June 2002
Volume 43, Issue 6
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Retina  |   June 2002
Cellular and Developmental Distribution of Human Homologues of the Drosophilia rdgB Protein in the Rat Retina
Author Affiliations
  • Donghua Tian
    From the Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
  • Sima Lev
    From the Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
Investigative Ophthalmology & Visual Science June 2002, Vol.43, 1946-1953. doi:
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      Donghua Tian, Sima Lev; Cellular and Developmental Distribution of Human Homologues of the Drosophilia rdgB Protein in the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2002;43(6):1946-1953.

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Abstract

purpose. The Nirs (Nir1, Nir2, and Nir3), human homologues of Drosophila retinal degeneration B (rdgB), have been considered candidate genes for human inherited retinal degeneration diseases. To gain a better understanding of their functions in the retina and their putative roles in retinal degeneration diseases, this study was undertaken to determine their distribution profile in developing and mature rat retinas.

methods. Specific antibodies against each of the Nir proteins were raised in rabbits and used in indirect immunofluorescence analysis to determine the distribution profile of the three proteins. Eyes from Wistar rats at various developmental stages (embryonic day [E]18 to postnatal day [P]16) were sectioned vertically and immunostained with anti-Nir antibodies. Coimmunostaining for Nirs and several specific cellular and subcellular markers was used to determine precisely the cellular and subcellular distribution of the Nirs. Sections were observed under a confocal laser microscope, and image analysis was performed with the standard operating software provided with the microscope.

results. Confocal microscopic analysis of Nir1 immunoreactivity revealed that it was predominantly expressed in premature Müller cells at birth and that it was upregulated during Müller cell maturation. In contrast, Nir2 and Nir3 were homogeneously distributed in undifferentiated neuroblasts and ganglion cells at birth and later became distinctly distributed in newly differentiated neuronal cells. From P4, Nir2 and Nir3 were highly expressed in neuronal cells and their processes, coinciding with the formation of synaptic layers and ongoing synaptogenesis. From P12, Nir2 was uniformly expressed in all classes of retinal neuronal cells, including ganglion cells, horizontal cells, amacrine cells, bipolar cells, and photoreceptor cells. In the adult rat retina, Nir2 was preferentially localized to the somata of all classes of retinal neurons, whereas Nir3 was highly expressed in the synaptic terminals. This specific localization of Nir3 was confirmed by double immunostaining with the presynaptic protein synaptosomal-associated protein (SNAP)-25. In photoreceptor cells, both Nir2 and Nir3 were found to be highly expressed in the inner segments but were not detectably expressed in the outer segments.

conclusions. These findings suggest that the three Nir proteins are highly expressed in the developing retina, each exhibiting a distinct distribution profile. The different distribution patterns of these closely related proteins during development and at maturity may reflect their different cellular functions in vivo and their different roles in retinal cell survival or degeneration.

