Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Mu¨ller’s Cells

In addition to expressing GATs, Mu¨ller’s cells in skate, baboon, and human express GABAA receptors (Figure 1). GABA-evoked currents in skate and human Mu¨ller’s cells show GABAA pharmacology, including enhancement by Zn2þ and, in human, enhancement by barbiturates and BZDs. In human, the GABAA response is depolarizing and sensitivity is higher at the endfoot, soma, and sclerad margin. The efflux of Cl– in response to GABAA activation on Mu¨ller’s cells could have a variety of consequences. For example, it could regulate extracellular Cl– homeostasis following Cl– influx into neuronal GABAA and GABAC receptors. It could also accelerate the clearance of extracellular GABA by stimulating uptake, which requires cotransport of Naþ and Cl–. This latter function could be critical given data in mouse showing that GABAA activation could play a role in ganglion cell death following oxidative stress. In rabbit, Mu¨ller’s cells synthesize and release the diazepam-binding inhibitor (acyl coenzyme A-binding protein, ACBP) in response to protein kinase A activation, mimicking intense neuronal activity. ACBP binds to an a subunit to reduce the Cl– current, and hence, the level of inhibition. The reduction of Cl– influx could reduce the osmotic entry of water into neurons, thereby preventing cell swelling. Thus, in mammals at least, Mu¨ller’s cells that extend across the thickness of the retina play a major role in regulating neuronal GABAergic transmission, with the additional consequence of maintaining extracellular ionic and osmotic balance.

See also: Information Processing: Amacrine Cells; Information Processing: Bipolar Cells; Information Processing: Ganglion Cells; Information Processing: Horizontal Cells; Neurotransmitters and Receptors: Dopamine Receptors.

Further Reading

Brecha, N. C. (1992). Expression of GABAA receptors in the vertebrate retina. In: Mize, R. R., Marc, R. E., and Sillito, A. M. (eds.) Progress in Brain Research, vol. 90, pp. 3–28. New York: Elsevier Science.

Cueva, J. G., Haverkamp, S., Reimer, R. J., et al. (2002).

Vesicular g-aminobutyric acid transporter expression in amacrine and horizontal cells. Journal of Comparative Neurology 445: 227–237.

Gasnier, B. (2004). The SLC32 transporter, a key protein for the synaptic release of inhibitory amino acids. Pflugers Archiv European Journal of Physiology 447: 756–759.

Jellali, A., Stussi-Garaud, C., Gasnier, B., et al. (2002). Cellular localization of the vesicular inhibitory amino acid transporter in the mouse and human retina. Journal of Comparative Neurology

449: 76–87.

Nelson, H. (1998). The family of Na2þ/Cl– neurotransmitter transporters.

Journal of Neurochemistry 71: 1785–1803.

Olsen, R. W. and Sieghart, W. (2009). GABAA receptors: Subtypes provide diversity of function and pharmacology. Neuropharmacology 56: 141–148.

Raiteri, M. (2008). Presynaptic metabotropic glutamate and

GABAB receptors. Handbook of Experimental Pharmacology 184: 373–407.

Schwartz, E. A. (2002). Transport-mediated synapses in the retina.

Physiological Reviews 82: 875–891.

Wa¨ssle, H., Koulen, P., Brandsta¨tter, J. H., Fletcher, E. L., and Becker, C. M. (1998). Glycine and GABA receptors in the mammalian retina. Vision Research 38: 1411–1430.

Yang, X-L. (2004). Characterization of receptors for glutamate and GABA in retinal neurons. Progress in Neurobiology 2004: 127–150.

Yazulla, S. (1986). GABAergic mechanisms in the retina. Progress in Retinal Research 5: 1–52.

Relevant Website

http://webvision.med.utah.edu – John Moran Eye Center, University of Utah, Webvision. The Organization of the Retina and Visual System.

Glossary

Atonal – Basic helix–loop–helix transcription factors involved in development. Atoh7, also called Ath5, is important in ganglion cell development.

Basic helix–loop–helix (bHLH) – A basic protein–DNA and helix–loop–helix protein–protein interaction domain found in some transcription factors.

Ganglion cell layer (GCL) – The innermost retinal cell layer, which contains retinal ganglion cell bodies and, in many vertebrates, displaced amacrine interneurons.

POU-domain transcription factors – A large group of transcription factors containing a bipartite DNAbinding domain called a POU domain.

Retinal progenitor cell (RPC) – A developing retinal cell that undergoes mitotic cell division to produce cells that can differentiate into retinal neurons or glia. Transitional cell – A retinal progenitor cell that is newly postmitotic, but not yet differentiated as a particular retinal neuron or glial cell type.

