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In Vivo Development of Retinal ON-Bipolar Cell Axonal Terminals Visualized in Nyx::MYFP Transgenic Zebrafish

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In Vivo Development of Retinal ON-Bipolar Cell Axonal Terminals Visualized in Nyx::MYFP Transgenic Zebrafish Visual Neuroscience ~2006!, 23, 833–843. Printed in the USA. Copyright © 2006 Cambridge University Press 0952-5238006 $16.00 DOI: 10.10170S0952523806230219 In vivo development of retinal ON-bipolar cell axonal terminals visualized in nyx::MYFP transgenic zebrafish ERIC H. SCHROETER,1 RACHEL O.L. WONG,1* and RONALD G. GREGG2* 1Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 2Department of Biochemistry and Molecular Biology, and Center for Genetics and Molecular Medicine, University of Louisville, Louisville, Kentucky (Received February 13, 2006; Accepted May 12, 2006! Abstract Axonal differentiation of retinal bipolar cells has largely been studied by comparing the morphology of these interneurons in fixed tissue at different ages. To better understand how bipolar axonal terminals develop in vivo,we imaged fluorescently labeled cells in the zebrafish retina using time-lapse confocal and two photon microscopy. Using the upstream regulatory sequences from the nyx gene that encodes nyctalopin, we constructed a transgenic fish in which a subset of retinal bipolar cells express membrane targeted yellow fluorescent protein ~MYFP!. Axonal terminals of these YFP-labeled bipolar cells laminated primarily in the inner half of the inner plexiform layer, suggesting that they are likely to be ON-bipolar cells. Transient expression of MYFP in isolated bipolar cells indicates that two or more subsets of bipolar cells, with one or two terminal boutons, are labeled. Live imaging of YFP-expressing bipolar cells in the nyx::MYFP transgenic fish at different ages showed that initially, filopodial-like structures extend and retract from their primary axonal process throughout the inner plexiform layer ~IPL!. Over time, filopodial exploration becomes concentrated at discrete foci prior to the establishment of large terminal boutons, characteristic of the mature form. This sequence of axonal differentiation suggests that synaptic targeting by bipolar cell axons may involve an early process of trial and error, rather than a process of directed outgrowth and contact. Our observations represent the first in vivo visualization of axonal development of bipolar cells in a vertebrate retina. Keywords: Zebrafish, Bipolar cell, Nyctalopin, Axonal development Introduction Cepko, 2005!, Bhlhb4 ~Bramblett et al., 2004!, Vsx1 ~Chow et al., 2004!, and Irx5 ~Cheng et al., 2005!, affect the differentiation of Retinal bipolar interneurons form the essential link between photo- bipolar cells. Immunolabeling for vesicular glutamate transporters receptors and the output layer of the retina, the ganglion cell layer. also provided interesting insights into when ON and OFF bipolar Synaptic connections in the inner retina between bipolar cells and cell axonal terminals differentiate ~Sherry et al., 2003!. Ultrastruc- the retinal ganglion cells are organized broadly into two major tural studies across many species have revealed when bipolar cells laminae ~reviewed by Wassle, 2004!. Connectivity between retinal make synapses in the OPL and IPL ~e.g., Olney, 1968; Dubin, cells that are depolarized by increased illumination ~ON-center 1970; Nishimura & Rakic, 1987; Crooks et al., 1995; Schmitt & cells! is restricted approximately to the inner half to two-thirds of Dowling, 1999!. the inner plexiform layer ~IPL!. Conversely, connections between Although much is known about bipolar cell development across retinal neurons that are hyperpolarized by increased illumination many species, how immature bipolar cells target their axons to the ~OFF-center cells! occupy the outer third to half of the IPL. Axon appropriate ON or OFF sublamina requires further study. To do so, terminals of ON and OFF bipolar cells thus stratify in distinct complete labeling of bipolar axonal terminals across development layers within the IPL. is necessary. This can be achieved in part, by immunostaining for Studies in the past have investigated the molecular and cellular various proteins specific to bipolar cells, but such an approach factors regulating bipolar cell development. For example, loss of generally leads to labeling of populations of cells, which limit transcription factors Chx10 ~Burmeister et al., 1996; Rowan & resolution of the morphology of individual terminals ~e.g., Miller et al., 1999; Gunhan-Agar et al., 2000, 2002; Kay et al., 2004!. Also the axonal terminals of developing bipolar cells are already laminated at the earliest ages when immunolabeling reveals their Address correspondence and reprint requests to: R.O.L. Wong, Department of Biological Structure, University of Washington, Seattle, morphology, making