All vertebrate retinas have the same basic laminar organization and physiological function. During retinal development, postmitotic cells generated in the germinative layer migrate laterally to form the striated, laminar pattern of the retina. 1 These cells differentiate into either neuronal or Müller glial cells. Neuronal cells can be subdivided into three different classes: light-sensitive photoreceptor neurons (cones and rods), interneurons (bipolar, horizontal, and amacrine cells), and retinal ganglion cells (RGCs). 2 Similar to many other neuronal tissues, the different cell types are generated at different stages of development. The RGCs appear first, followed by the horizontal cells, the cone photoreceptors, amacrine cells, bipolar cells, and finally the rod photoreceptor cells. 2 3 The light-sensitive photoreceptor cells are responsible for light absorption and propagation of a phototransduction cascade. The light signal is transmitted from the photoreceptor cells through the bipolar cells and ganglion cells to the visual cortex in the brain. This signaling pathway consists of a series of amplification signals that enable detection of a single photon of light. Disruption of any step in this pathway may cause visual impairment and blindness. A key step in understanding the molecular basis of visual impairment is to identify proteins that are involved in phototransduction, photoreceptor cell metabolism, or structural support of photoreceptors 4 5 or that are required for retinal morphogenesis and differentiation. Several genes that are essential for mammalian eye development, such as the paired-like homeobox genes, have been implicated in human inherited retinal degeneration disease and blindness. The homeobox Crx gene, which causes an autosomal dominant form of cone-rod dystrophy (CRD), 6 7 was first considered a candidate gene for CRD, because of its chromosomal localization. Other genes involved in inherited retinal degeneration diseases have also been considered candidates, based on their chromosomal localization or on their homology to genes involved in retinal degeneration in other organisms. 8 9 Mutations of several human homologues of Drosophila phototransduction genes, including rhodopsin and arrestin, have been shown to induce retinal degeneration in both species. 10 11  
The power of Drosophila genetics has led to the isolation and characterization of genes encoding proteins expected to participate in the visual transduction pathway. 12 The retinal degeneration B (rdgB) gene was one of the first Drosophila retinal degeneration mutants identified. rdgB mutant flies exhibit light-enhanced retinal degeneration and defects in the electroretinogram (ERG). The morphology of its six peripheral photoreceptor cells (R1–R6) is normal in the dark, but a single flash of light triggers irreversible retinal degeneration. 13 14 We have recently isolated a novel family of human genes, which are the mammalian homologues of the Drosophila rdgB. These genes (Nir1, Nir2, and Nir3) are highly expressed in the adult rat retina, 15 and two of them have been mapped to chromosomal regions associated with human inherited retinal degeneration diseases. 15 16 17 Nir1 was mapped to human chromosome 17p13.1 near the marker D17S938. 15 The D17S938 locus is linked to at least three retinal disorders: Leber congenital amaurosis (LCA), 18 cone–rod dystrophy (CORD5), 19 and autosomal dominant retinitis pigmentosa (adRP). 20 The Nir2 gene (also known as H-rdgB) has been mapped to human chromosome 11q13.1. 21 22 The 11q13.1 locus is in the immediate vicinity of four human retinal diseases, including recessive Bardet-Biedle syndrome-1, 23 dominant vitelliform macular dystrophy (Best disease), 24 25 dominant Criswick-Schepens syndrome, 26 and dominant neovascular inflammatory vitreoretinopathy. 27 Based on these chromosomal localizations and their homology to the Drosophila rdgB, the Nirs can be considered candidate genes for inherited human retinal degeneration diseases. 
To gain a better understanding of Nir functions in the retina and their putative roles in retinal degeneration, we determined the precise distribution profile of the three Nir proteins in the developing and adult rat retina. Rat retinal sections at different developmental stages (embryonic day [E]18 to postnatal day [P]16) were either immunostained with anti-Nir antibodies or were costained with several cell- and subcell-specific markers. The results of these immunolocalization studies were analyzed by confocal laser scanning microscopy. We found that Nir1 was predominantly expressed in Müller cells at birth and that its expression was upregulated during Müller cell maturation. In contrast, Nir2 and Nir3 were mainly localized to various retinal neurons, with an enhanced expression in the inner segments of photoreceptor cells. Nir2 was preferentially localized to the somata of all classes of retinal neurons, whereas Nir3 was highly expressed in the synaptic terminals. The different distribution patterns of these closely related Nir proteins during retinal development suggest that they may have different cellular functions. 
Materials and Methods
Rats
All experimental procedures were performed in conformity with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wistar rats at ages ranging from E18 to P16 and adult rats were obtained from the Animal Breeding Unit of the Weizmann Institute of Science. The day of birth was designated as P0. To ensure uniformity in the developmental studies, we used littermates. 
Antibodies
Polyclonal antibodies against Nir1, Nir2, and Nir3 were raised in rabbits immunized with KLH-conjugated synthetic peptides corresponding to amino acids 965 to 974 of Nir1, 287 to 301 of Nir2, and 652 to 666 of Nir3. All polyclonal antibodies were affinity purified from rabbit antiserum by using the cognate antigens immobilized on agarose beads. Monoclonal antibody against synaptosomal-associated protein (SNAP)-25 were purchased from Chemicon (Temecula, CA), monoclonal antibodies against glial fibrillary acidic protein (GFAP) or parvalbumin and polyclonal antibody against protein kinase Cα (PKCα) from Sigma (St. Louis, MO), polyclonal antibody against calbindin from Swant (Bellinzona, Switzerland), and anti-HA monoclonal antibody from Roche Molecular Biochemicals (Mannheim, Germany). Monoclonal antibody against rhodopsin was kindly provided by Robert S. Molday (University of British Columbia, Canada) and biotinylated peanut agglutinin (biotin-PNA) and fluorescein-labeled avidin by Melvin I. Simon (California Institute of Technology, Pasadena, CA). 