Retinal Ganglion Cell Formation

Ganglion cells are the first neurons generated by retinal progenitor cells (RPCs) in the developing optic cup. Retinal ganglion cells (RGCs) differentiate first in all vertebrates. In birds and mammals, retinal neurogenesis begins in the center of the cup with the cell cycle exit of a group of RPCs near the optic stalk. Many, but not all, early neurons differentiate as RGCs. From this initiation zone, RGC genesis spreads outward to the periphery of the optic cup. However, in zebrafish, retinal neuron formation initiates in the ventral, nasal cup and propagates around the circumference of the eye as a wave. Ganglion cell layer (GCL) formation is the initial step of optic cup lamination, which results in a mature retina organized into three cell layers (outer nuclear layer, inner nuclear layer, and GCL) separated by two synaptic layers (outer and inner plexiform layers).

Over the past 15 years, considerable progress has been made toward understanding of how mitotically active RPCs choose the RGC fate, terminally differentiate as RGC neurons, extend their axons, and establish synaptic connections in the brain. Yet, this gene network remains

incomplete, with more work needed to define RGC formation in molecular terms. Within the hierarchy, both intrinsic transcription factors and extrinsic signaling pathways provide integrated regulation of progenitor development into an RGC. While this article emphasizes intrinsic regulation of RGC fate specification, a short description of extrinsic signaling pathways known to regulate RGC development is also included.

Atoh7/Ath5 Function is Critical for RGC Development

The onset of retinal neurogenesis in a subset of optic cup RPCs is characterized by the activation of the basic helix–loop–helix (bHLH) transcription factor Atoh7/ Ath5. The bHLH factors regulate many characteristics of retinal neuron formation. The encoded proteins contain both a basic DNA-binding and helix–loop–helix protein dimerization domains. These factors are expressed by RPCs, in which one or more cells ultimately becomes a neuron. Atoh7/Ath5 occupies a critical node in the RGC gene hierarchy, since zebrafish or mouse mutants lacking the gene have a profound to total loss of RGC differentiation and abnormal optic nerve formation. Adult mutant animals are viable, but have no optic nerves or chiasmata. Overexpression of frog and chick Ath5 during retinal development induces ectopic RGCs, but mammalian Atoh7 does not seem to have this ability. This difference correlates with the observation that, in the mouse retina, Atoh7-expressing cells (the Atoh7 retinal lineage) give rise to all seven major classes of retinal neurons and glia in the adult retina, and do not strictly produce RGCs. This indicates that Atoh7 function is needed to acquire the potential to become an RGC, but is insufficient to commit a cell to this fate. During embryonic retinal neurogenesis, the Atoh7 lineage is hypothesized to predominantly give rise to RGCs, but after the peak of RGC genesis, Atoh7-expressing cells are better able to adopt other retinal fates. This change in developmental ability is presumably due to input from signals secreted by differentiated RGCs and possibly other (non-Atoh7) retinal lineages. Such signals may either promote the adoption of non-RGC retinal neuron or glial fates, block RGC fates, or both.

One role for Atoh7 in the mouse retina is to facilitate cell cycle exit, since mutant cells appear to stall at a cell cycle checkpoint, unable to either resume mitosis or differentiate as RGCs. Eventually, these cells erroneously adopt the last retinal fate, that of Mu¨ller glia. Mouse

retinal cells expressing Atoh7 fit the definition of transitional cells, because they are nonmitotic, migratory RPCs that commit to a particular fate, but remain undifferentiated when they express Atoh7. Atoh7+ cells co-express the cyclin kinase inhibitor Cdkn1b/p27Kip1, which has multiple functions, including promoting exit from mitosis of cells at the G1/G0 checkpoint. Without Atoh7, the percentage of Cdkn1bþ cells fluctuates in both number and arrangement during the peak of RGC genesis. However, questions remain about the relationship between Atoh7 and Cdkn1b. Does mouse Atoh7 directly regulate the activation of Cdkn1b, or do Atoh7 and Cdkn1b act cooperatively to drive RPCs out of mitosis? The latter possibility has been proposed for frog Ath5 and cyclin-dependent kinase inhibitor xic1 (p27Xic1), although it is unclear whether this represents protein–protein interactions, coordinate regulation of shared downstream genes, or cross-regulation. Alternatively, the shifts in mammalian cyclin-dependent kinase inhibitor 1B (p27Kip1) expression in Atoh7 mutant eyes may be correlative, but not indicative, of either type of interaction. Since cyclin-dependent kinase inhibitor 1B (Cdkn1b) transcription, translation, and protein stability are independently regulated during development, distinguishing among these possibilities will require further in-depth analyses.