it difficult to assess bipolar cell structure WA 98125. E-mail: [email protected] prior to axonal stratification. Golgi techniques applied to fixed *The authors contributed equally. retinas at different ages have, however, enabled comparison of the 833 834 E.H. Schroeter et al. axonal and dendritic morphology of bipolar cells across develop- et al., 2000; Gregg et al., 2003!, although only exons 2 and 3 ment ~Quesada et al., 1981; Quesada & Genis-Galvez, 1985!. But, contain coding sequence. In silico analyses of zebrafish se- direct comparison of developmental changes of individual cells or quences also identified a gene that generates a predicted cDNA within a subtype of bipolar cells is difficult with this approach. sequence encoding the zebrafish nyctalopin protein ~Accession # While much insight has been gained from studies utilizing XM_692177!. Experimental analyses of cDNA clones from zebra- fixed tissue, live imaging approaches are required to reveal the fish indicate the nyx gene in this species also contains 3 exons dynamic behaviors underlying structural changes resulting in the ~data not shown! encoding a predicted protein of 449 amino mature morphology. In recent years, it has become feasible to label acids that shares 51% identity with the mouse protein. A DNA and visualize live retinal cells by driving expression of fluorescent fragment that extended 1482 bp upstream of the 3' end of proteins using ubiquitous or cell-specific promoters ~reviewed by exon 2 ~position 25 in the ORF, Acc # XM_692177! was cloned Lohmann et al., 2005; Morgan et al., 2005; Mumm et al., 2005!. by PCR ~primers: AC-CGGCAATATTGATGATGA; GAAACG- Most recently, bipolar cell development has been studied in retinal CAAGAAATAAGCATGA! from genomic DNA. This fragment explants from transgenic mice in which ON bipolar cells express was cloned into a modified pCS2ϩ Gal4/VP16 plasmid ~Koster green fluorescent protein ~GFP! under the control of the mGluR6 & Fraser, 2001! resulting in pZNYX-GalVP16. The final con- promoter ~Morgan et al., 2006!. But, for technical reasons, it is not struct is shown schematically in Fig. 1A. Intron 1 was included yet possible to follow the in vivo development of retinal bipolar in the hope that providing a splice site would improve in vivo cells in mammals. Such observations, however, are readily achieved expression of the Gal-VP16 protein. The Gal40VP16 expression using zebrafish. system was used to amplify expression levels since nyx mRNA The combination of rapid embryonic development of the ze- levels in mouse retina are low ~Gregg et al., 2003!. This driver brafish retina and the transparency of the embryos permit visual- plasmid is also modular, allowing us to use it with reporter ization, and time-lapse imaging of retinal cells marked by fluorescent plasmids containing the 14X UAS E1b promoter ~Koster & proteins during the period when their circuitry is forming. For Fraser, 2001!. Three different reporter plasmids expressing dif- example, using stable transgenic zebrafish lines in which subsets ferent fluorescent proteins were used in the present study. pUAS- of amacrine cells express various spectral variants of green fluo- MYFP and pUAS-MCFP express membrane-targeted versions of rescent protein, recent studies have determined how amacrine cell EYFP and ECFP respectively and were created by cloning the neurites ramify within the IPL early in development ~Kay et al., 14XUAS E1b promoter ~Koster & Fraser, 2001! into pEYFP-N1 2004; Godinho et al., 2005!. Here, we explored how retinal bipolar or pECFP-N1 ~Clontech!, replacing the CMV promoter, along cells in zebrafish obtain their appropriate stratification level in the with the first 20 amino acids of the zebrafish GAP43 gene fused IPL and form bouton-like terminals with maturation. to the amino terminus of the FP coding sequence. pUAS- To label bipolar cells, we searched for a suitable promoter to DsRedExpress was a generous gift from Martin Meyer ~Stanford drive expression of the fluorescent proteins. The nyx gene encodes University! and has the 14XUAS E1b promoter cloned into a small leucine rich proteoglycan that is mutated in human X-linked pDsRed-Express-1 ~Clontech!. Congenital Stationary Night Blindness ~CSNB1, Bech-Hansen et al., 2000; Pusch et al., 2000!. Loss of nyctalopin in mice results Injections in loss of the ERG b-wave ~Gregg et al., 2003!, which is derived from signaling through ON bipolar cells. We show here that Injections of DNA and imaging were performed essentially as de- sequences from the zebrafish nyx promoter are able to drive scribed previously ~Lohmann et al., 2005!. Briefly, DNA was di- expression in a subset of ON bipolar cells. We generated a trans- luted in 1X Danieau’s solution ~58 mM NaCl, 7 mM KCl, 0.6 mM genic line in which morphologically defined ON-bipolar cells Ca~NO3!2, 0.4 mM MgSO4, 5 mM HEPES, pH 6.8! at a final
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