Immunoprecipitation and Western Blot Analysis
Rat retinas were isolated and homogenized in lysis buffer containing 1% Triton X-100, 10% glycerol, 120 mM NaCl, 25 mM HEPES (pH 7.4), 1 mM EGTA, 20 mM NaF, 0.75 mM MgCl2, 0.1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/mL leupeptin, and 10 μg/mL aprotinin. Immunoprecipitation and immunoblotting with the different anti-Nirs antibodies was performed essentially as previously described. 28  
Tissue Preparation and Immunofluorescence
Wistar rats were killed at various developmental stages, beginning at E18, either by cervical dislocation under chloral hydrate anesthesia or by decapitation (E18–P6). Eyes were immediately enucleated, quick frozen in optimal cutting temperature OCT compound (Sakura Finetek, Tokyo, Japan), and stored at −80°C. Retinal sections of 15-μm thickness were prepared on a cryostat, mounted on gelatin-coated slides, and fixed, either with 4% paraformaldehyde in 0.1 M phosphate buffer for 20 minutes at room temperature (RT) or with 95% ethanol for 5 minutes at −20°C (specifically for anti-Nir1 polyclonal antibody). The sections were then washed three times for 5 minutes each in sodium phosphate-buffered saline (PBS) and laid flat in a dark, moist box for all subsequent incubations. Sections were incubated in blocking buffer (1% glycine, 10% goat serum, 2% bovine serum albumin, and 0.1% Triton X-100 in PBS) for 2 hours at RT and then incubated with various primary antibodies for 12 hours at 4°C. After several washes in 1× PBS and 0.1% Triton X-100, sections were incubated for 1 hour at RT with either Cy3-conjugated goat anti-rabbit IgG (2.0 μg/mL; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) or a mixture of donkey anti-mouse IgG (Alexa 488; 1 μg/mL; Molecular Probes, Inc., Eugene, OR) and Cy3-conjugated goat anti-rabbit IgG or Cy3-conjugated goat anti-rabbit IgG and fluorescein-labeled avidin. After they were washed, the slides were mounted in polyvinyl alcohol (Moviol 488; Calbiochem, La Jolla, CA) and examined by confocal fluorescence microscopy. 
Confocal Microscopy and Image Processing
Sections were observed under a confocal laser microscope (model 510; Carl Zeiss, Oberkochen, Germany) equipped with filters for fluorescein and Cy3 epifluorescence. Both 488- and 543-nm laser beams were used for excitation, and 505- to 530-nm band-pass and 560-nm long-pass filters for emission. Image analysis was performed using the standard operating software provided with the microscope (ver. 2.01; Carl Zeiss). All the images were taken under the same conditions and from approximately the same region of the retina (middle of the nasal side of vertical sections crossing the optic nerve). 
Results
Distribution Profiles of Nir Proteins in the Adult Rat Retina
We have shown that the Nirs (Nir1, Nir2, and Nir3) are highly expressed in the adult rat retina. 15 In the present study, we characterized the distribution profile of the Nirs at various developmental stages of the rat retina. An indirect immunofluorescence analysis was performed with affinity-purified polyclonal antibodies against Nirs. These antibodies, which had been raised in rabbits, as described in the Materials and Methods section, recognized a single protein of the expected size from either transfected HEK293 cells expressing Nir1, Nir2, or Nir3 (Fig. 1A) or from rat retinal lysates (Fig. 1B) . The specificity of these antibodies was analyzed by immunoblot analysis (Fig. 1A) , immunoprecipitation, or immunofluorescence analysis (data not shown), and no cross-reactivity was detected (Fig. 1A) . Although these antibodies were raised against the human proteins, each of them recognized a single protein from rat brain (data not shown) or rat retina (Fig. 1B) . These rat proteins exhibited apparent molecular weights similar to those of the human proteins, and were also detected by additional anti-Nir antibodies that were raised against different portions of the human proteins (data not shown). Several of them have been described. 15 Thus, the anti-Nir antibodies used in this study recognized both the human and the rat Nir proteins. 
To characterize the distribution of the Nirs in the adult rat retina, vertical retinal sections were immunostained with anti-Nir1, -Nir2, or -Nir3 antibodies and analyzed by confocal microscopy. The results shown in Figure 2 demonstrate the different distribution patterns of the three Nirs in the adult rat retina. Nir1 immunoreactivity exhibited brightly labeled end feet and radial fibers that extended from the inner to the outer limiting membrane, a characteristic distribution pattern of Müller cells. A low level of Nir1 expression was also detected in photoreceptor cells, particularly in their cell bodies and inner segments (IS). Nir1 immunoreactivity was also observed in the retinal pigment epithelium (RPE) and was evident in the inner (IPL) and outer plexiform layers (OPL). The localization of Nir1 in Müller cells was further confirmed by double-immunostaining analysis using antibodies against Nir1 and GFAP. It has been shown that GFAP is restrictively localized to the inner portion of Müller cells in normal adult rat retina. 29 30 31 Therefore, GFAP was used as a marker for Müller cells. The confocal images shown in Figure 3A clearly demonstrate the overlapping between Nir1 and GFAP in the inner half of Müller cells and their end feet, suggesting that Nir1 is highly expressed in Müller cells. Nevertheless, Nir1 immunoreactivity was not restricted to Müller cells, but rather appeared in other cell types distinct from GFAP immunoreactivity (Fig. 3A)
In contrast to Nir1, Nir2 was abundantly expressed throughout the retina, including the RPE and all classes of neuronal cells (Fig. 2B) . It was highly expressed in the IS of photoreceptor cells, but could not be detected in the outer segments (OS), as determined by double-immunostaining analysis with antibodies against rhodopsin, a marker for the OS of rod photoreceptor cells 32 and Nir3 (data not shown). Its immunoreactivity was mainly localized to the somata of photoreceptor cells and the inner nuclear layer (INL) neurons. Within the INL, Nir2 immunoreactivity was detected in all classes of neurons, including amacrine, horizontal, and bipolar cells. Its expression in amacrine cells was confirmed by double-immunostaining of retinal sections with antibodies against Nir2 and parvalbumin, a marker for amacrine cells. 33 The confocal images shown in Figure 3D indicate that Nir2 and parvalbumin immunoreactivities overlapped in parvalbumin-positive amacrine cells. Nir2 was also detected in horizontal and bipolar cells by immunostaining of serial sections for calbindin and PKCα (Figs. 3E 3G) . These two proteins are specific markers for horizontal and bipolar cells, respectively. 33 Calbindin immunoreactivity was obtained in horizontal cells localized in the uppermost portion of the INL (Fig. 3E) , consistent with previous reports. Nir2 immunoreactivity was obtained in similar cells (Fig. 3F) . Similarly, Nir2 immunoreactivity was detected in cells that exhibited morphologic characteristics of bipolar cells, as determined by parallel immunostaining of serial sections for PKCα (Fig. 3G)