Integrated Regulation of Atoh7/Ath5

Retinal Expression

Many proneural bHLH genes autoregulate their expression, including fruit fly atonal and mouse Atoh1, the most closely related bHLH factors to Atoh7. However, multiple

lines of evidence show that Atoh7/Ath5 does not regulate its own expression. Atoh7/Ath5 appears in the optic cup slightly ahead of other proneural bHLH factors and has a very dynamic expression pattern first detectable in a subset of transitional cells that give rise to the earliest retinal neurons. Therefore, determining which transcription factors and signal transduction pathways directly influence Atoh7/Ath5 expression is essential for comprehending how broadly acting factors regulate the precise spatiotemporal patterns of retinal neurons. Among the early optic cup transcription factors, only Pax6 has been shown to directly activate retinal expression of Atoh7/Ath5 via binding to its highly conserved enhancer. Because Atoh7/Ath5 expression initiates in a subset of Pax6expressing cells, other factors must simultaneously restrict Atoh7/Ath5 expression to newly postmitotic transition cells (Figure 1). Another equally important aspect of Atoh7/Ath5 regulation is its turnover, since Atoh7 mRNA disappears abruptly from transitional cells at their entry into the GCL. Therefore, repression is just as important as initial activation for the proper regulation of Atoh7/Ath5 (Figure 1).

There are several factors that genetically suppress Atoh7/Ath5 expression, including hairy and enhancer of split 1 (Hes1), neurogenic differentiation 1 (Neurod1), and neurogenic differentiation 4 (Neurod4/Ath3). Because Hes1 is a transcriptional repressor, it is not surprising that it blocks Atoh7 expression. Hes1 similarly suppresses the expression of other retinal proneural bHLH factors like neurogenin 2 (Neurog2) and achaete-scute complex homolog 1 (Ascl1). In the absence of Hes1, Atoh7, Neurog2, and Ascl1 are each prematurely activated in the mouse optic cup. Although Hes1 regulates the overall

timing of neurogenesis, other regulatory inputs control the precise onset of each proneural bHLH gene. The timing function of Hes1 reflects its role as a downstream effector of Notch signaling, although not all Hes1 functions are Notch dependent. Other factors that suppress Atoh7 expression are Neurod1 and Neurod4, which do so synergistically. Embryonic mouse retinas that are mutant for both genes have expanded Atoh7 expression and a dramatic increase in RGCs, along with a profound loss of amacrine neurons. Thus, Neurod1 and Neurod4 repress Atoh7 expression (directly or indirectly) in RPCs that normally give rise to amacrine cells.

Multiple extrinsic signals influence vertebrate Atoh7/ Ath5 expression, particularly for RGC formation. These include fibroblast growth factor 3 (Fgf3), fibroblast growth factor 8 (Fgf8), one eyed pinhead/nodal-like (oep/nodallike), notch homolog 1 (Notch1), growth differentiation factor 11 (Gdf11), and sonic hedghog (Shh) signaling pathways (Figure 1). As shown in the zebrafish and chick retina, Fgf and oep signaling are required for Ath5 activation, while Notch (chick, frog, mouse), Gdf11 (mouse), and Shh (zebrafish, chick, mouse) each suppresses Atoh7/Ath5 expression. Intriguingly, chick Fgf3 and Fgf8 are both expressed in the central optic cup, just prior to the onset of retinal neurogenesis and Fgf protein-coated beads induce Ath5 expression. Although no role during early mouse retinal development has been reported for the orthologous genes, the secretion of stimulatory signals from the central optic cup (Fgf3 or 8) and/or optic stalk (Fgf8 and oep) would be sufficient to explain the onset of Atoh7/Ath5 expression in a restricted retinal domain. By contrast, the extrinsic pathways that negatively regulate Atoh7/Ath5 expression emanate from two distinct sources, RPCs and RGCs. The Notch pathway functions in multiple and complex ways during neurogenesis, but its classic role is to transduce a competitive signal between two adjacent progenitors, in which one cell (expressing more ligand) laterally inhibits the other (expressing more receptor) from differentiating. Therefore, Notchþ cells remain proliferative by suppressing neuron promoting factors like Atoh7. The other two signals, Gdf11 and Shh, are made and secreted by differentiated RGCs to provide negative feedback regulation to RPCs so that the number of neurons is not drastically overproduced. But, to date none of these pathways have been shown to directly activate or repress Atoh7/Ath5 transcription.

