potentials. The large outward current in external nitrate has the same ionic dependence, apparent affinity for L-glutamate, and pharmacology as the glutamate transporter previously reported to exist in these cells. Varying the concentration of external nitrate shifts the reversal potential in a manner consistent with a conductance permeable to nitrate. Together, these results suggest that the glutamate transporter in retinal glial cells is associated with an anionic conductance. This anionic conductance may be important for preventing a reduction in the rate of transport due the depolarization that would otherwise occur as a result of electrogenic glutamate uptake.

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.

Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase.

The phosphoprotein phosducin (Pd) regulates many guanine nucleotide binding protein (G protein)-linked signaling pathways. In visual signal transduction, unphosphorylated Pd blocks the interaction of light-activated rhodopsin with its G protein (Gt) by binding to the beta gamma subunits of Gt and preventing their association with the Gt alpha subunit. When Pd is phosphorylated by cAMP-dependent protein kinase, it no longer inhibits Gt subunit interactions. Thus, factors that determine the phosphorylation state of Pd in rod outer segments are important in controlling the number of Gts available for activation by rhodopsin. The cyclic nucleotide dependencies of the rate of Pd phosphorylation by endogenous cAMP-dependent protein kinase suggest that cAMP, and not cGMP, controls Pd phosphorylation. The synthesis of cAMP by adenylyl cyclase in rod outer segment preparations was found to be dependent on Ca2+ and calmodulin. The Ca2+ dependence was within the physiological range of Ca2+ concentrations in rods (K1/2 = 230 +/- 9 nM) and was highly cooperative (n app = 3.6 +/- 0.5). Through its effect on adenylyl cyclase and cAMP-dependent protein kinase, physiologically high Ca2+ (1100 nM) was found to increase the rate of Pd phosphorylation 3-fold compared to the rate of phosphorylation at physiologically low Ca2+ (8 nM). No evidence for Pd phosphorylation by other (Ca2+)-dependent kinases was found. These results suggest that Ca2+ can regulate the light response at the level of Gt activation through its effect on the phosphorylation state of Pd.

Determination of the transiently lowered pKa of the retinal Schiff base during the photocycle of bacteriorhodopsin.

Reprotonation of the transiently deprotonated retinal Schiff base in the bacteriorhodopsin photocycle is greatly slowed when the proton donor Asp-96 is removed with site-specific mutagenesis, but its rate is restored upon adding azide or other weak acids such as formate and cyanate. As expected, between pH 3 and 7 the rate of Schiff base protonation in the photocycle of the D96N mutant correlates with the concentrations of the acid forms of these agents. Dissection of the rates in the biexponential reprotonation kinetics of the Schiff base between pH 7 and 9 yielded calculated rate constants for the protonation equilibrium. Their dependencies on pH and azide or cyanate concentrations are consistent with both earlier suggested mechanisms: (i) azide and other weak acids may function as proton carriers in the protonation equilibrium of the Schiff base, or (ii) the binding of their anionic forms may catalyze proton conduction to and from the Schiff base. The measured rate constants allow the calculation of the pKa of the Schiff base during its reprotonation in the photocycle of D96N. It is 8.2-8.3, a value much below the pKa determined earlier in unphotolyzed bacteriorhodopsin.

Molecular biology of retinal ganglion cells.

Retinal ganglion cells are the output neurons that encode and transmit information from the eye to the brain. Their diverse physiologic and anatomic properties have been intensively studied and appear to account well for a number of psychophysical phenomena such as lateral inhibition and chromatic opponency. In this paper, we summarize our current view of retinal ganglion cell properties and pose a number of questions regarding underlying molecular mechanisms. As an example of one approach to understanding molecular mechanisms, we describe recent work on several POU domain transcription factors that are expressed in subsets of retinal ganglion cells and that appear to be involved in ganglion cell development.

Multineuronal codes in retinal signaling.

The visual world is presented to the brain through patterns of action potentials in the population of optic nerve fibers. Single-neuron recordings show that each retinal ganglion cell has a spatially restricted receptive field, a limited integration time, and a characteristic spectral sensitivity. Collectively, these response properties define the visual message conveyed by that neuron's action potentials. Since the size of the optic nerve is strictly constrained, one expects the retina to generate a highly efficient representation of the visual scene. By contrast, the receptive fields of nearby ganglion cells often overlap, suggesting great redundancy among the retinal output signals. Recent multineuron recordings may help resolve this paradox. They reveal concerted firing patterns among ganglion cells, in which small groups of nearby neurons fire synchronously with delays of only a few milliseconds. As there are many more such firing patterns than ganglion cells, such a distributed code might allow the retina to compress a large number of distinct visual messages into a small number of optic nerve fibers. This paper will review the evidence for a distributed coding scheme in the retinal output. The performance limits of such codes are analyzed with simple examples, illustrating that they allow a powerful trade-off between spatial and temporal resolution.

Molecular mechanism of protein-retinal coupling in bacteriorhodopsin.

Bacteriorhodopsin is a membrane protein that functions as a light-driven proton pump. Each cycle of proton transport is initiated by the light-induced isomerization of retinal from the all-trans to 13-cis configuration and is completed by the protein-driven reisomerization of retinal to the all-trans configuration. Previous studies have shown that replacement of Leu-93, a residue in close proximity to the 13-methyl group of retinal, by alanine, resulted in a 250-fold increase in the time required to complete each photocycle. Here, we show that the kinetic defect in the photocycle of the Leu-93-->Ala mutant occurs at a stage after the completion of proton transport and can be overcome in the presence of strong background illumination. Time-resolved retinal-extraction experiments demonstrate the continued presence of a 13-cis intermediate in the photocycle of the Leu-93-->Ala mutant well after the completion of proton release and uptake. These results indicate that retinal reisomerization is kinetically the rate-limiting step in the photocycle of this mutant and that the slow thermal reisomerization can be bypassed by the absorption of a second photon. The effects observed for the Leu-93-->Ala mutant are not observed upon replacement of any other residue in van der Waals contact with retinal or upon replacement of Leu-93 by valine. We conclude that the contact between Leu-93 and the 13-methyl group of retinal plays a key role in controlling the rate of protein conformational changes associated with retinal reisomerization and return of the protein to the initial state.

Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.

Intrinsic changes in developing retinal neurons result in regenerative failure of their axons.

The failure of mature mammalian central nervous system axons to regenerate after transection is usually attributed to influences of the extraneuronal milieu. Using explant cocultures of retina and midbrain tectum from hamsters, we have found evidence that these influences account for failure of regrowth of only a small minority of retinal axons. For most of the axons, there is a programmed loss of ability to elongate in the central nervous system. We show that there is a precipitous decline in the ability of retinal axons to reinnervate tectal targets when the retina is derived from pups on or after postnatal day 2, even when the target is embryonic. By contrast, embryonic retinal axons can regrow into tectum of any age, overcoming growth-inhibiting influences of glial factors.

The kinetics of quantal transmitter release from retinal amacrine cells.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Genetic engineering of vein grafts resistant to atherosclerosis.

Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.