Nir3, which was also abundantly expressed throughout the retina, exhibited a distinct distribution pattern compared with Nir1 or Nir2 (Fig. 2C) . It was highly expressed in the inner segments of photoreceptor cells, but less expressed in photoreceptor and other neuronal cell bodies. It was detected in the RPE, but was not present in the OS, as determined by double-immunostaining analysis with antibodies against Nir3 and rhodopsin (data not shown). However, its immunoreactivity was distinctly enhanced in the IPL and OPL and was visualized in long processes within the IPL. To determine whether the expression of Nir3 in the OPL reflects its accumulation in synaptic terminals, double-staining for Nir3 and either SNAP-25 or peanut agglutinin (PNA) was performed. SNAP-25, a presynaptic membrane protein that specifically localizes to the retinal neuronal cell somata and synaptic terminals, 34 35 was highly expressed in the OPL. PNA, which binds to the specific oligosaccharides of cone pedicles in the OPL, also accumulated in the OPL. 36 37 Double-staining of these two markers with anti-Nir3 antibodies revealed prominent overlapping, suggesting strongly that Nir3 is abundantly expressed in synaptic terminals (Fig. 4) . However, in similar costaining studies with anti-Nir2 antibodies, weak immunoreactivity of Nir2 was detected in the OPL, and accordingly, negligible overlapping with either SNAP-25 or PNA was found. In addition to the profound accumulation of Nir3 in synaptic terminals, Nir3 immunoreactivity was diffusely distributed in the outer portion of the INL, but slightly enhanced in the innermost portion of INL, in which it exhibited a putative amacrine cell morphology. 
Expression of Nir1 in the Developing Retina
To determine the expression profile of Nir1 during rat retinal development, retinal sections of rat at E18 through P16 were immunostained with anti-Nir1 antibody. At E18, the earliest age studied, moderate levels of Nir1 expression were detected throughout the retina (Fig. 5A) . From P0 to P2, Nir1 was abundantly expressed throughout the neuroblast and ganglion cell layers (NBL and GCL, respectively), exhibiting a pattern of distribution that resembled that obtained in a prior study with RT10F7, an antibody that specifically recognizes Müller cells. 38 At P4, a discontinuous Nir1-positive band was detected in the outer portion of the retina that most likely represents the premature OPL. At this stage of development, the NBL was divided into the INL and the outer nuclear layer (ONL) as a result of the OPL’s formation (Fig. 5D) . The expression of Nir1 was enhanced in the OPL from P6 to P8 (Figs. 5E 5F) . From P12 to P16, Nir1 immunoreactivity revealed rootlike structures in the GCL and continuous radial processes throughout the retina, which were probably the end feet and radial fibers of the Müller cells (Figs. 5G 5H) . To support this hypothesis, double-immunostaining analysis was applied with antibodies against Nir1 and GFAP. The confocal images shown in Figure 6 demonstrate that Nir1 and GFAP immunoreactivities overlapped and exhibited typical Müller cell morphology. Nevertheless, the immunolocalization of the two proteins was not identical, because Nir1 immunoreactivity was also detected in the IS, whereas no GFAP immunoreactivity was detected in that region. 
Expression of Nir2 in the Developing Retina
Next, we characterized the distribution of Nir2 during retinal development. Nir2 immunoreactivity was markedly different from that of Nir1. At E18, Nir2 immunoreactivity was homogeneously distributed throughout the retina, with a slightly stronger intensity in both the inner and outer portions (Fig. 7A) . At P0, Nir2 immunoreactivity appeared in the ganglion cells (defined by their localization and morphology), preferentially localized to the perikaryotic region of their somata (Fig. 7B) . At P2, the expression of Nir2 in ganglion cells was enhanced. Several rows of cells situated directly adjacent to the IPL (presumptive amacrine cells) were strongly immunoreactive for Nir2 (Fig. 7C) . At P4, distinct neuronal cells were detected by Nir2 immunostaining. In the outer retina, Nir2 immunoreactivity displayed a discontinuous band and typical horizontal cells, sending horizontally oriented processes into the discontinuous band (Fig. 7D) . From P6 Nir2 immunoreactivity was detected in the GCL and the ONL (Fig. 7E) , and its immunoreactivity was enhanced in the IPL and OPL at P8 (Fig. 7F) . At P12, bipolar cells, which are thought to develop at this stage, were strongly immunoreactive for Nir2 (Fig. 7G) . At P16, the distribution of Nir2 was similar to its adult pattern (Fig. 7H)
Expression of Nir3 in the Developing Retina
Whereas Nir1 and Nir2 were distinctly distributed during rat retinal development, the distribution of Nir3 was very similar to the pattern of Nir2 at early stages of development (E18–P2). From E18 to P0, Nir3 was homogeneously distributed throughout the retina (Figs. 8A 8B) . At P2, the ganglion cells and the outermost portion of the retina were strongly immunoreactive for Nir3 (Fig. 8C) . Similar to Nir2, Nir3 immunoreactivity also exhibited a distinct discontinuous band in the outer portion of the retina, which represented the putative premature OPL. However, no putative horizontal cells were revealed (Fig. 8D) . From P6 to P8, Nir3 immunoreactivity was enhanced in the OPL and INL, and cells that were strongly immunoreactive for Nir3 were revealed in the GCL (Figs. 8E 8F) . At P12, the IS, the OPL, and the inner portion of the INL, which contain mainly amacrine cells, were intensively immunoreactive for Nir3 (Fig. 8G) . At P16, Nir3 was distributed similar to its adult pattern, preferentially localized to the IS and synaptic terminals (Fig. 8H)