Atoh7/Ath5 Activates Pou4f/Brn3

Expression in RGCs

Atoh7/Ath5 activation of Pou4f/Brn3b expression is the principal molecular pathway directing RGC specification and differentiation. Studies in the chick, frog, and mouse eye all suggest that this activation is direct, although definitive biochemical assays have not yet been performed.

In the mouse optic cup, Atoh7 and Pou4f2 expression are 90% overlapping, with Atoh7 appearing slightly earlier than Pou4f2 in cells completing their final mitosis, migrating toward the GCL, and differentiating (Figure 2). Importantly, Atoh7 is abruptly turned off as nascent RGCs enter the GCL, while Pou4f2 expression is maintained by mature RGCs, even into adulthood.

The Pou4f/Brn subfamily of POU-domain transcription factors is comprised of Pou4f1/Brn3a, Pou4f2/Brn3b, and Pou4f3/Brn3c. Each of these genes acts as key regulators of sensory neuron development, particularly projection neurons. But, each POU-domain factor is required by different kinds of projection neurons. Although a particular class of projection neuron can express all three Pou4f factors, the one that is activated first performs the critical functions. For example, in the retina, all three factors are present in overlapping subsets of differentiated RGCs, but Pou4f2/Brn3b expression initiates expression

Atoh7LacZ

Pou4f2

GCL

(b)

Figure 2 Atoh7 and Pou4f2 expression during RGC development. (a) E11.5 Atoh7LacZ/þ embryo expressing b-galactosidase in the optic cup (detected through a chromagenic reaction that turns cells in the eye blue).

(b) Anti-b-galactosidase (which detects cells with Atoh7LacZ expression) and anti-Pou4f2 double immunofluorescent staining of E13.5 central optic cup section. Arrows point to Atoh7expressing cells completing mitosis, which then migrate to the GCL. During migration, these cells initiate Pou4f2 expression

(in yellow). Atoh7/Pou4f2 co-expressing cells are in white. Pou4f2 activates downstream target genes for RGC differentiation, axon outgrowth/guidance or survival. The bacterial b-galactosidase has a long half-life in mouse tissues, thus is present in mature RGCs, while endogenous Atoh7 is absent from the GCL (bracket).

about 2 days ahead of the others. The loss of either Pou4f1/Brn3a or Pou4f3/Brn3c has no effect on RGC development, although trigeminal and dorsal root ganglia critically require Pou4f1, and cochlear and vestibular neurons need Pou4f3. However, Pou4f2/Brn3b is a key regulator of RGC development, and mutant adult eyes have extremely thin optic nerves lacking 70% of the RGCs. In contrast to Atoh7 mutants, Pou4f2 mutants have differentiated RGCs that extend axons through the optic nerve, but subsequently fail to innervate the correct regions of the brain and die because these cells lack a critical survival factor. Although Pou4f1 and Pou4f3 seem dispensable for retinal development, they act redundantly with Pou4f2. Pou4f1–/–;Pou4f2–/– and Pou4f3–/–;Pou4f2–/– double mutants have a more profound loss of RGCs than Pou4f2 single mutants. Moreover, overexpression of individual Pou4f genes in chick RPCs demonstrated that each is fully capable of inducing RGCs. Finally, Pou4f3 rescues the loss of Pou4f2 in retinal explant cultures; and targeted replacement of Pou4f1 into the Pou4f2 locus restores RGC development and survival.

Pou4f2 regulates two distinct molecular pathways during RGC development. In the first, Pou4f2 turns on unknown downstream genes within the earliest differentiating RGCs. This subset of RGCs can develop as if Pou4f2 independent, because in Pou4f2 mutants Pou4f1 and Pou4f3 expression rescues them. The second, Pou4f2dependent pathway produces the bulk of RGCs at later embryonic ages. Here, Pou4f2 regulates a distinct set of downstream genes from those found in the earliest RGCs, including the transcription factors distal-less homeobox 1 and 2 (Dlx1 and Dlx2). When Pou4f2 function is removed from the mouse optic cup, the early RGCs persist, due to Pou4f1 and Pou4f3 compensation. However, the larger, Pou4f2-dependent RGC population cannot complete neuronal differentiation, correctly innervate the brain or express survival factors. This results in mutant eyes with abnormally thin optic nerves. Interestingly, these circumstances point to the Pou4f2 mutant mouse as an excellent system in which to study optic nerve regeneration. For instance, could ex vivo cultured (and introduced) RPCs, or early differentiating RGCs, use the Pou4f2 mutant reduced optic nerve, as a substrate for projecting their axons to the correct regions of the brain? Or, could Pou4f1 and Pou4f3 expression be manipulated (activated or derepressed) within the second, later group of developing RGCs to rescue their outgrowth and survival defects?