Discussion
The rdgB mutant fly was one of the first Drosophila retinal degeneration mutants identified. 8 13 14 39 Extensive genetic, biochemical, and electrophysiological studies suggest that rdgB plays an important role in the phototransduction cascade. Nevertheless, the actual function of rdgB in this pathway remains largely unknown. Recently, several mammalian homologues of rdgB have been cloned by different research groups. 15 16 17 22 40 41 These mammalian proteins share high sequence homology with the Drosophila rdgB and exhibit similar organization of structural domains. The high conservation of these proteins throughout evolution suggests that they may also be functionally related. Indeed, it has been shown that mrdgB1 can rescue the phenotype of rdgB mutant flies, 17 implying that rdgB and its mammalian counterparts implement similar functions in vivo. 
In the present report, we describe the cellular and developmental distribution of Nir proteins in the developing and mature rat retina, using specific antibodies against the Nirs. Our study showed that the three Nir proteins were abundantly expressed in the developing rat retina, with each exhibiting a unique distribution pattern. Nir1 was predominantly detected in Müller cells at early developmental stages, and its expression was upregulated during Müller cell maturation (Figs. 5 6) . In contrast, Nir2 and Nir3 were extensively detected in retinal neuronal cells throughout various developmental stages (Figs. 7 8) . Their expression in neuronal cells and processes coincided with the formation of the synaptic layers and ongoing synaptogenesis. However, there was no transient expression of the Nirs in any particular cell type. Instead, from E18, the earliest sample studied, the expression of Nir proteins was continuously detected and slightly upregulated, in accordance with the process of retinal maturation. Therefore, it is unlikely that the Nirs are necessary for transient developmental events, such as retinal neuritogenesis, synapse formation, and differentiation of retinal neurons. 
From P8, Nir3 was predominantly localized to synaptic terminals (Fig. 8) . Double-immunostaining for Nir3 and the two synaptic terminals markers, SNAP-25 and PNA, confirmed the localization of Nir3 in this subcellular compartment (Fig. 4) . In contrast, Nir2 was hardly detected in the synaptic terminals, but rather was highly expressed in photoreceptor, amacrine, bipolar, and horizontal cell bodies. In photoreceptor cells, Nir2 and Nir3 immunoreactivities were highly detected in the inner segments. Although Nir1 was also localized to this particular region, its immunoreactivity was much weaker than that of Nir2 or Nir3. 
The accumulation of Nir2 and Nir3 in the IS of photoreceptor cells and the profound localization of Nir3 in synaptic terminals suggest that these proteins may play a role in these regions. Both the IS and the synaptic terminals are known as regions in which dynamic trafficking processes and high membrane turnover occur. Therefore, Nir2 and Nir3 may be involved in these cellular processes. However, both Nir2 and Nir3, but not Nir1, contain a conserved N-terminal phosphatidylinositol (PI)-transfer domain. 15 This domain can transfer PI and phosphatidylcholine between membrane bilayers in vitro, similar to soluble PI-transfer proteins. The PI-transfer proteins play essential roles in intracellular vesicle trafficking and inositol lipid metabolism. 42 In mammalian cells, they have been implicated in protein transport, phospholipid signal transduction, vesicle budding, and exocytosis. Although the function of the PI-transfer domains of Nir2 and Nir3 has not yet been elucidated in vivo, recent reports suggest that this domain is critical for rdgB function in flies. 43 It has been proposed that rdgB is required for membrane turnover in the photoreceptors of compound eyes, 44 and its PI-transfer domain is crucial for this function. Similar to invertebrates, vertebrate photoreceptors have a very high rate of membrane turnover. Phospholipids and proteins used for OS disc assembly are synthesized, processed in the IS, and transported to and fused with a special region of connecting cilium. Thus, the accumulation of Nir2 and Nir3 in the IS suggests that they may be involved in the process of OS membrane renewal of mammalian photoreceptors, and that their PI-transfer domain may be crucial in this function. In addition, the accumulation of Nir3 in synaptic terminals and its colocalization with SNAP-25 (Fig. 4) , a protein that plays an essential role in the vesicle–plasma membrane fusion process, suggests that Nir3 may also play a role in synaptic vesicle trafficking in neurons. 
Because membrane renewal is vital for photoreceptor cell function, disruption of this orderly flow of membrane from the IS to the OS may cause photoreceptor degeneration. Indeed, Nir2 was mapped to human chromosome 11q13, a region known to contain several retinopathy loci. 27 It could therefore be that mutations within the Nir2 gene affect membrane turnover of human photoreceptors and thereby induce retinal degeneration. 
Unlike Nir2 and Nir3, Nir1 does not possess the PI-transfer domain, 15 and its immunoreactivity was predominantly localized to Müller cells (Figs. 5 6) . A high expression level of Nir1 was detected in the retina at E18, the earliest stage studied, exhibiting brightly labeled end feet and radial fibers that extended from the inner to the outer limiting membrane, coursing along the blast cells. Müller cell processes and trunks are thought to be required for guidance of neuronal cell migration and direct neurite differentiation. These cells nourish retinal neurons and also provide support and stabilization of retinal synapses. 45 Although the function of Nir1 in Müller cells is not yet clear, its increased expression in these cells during the early days after birth suggests its putative role in Müller cell differentiation and/or function. 
Taken together, the remarkable conservation of amino-acid sequence and domain topology among the Nir/rdgB family members and the striking phenotype of rdgB mutant flies suggest that this family of proteins possesses important cellular functions that have yet to be determined. In this study, we have characterized the expression profiles of Nir proteins in the developing and mature rat retina. Our findings indicate that each of them exhibits a unique pattern of distribution that may reflect their different physiological functions in vivo. 
 
Figure 1.
 
Anti-Nir antibodies specifically recognize the human and rat Nir proteins. Polyclonal antibodies against Nir1, Nir2, and Nir3 were raised in rabbits. (A) HA-tagged Nir1, Nir2, and Nir3 expressed in HEK293 cells were immunoprecipitated with anti-HA antibodies, resolved by SDS-PAGE, and immunoblotted with anti-HA, anti-Nir1, anti-Nir2, or anti-Nir3 antibodies. As shown, all the three Nir proteins were recognized by anti-HA antibodies, but each of them was only recognized by anti-Nir1, -Nir2, or -Nir3 antibodies, respectively. (B) Rat retinal lysate was prepared, and Nir1, Nir2, or Nir3 was immunoprecipitated by anti-Nir antibodies, as indicated. Preimmune (P.I.) serum was used as the control. After immunoprecipitation the samples were washed, resolved by SDS-PAGE, and immunoblotted as; indicated.
Figure 1.
 
Anti-Nir antibodies specifically recognize the human and rat Nir proteins. Polyclonal antibodies against Nir1, Nir2, and Nir3 were raised in rabbits. (A) HA-tagged Nir1, Nir2, and Nir3 expressed in HEK293 cells were immunoprecipitated with anti-HA antibodies, resolved by SDS-PAGE, and immunoblotted with anti-HA, anti-Nir1, anti-Nir2, or anti-Nir3 antibodies. As shown, all the three Nir proteins were recognized by anti-HA antibodies, but each of them was only recognized by anti-Nir1, -Nir2, or -Nir3 antibodies, respectively. (B) Rat retinal lysate was prepared, and Nir1, Nir2, or Nir3 was immunoprecipitated by anti-Nir antibodies, as indicated. Preimmune (P.I.) serum was used as the control. After immunoprecipitation the samples were washed, resolved by SDS-PAGE, and immunoblotted as; indicated.
Figure 2.
 