Pou4f2/Brn3b Controls Numerous

RGC Processes

Multiple studies indicate that Pou4f2 is strictly a transcriptional activator, with numerous potential downstream target genes. The list of identified downstream genes

includes Pou4f2 itself (autoregulation), Pou4f1 (crossregulation), the transcription factor eomesodermin homolog (Eomes), the anti-apoptosis factor B-cell lymphoma-2 associated protein (Bcl2), the signaling molecule Shh and multiple cytoplasmic or cell surface proteins, including actin-binding LIM protein 1 (Ablim1), which participate in axon outgrowth, guidance, or vesicular trafficking at synapses. Among these Eomes, Ablim1, and Shh are described here as examples that highlight the diverse set of genes that Pou4f2 directly or indirectly regulates in nascent RGCs. First, Eomes is a transcription factor with a T-box DNA-binding protein motif. During the period of maximal Pou4f2 embryonic expression, Pou4f2 directly activates Eomes transcription in differentiated RGCs. Eomes is also expressed by a subset of inner nuclear retinal cells, potentially amacrine cells, but the role of this factor in the inner nuclear layer remains unexplored. When Eomes function was removed during retinal development, mutant mice have thinner than normal optic nerves, which reflect a postnatal loss of RGCs. Although initial RGC development does not require Eomes activity, myelination of RGC nerve fibers does, and this defect causes elevated RGC death in Eomes mutants. This myelination defect is very similar to the one that has been described for Pou4f2 mutant mice.

Pou4f2 function is also necessary for the proper expression of proteins that direct neurite outgrowth, axon guidance, and neuronal function at synapses. One such gene is Ablim1, encoding an actin-binding protein, with a LIM protein–protein interaction domain. Misexpression of a mutant form of Ablim1 during chick optic nerve development causes RGC axon guidance defects. But paradoxically, Ablim1 mutant mice have no retinal or optic nerve defects. This implies that Ablim1 acts redundantly with another molecule during RGC axon outgrowth. Because there are two other Ablim genes in mammals, future studies are needed to test the role of these genes, singly and in combination with each other and Ablim1, to understand if Pou4f2 regulation of Ablim expression is sufficient to explain the RGC axon guidance defects of Pou4f2 mutant mice. Finally, the search for additional genes regulated by Pou4f2 uncovered the secreted signaling molecule Shh.

Islet1 Acts Parallel to Pou4f2

During RGC Development

Although Atoh7 activation of Pou4f2 deploys an important molecular mechanism for generating RGCs, other factors also act at this same step in the genetic hierarchy of RGC. These genes, which largely encode transcription factors, require Atoh7/Ath5, but not Pou4f2, for their activation. There are several possibilities for how these additional factors act during RGC genesis. They may

regulate a separate set of target genes, cross-regulate or interact with Pou4f2 and its downstream targets, or both. Several candidates that appear to act parallel with Pou4f2 are lim-homeodomain protein 1, Islet1 (Isl1), transducinlike enhancer of split 1 homolog (Tle1), and myelin transcription factor (Myt1). At present only Isl1 is known to function during RGC formation, and retinal-specific deletion of Isl1 results in adult mice lacking 95% of RGCs. This profound loss is more like the Atoh7 phenotype than it is to that of Pou4f2, with the main difference being the developmental age when RGCs are affected. The Isl1 transcription factor contains both a protein– protein interaction LIM-domain and a DNA-binding homeo-domain. Isl1 function is not only important during development in multiple tissues, like the spinal cord and pancreas, but also for cholinergic amacrine and bipolar neuron genesis in the retina. In optic cups devoid of Isl1 function, RGCs are specified and differentiate on schedule, including activating expression of Pou4f2 and other differentiation markers. But by birth, most RGCs downregulate Pou4f2 expression and then rapidly die. Closer examination of Isl1 mutant RGCs showed both aberrant axon projections and reduced Ablim1 expression, reminiscent of Pou4f2 mutants. Importantly, Pou4f2 and Isl1 act synergistically to control the activation of shared downstream genes, like Pou4f2, Pou4f1, and Shh. Thus, more work is needed to determine which genes Isl1 and Pou4f2 regulate in common versus those controlled by one factor or the other. Since presumably Tle1 and Myt1 also regulate similar processes during RGC formation, working out the combinatorial code of which transcription factors regulate each downstream gene is still needed.