Distribution profile of the three Nir proteins in the adult rat retina. Vertical cryostat sections (15 μm) of adult rat retina were immunostained with anti-Nir1, anti-Nir2, or anti-Nir3 affinity-purified polyclonal antibodies. Micrographs were taken in approximately the same region of the retina. (A) Immunofluorescence image of Nir1 immunoreactivity shows the radial fibers, branchlets, somata, and end feet of putative Müller cells. (B) Immunofluorescence image of Nir2 distribution. Nir2 immunoreactivity was detected throughout the retinal layers, preferentially localized to the IS of photoreceptor cells and the somata of various retinal neuronal cells. (C) Immunofluorescence image of Nir3 distribution. Intensive immunoreactivity of Nir3 was detected in various layers of the retina, predominantly localized in the IS of the photoreceptor cells and the OPL. Scale bar, 50 μm.
Figure 2.
 
Distribution profile of the three Nir proteins in the adult rat retina. Vertical cryostat sections (15 μm) of adult rat retina were immunostained with anti-Nir1, anti-Nir2, or anti-Nir3 affinity-purified polyclonal antibodies. Micrographs were taken in approximately the same region of the retina. (A) Immunofluorescence image of Nir1 immunoreactivity shows the radial fibers, branchlets, somata, and end feet of putative Müller cells. (B) Immunofluorescence image of Nir2 distribution. Nir2 immunoreactivity was detected throughout the retinal layers, preferentially localized to the IS of photoreceptor cells and the somata of various retinal neuronal cells. (C) Immunofluorescence image of Nir3 distribution. Intensive immunoreactivity of Nir3 was detected in various layers of the retina, predominantly localized in the IS of the photoreceptor cells and the OPL. Scale bar, 50 μm.
Figure 3.
 
Localization of Nir1 and Nir2 in different retinal cells. (A) Vertical retinal sections were double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. The merged confocal image shows the red immunostaining for Nir1 and the overlap with GFAP appears in yellow. Although Nir1 immunoreactivity extensively overlapped with GFAP (yellow), Nir1 was also expressed in other cell types (red) that were not labeled with anti-GFAP antibody. Nir2 was expressed in amacrine cells. (BD) Vertical retinal sections were double immunostained with anti-Nir2 polyclonal antibody and anti-parvalbumin monoclonal antibody. The distribution of Nir2 (B; red) and parvalbumin (C; green) are shown, along with the merged confocal image demonstrating the overlapping immunostaining (D; yellow). (EG) Serial retinal sections were immunostained with anti-calbindin (E), anti-Nir2 (F), or anti-PKCα (G) polyclonal antibodies. Both Nir2 and calbindin immunoreactivities appeared in horizontal cells (E, F, arrows), whereas Nir2 immunoreactivity outlined cells with morphology similar to that obtained by PKCα immunoreactivity (G). Scale bar, 25 μm.
Figure 3.
 
Localization of Nir1 and Nir2 in different retinal cells. (A) Vertical retinal sections were double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. The merged confocal image shows the red immunostaining for Nir1 and the overlap with GFAP appears in yellow. Although Nir1 immunoreactivity extensively overlapped with GFAP (yellow), Nir1 was also expressed in other cell types (red) that were not labeled with anti-GFAP antibody. Nir2 was expressed in amacrine cells. (BD) Vertical retinal sections were double immunostained with anti-Nir2 polyclonal antibody and anti-parvalbumin monoclonal antibody. The distribution of Nir2 (B; red) and parvalbumin (C; green) are shown, along with the merged confocal image demonstrating the overlapping immunostaining (D; yellow). (EG) Serial retinal sections were immunostained with anti-calbindin (E), anti-Nir2 (F), or anti-PKCα (G) polyclonal antibodies. Both Nir2 and calbindin immunoreactivities appeared in horizontal cells (E, F, arrows), whereas Nir2 immunoreactivity outlined cells with morphology similar to that obtained by PKCα immunoreactivity (G). Scale bar, 25 μm.
Figure 4.
 
Nir3 is highly expressed in synaptic terminals. Vertical cryostat sections (15 μm) were taken in adult rat retina and double immunostained with anti-Nir2 or -Nir3 polyclonal antibodies and anti-SNAP-25 monoclonal antibody or biotin-PNA. Intensive immunoreactivity of Nir3 was detected in the OPL, where it colocalized with PNA and SNAP-25. In contrast, weak immunoreactivity of Nir2 was observed in the OPL and colocalization with either PNA or SNAP-25 was hardly detectable. Scale bar, 25 μm.
Figure 4.
 
Nir3 is highly expressed in synaptic terminals. Vertical cryostat sections (15 μm) were taken in adult rat retina and double immunostained with anti-Nir2 or -Nir3 polyclonal antibodies and anti-SNAP-25 monoclonal antibody or biotin-PNA. Intensive immunoreactivity of Nir3 was detected in the OPL, where it colocalized with PNA and SNAP-25. In contrast, weak immunoreactivity of Nir2 was observed in the OPL and colocalization with either PNA or SNAP-25 was hardly detectable. Scale bar, 25 μm.
Figure 5.
 