Conclusion

As the genetic hierarchy of RGC development is deciphered, these studies draw us increasingly closer to understanding the sequence of steps for directing RPCs

out of the cell cycle to differentiate as RGCs, and how these neurons make appropriate connections in the brain.

See also: Coordinating Division and Differentiation in Retinal Development; Embryology and Early Patterning; Eye Field Transcription Factors; Histogenesis: Cell Fate: Signaling Factors; Information Processing: Ganglion Cells; Intraretinal Circuit Formation; Photoreceptor Development: Early Steps/Fate; Retinal Histogenesis.

Further Reading

Brown, N. L., Patel, S., Brzezinski, J., and Glaser, T. (2001). Math5 is required for retinal ganglion cell and optic nerve formation. Development 128: 2497–2508.

Erkman, L., Yates, P. A., McLaughlin, T., et al. (2000). A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system. Neuron 28: 779–792.

Hatakeyama, J. and Kageyama, R. (2004). Retinal cell fate determination and bHLH factors. Seminars in Cell and Developmental Biology 15: 83–89.

Kanekar, S., Perron, M., Dorsky, R., et al. (1997). Xath5 participates in a network of bHLH genes in the developing Xenopus retina. Neuron 19: 981–994.

Kay, J. N., Finger-Baier, K. C., Roeser, T., Staub, W., and Baier, H. (2001). Retinal ganglion cell genesis requires lakritz, a zebrafish atonal homolog. Neuron 30: 725–736.

Livesey, F. J. and Cepko, C. L. (2001). Vertebrate neural cell-fate determination: Lessons from the retina. Nature Review Neuroscience 2: 109–118.

Marquardt, T. (2003). Transcriptional control of neuronal diversification in the retina. Progress in Retinal and Eye Research 22: 567–577.

Mu, X., Fu, X., Beremand, P. D., Thomas, T. L., and Klein, W. H. (2008). Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2.

Proceedings of the National Academy of Sciences of the United States of America 105: 6942–6947.

Pan, L., Deng, M., Xie, X., and Gan, L. (2008). ISL1 and BRN3B coregulate the differentiation of murine retinal ganglion cells. Development 135: 1981–1990.

Rapaport, D. H. (2006). Retinal neurogenesis. In: Sernagor, E., Eglen, S., Harris, B., and Wong, R. (eds.) Retinal Development, pp. 30–58. New York: Cambridge University Press.

Wang, S. W., Kim, B. S., Ding, K., et al. (2001). Requirement for Math5 in the development of retinal ganglion cells. Genes and Development 15: 24–29.

Glossary

Electroretinogram (ERG) – In vivo extracellular recording of the voltage response to light of the entire retina.

G protein – Guanine nucleotide-binding protein, a ubiquitous biological switch that is turned on by receptor-activation-induced exchange of bound guanosine diphosphate (GDP) with cytoplasmic guanosine triphosphate (GTP) and turned off by hydrolysis of the bound GTP.

Phosphoinositide (PI) signaling – Also designated as inositol-lipid signaling, it is a ubiquitous enzymatic cascade that uses phospholipids or their products for signaling.

Phospholipase C (PLC) – A superficial membranebound enzyme that hydrolyzes a minor plasma membrane phospholipid, phosphatidylinositol- 4,5-bisphosphate (PIP2), and produces two messengers: inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG).

Photochemical cycle – A multistep process that begins by absorption of a photon by the lightsensitive receptor, rhodopsin, which undergoes multiple modifications and ends by the regeneration of the original molecule.

Phototransduction – The process by which absorption of photons by light-sensitive receptors lead to production of electrical signal comprehensive to the nervous system.

Prolonged depolarizing afterpotential (PDA) –

The light-induced electrical signal that continues in the dark long after light is turned off, produced in invertebrate photoreceptor cells. The PDA phenomenon has been widely exploited to screen for visual defective Drosophila mutants.

Rhabdomere – The photosensitive organelle of Drosophila photoreceptors which is composed of tightly packed microvilli.

Transient receptor potential (TRP) channels –

A family of cation channel proteins that mediate a large variety of physical stimuli such as light, temperature, and chemical compounds.

Phototransduction is a process by which light is converted into electrical signals understood by the central nervous system. Initial studies of invertebrate phototransduction

used Balanus and Limulus as the preparations of choice, due to their extremely large photoreceptor cells and the assumption that they constitute a simplified model system that represents phototransduction in general. However, later studies revealed that although both invertebrate and vertebrate photoreceptors use rhodopsin (R) as a receptor and a G (guanine nucleotide-binding)-protein- coupled signaling cascade to produce the electrical response to light; the rest of their enzymatic cascade of vision is entirely different. Phototransduction in vertebrate rods and cones uses cyclic guanosine monophosphate (cGMP) phosphodiesterase as an effector enzyme and cGMP-gated ion channels as its target. In contrast, phototransduction in the microvillar photoreceptors of invertebrates (photoreceptors in which the photosensitive organelle is composed of microvilli – the rhabdomere) uses a phosphoinositide (PI) signaling cascade, which is characterized by phospholipase C (PLC) as the effector enzyme and, at least in Drosophila, the TRP channels as its target. Nevertheless, phototransduction in invertebrate microvillar photoreceptors has become a model system for investigating inositol-lipid signaling and its role in TRP channel regulation and activation. The great advantage of using invertebrate microvillar photoreceptors is the accessibility of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics. The latter feature is mainly attributed to Drosophila melanogaster as a preferred preparation.

Although the phototransduction cascade of invertebrate microvillar photoreceptors is clearly different from that of vertebrate rods and cones, it may be similar to that in the intrinsically photosensitive retinal ganglion cells (RGCs) in vertebrates. The light-sensitive RGCs contain the visual pigment melanopsin and provide photic input to the circadian pacemaker of the master circadian clock. Both Drosophila photoreceptors and the light-sensitive RGCs are characterized by a bi-stable R that initiates a phototranduction cascade with TRP channels as the final target. It is now widely accepted that the detailed knowledge that has been obtained through studies of Drosophila photoreceptors constitute guidelines for research of the melanopsin-containing RGCs, and that a striking commonality has been found in both cell types.

Extensive genetic studies of the fruit fly, D. melanogaster, initiated at the turn of the twentieth century, by the Nobel prize laureate, Thomas Hunt Morgan, and greatly advanced by many other laboratories, has established the Drosophila as an extremely useful experimental model for genetic

dissection of complex biological processes. The relatively small size of the Drosophila genome, ease of growth, rapid generation time, and high fecundity make this system ideally suited for screening large numbers of mutagenized individual flies for defects in virtually any phenotypically observable or measurable trait. The creation of balancer chromosomes – containing dominant markers and multiple inversions – which prevent recombination with the native chromosomes, allows any mutation – once recognized – to be rapidly isolated and maintained. Importantly, germ line transformation, using P-element transposition, combined with the availability of tissue-specific promoters, allows the introduction of cloned genes into specific cells of the

organism. This provides a way to study in vitro modified gene products in their native cellular environment. These powerful molecular genetic tools, combined with the available genome sequence, allow screening for mutants defective in critical molecules, while devoid of a priori assumptions. Indeed, this methodology has produced large numbers of mutants defective in novel proteins the existence of which would have been difficult to otherwise predict. The following sections emphasize the unique contribution of the research in Drosophila photoreceptors, which has led to the discovery of novel molecules and processes important not only for phototransduction but also for many other biological mechanisms (Figure 1).

The Drosophila Phototransduction

Cascade

Genetic Screens for Mutants Defective in Phototransduction Proteins

A genetic screen is a procedure to identify and select mutated individuals that possess a phenotype with a specific malfunction in a specific trait. This method requires a large number of mutated individuals, usually obtained by the use of mutagens or by random DNA insertions (transposons) and a simple, but powerful, isolation procedure. Additionally, this method requires genetic tools to isolate and maintain the stability of the mutation through generations. These requirements make Drosophila an ideal organism for genetic screens. Wide genetic screens targeting the two autosomes (II and III) and the X chromosome of Drosophila, have produced many mutants with identified phenotypes linked specifically to genes involved in visual behavior or photoreceptor cell function. These screens used defects in a number of visual functions such as optomotor response, phototaxis, and electrophysiological response to light. The isolation of mutants, specifically defective in the visual transduction pathway initially made use of the electroretinogam (ERG), which measures the electrical activity of the eye at the corneal level (Figure 1(a)). However, the ERG methodology failed to isolate the large numbers of mutants expected from a multistep and complex process such as the phototransduction cascade. The main reasons for this failure are that the phototransduction proteins are highly abundant and the upper limit in the depolarization signal is reached even when only a small fraction of the signaling molecules are excited. Therefore, mutations causing even a significant reduction in concentration or subtle malfunction of the phototransduction components could not be identified by this method. This necessitated the employment of a more sensitive, and yet simple, method for isolation of mutants.

In order to find mutants that are specifically affected in the phototransduction cascade and to identify those with subtle phenotypes, Pak and colleagues employed the electrical phenomenon devised by Hillman Hochstein and Minke designated prolonged depolarizing afterpotential (PDA; Figure 2(a), upper trace). This method is based on a large net photoconversion of R to its dark stable photoproduct metarhodopsin (M) with a minimal conversion back to R (Figure 3), which brings the capacity of the phototransduction process to its upper limit. PDA is achieved in the fly by genetically removing the red screening pigment (Figure 1(a)) and by applying blue light, which is preferentially absorbed by the R state of the Rh1 photopigment. Rh1 is the photopigment expressed in photoreceptors R1–R6 in the Drosophila eye (Figure 1(a) and (b)). A large net photoconversion of R to M (Figure 3) prevents phototransduction termination at the photopigment level.

As a result, excitation is sustained long after the light is turned off, and the PDA-producing cells are unable to respond further (termed inactivation, Figure 2(a), upper trace). Subsequently, application of orange light reconverts the activated M back to R and terminates the sustained excitation after the light is turned off. Since the PDA tests the maximal capacity of the photoreceptor cell to maintain excitation for an extended period and is strictly dependent on the presence of high concentrations of R and other signaling molecules, it detects even minor defects in R biogenesis, or exhaustion of critical signaling molecules. Thus, defects in the PDA easily reveal deficiencies in the concentration of phototransduction components. Indeed, the PDA screen yielded a plethora of novel and interesting visual mutants. One group of PDA mutants exhibited a loss in several features of the PDA. They were termed nina mutants, which stands for neither inactivation nor afterpotential (Figure 2(a), lower trace). Most nina mutants were caused by reduced levels of R. The second group of PDA mutants lost the ability to produce the voltage response associated with the PDA, but were still inactivated by strong blue light and the inactivation could be relieved by orange light (Figure 2(a), middle trace). These mutants, consisting of seven allelic groups, are termed inaA–G, which stand for inactivation but no afterpotential (no PDA-voltage response). The ina mutants were found to have normal R levels but are deficient in proteins associated with the function of the TRP channel. The nina and ina mutants have led to the identification of most of the crucial components of Drosophila phototransduction, many of which are novel proteins of general importance for many cells and tissues.

The Photochemical Cycle and the

Mechanism Underlying Termination

of M Activity

The ninaE mutant, having reduced Rh1 opsin levels, was isolated by Pak and colleagues using the PDA screen. R belongs to the super family of seven transmembrane G-protein-coupled receptors. It is activated by the absorption of a photon that isomerizes the chromophore (11-cis 3-hydroxy retinal), resulting in a conformational change of the opsin molecule and production of the physiologically active M state of the photopigment. To ensure high sensitivity, high temporal resolution, and low dark noise of the photoresponse, the active M has to be quickly inactivated and recycled (Figure 3). The latter requirement is achieved, in invertebrates, by two means: the absorption of an additional photon by the dark stable M – which photoconverts M back to R – or by a multistep photochemical cycle (Figure 3). The termination of vertebrate M activity is a two-step process initiated by M phosphorylation, followed by the binding of the protein arrestin (ARR) to phosphorylated M. Invertebrate M also undergoes

light-dependent phosphorylation (Figure 3; Mpp), but this process is not required for response termination. This was

demonstrated in experiments which showed that two Drosophila transgenic mutants, P[Rh1D356] and P[Rh1S to A], ex-

pressing Rs that lack the putative phosphorylation sites exhibit normal inactivation and ARR binding. However, as in the photoreceptors of vertebrates, the binding of ARR to M in Drosophila photoreceptors inactivates M (Figure 3). Drosophila has two protein homologs to the vertebrate ARR, both undergo light-dependent phosphorylation: the 49-kDa ARR (ARR2) and the 39-kDa ARR (ARR1). The phosphorylation of ARR2 is evident at dim light, while phosphorylation of ARR1 requires stronger light intensities. Both arrestins are phosphorylated by a

Ca2+-calmodulin-dependent protein kinase II (CaMK II). ARR phosphorylation is required for the dissociation of ARR from the phosphorylated M (Mpp) upon photoconversion and for preventing endocytotic internalization of the ARR2–Mpp complex (Figure 3).

Based on an understanding of the photochemical cycle, Minke and Selinger explained the PDA phenomenon as follows: cellular ARR2 is present at a concentration which is insufficient to inactivate all the M generated by a large net photoconversion of R to M, leaving an excess of M persistently active in the dark. This explanation easily accounts for the elimination of the PDA response by mutations or by carotenoid deprivation which reduce the cellular level of R. It also accounts for the need to