Expression of Nir1 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir1 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18, diffuse Nir1 immunoreactivity was detected throughout the retina (A). From P0 to P2, Nir1 immunoreactivity was enhanced in the GCL (B, C). At P4, the premature OPL was visualized with Nir1 immunoreactivity (D). From P6 to P8, Nir1 immunoreactivity gradually displayed the distinct distribution pattern of Müller cells (E, F). From P12 to P16, Nir1 immunoreactivity was significantly enhanced in Müller cells (G, H). Scale bar, 30 μm.
Figure 5.
 
Expression of Nir1 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir1 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18, diffuse Nir1 immunoreactivity was detected throughout the retina (A). From P0 to P2, Nir1 immunoreactivity was enhanced in the GCL (B, C). At P4, the premature OPL was visualized with Nir1 immunoreactivity (D). From P6 to P8, Nir1 immunoreactivity gradually displayed the distinct distribution pattern of Müller cells (E, F). From P12 to P16, Nir1 immunoreactivity was significantly enhanced in Müller cells (G, H). Scale bar, 30 μm.
Figure 6.
 
Localization of Nir1 in the Müller cells. Vertical cryostat sections (15 μm) were taken from rat retina at P12 and double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. Both Nir1 (A; red) and GFAP (B; green) immunoreactivity displayed typical Müller cell morphology, and were well colocalized in Müller cells (C, yellow). Scale bar, 30 μm.
Figure 6.
 
Localization of Nir1 in the Müller cells. Vertical cryostat sections (15 μm) were taken from rat retina at P12 and double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. Both Nir1 (A; red) and GFAP (B; green) immunoreactivity displayed typical Müller cell morphology, and were well colocalized in Müller cells (C, yellow). Scale bar, 30 μm.
Figure 7.
 
Expression of Nir2 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir2 polyclonal antibody. Immunofluorescence images were taken from approximately the same region of the retina. From E18 to P0, Nir2 immunoreactivity was present throughout the retinal layers (A, B). At P2, newly differentiated photoreceptor, amacrine, and ganglion cells were observed (C). At P4, the OPL and horizontal cells (arrows) were detected (D). From P6 (E) to P8 (F), the immunoreactivity of Nir2 in horizontal and amacrine cells was enhanced. At P12, Nir2 immunoreactivity displayed distinct horizontal cell morphology (arrows) and Nir2-positive bipolar cells were observed (G). At P16, the expression pattern of Nir2 was virtually the same as the adult profile (H). Scale bar, 30 μm.
Figure 7.
 
Expression of Nir2 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir2 polyclonal antibody. Immunofluorescence images were taken from approximately the same region of the retina. From E18 to P0, Nir2 immunoreactivity was present throughout the retinal layers (A, B). At P2, newly differentiated photoreceptor, amacrine, and ganglion cells were observed (C). At P4, the OPL and horizontal cells (arrows) were detected (D). From P6 (E) to P8 (F), the immunoreactivity of Nir2 in horizontal and amacrine cells was enhanced. At P12, Nir2 immunoreactivity displayed distinct horizontal cell morphology (arrows) and Nir2-positive bipolar cells were observed (G). At P16, the expression pattern of Nir2 was virtually the same as the adult profile (H). Scale bar, 30 μm.
Figure 8.
 
Expression of Nir3 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir3 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18 (A), Nir3 immunoreactivity was observed throughout the retina, with a stronger intensity over the GCL and the outermost portion of the NBL. At P0 (B) to P2 (C), Nir3 immunoreactivity was enhanced in the GCL. At P4 (D), similar to the profile of Nir1 and Nir2, the premature OPL was detected. Sections of retinas at P6 (E), P8 (F), P12 (G), and P16 (H) show that Nir3 immunoreactivity in the IS, the OPL, and the outer portion of the IPL was significantly enhanced. Nir3 was also detected to a lesser extent in retinal neuronal cells localized in the ONL and INL. Scale bar, 30 μm.
Figure 8.
 
Expression of Nir3 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir3 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18 (A), Nir3 immunoreactivity was observed throughout the retina, with a stronger intensity over the GCL and the outermost portion of the NBL. At P0 (B) to P2 (C), Nir3 immunoreactivity was enhanced in the GCL. At P4 (D), similar to the profile of Nir1 and Nir2, the premature OPL was detected. Sections of retinas at P6 (E), P8 (F), P12 (G), and P16 (H) show that Nir3 immunoreactivity in the IS, the OPL, and the outer portion of the IPL was significantly enhanced. Nir3 was also detected to a lesser extent in retinal neuronal cells localized in the ONL and INL. Scale bar, 30 μm.
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Figure 1.
 
Anti-Nir antibodies specifically recognize the human and rat Nir proteins. Polyclonal antibodies against Nir1, Nir2, and Nir3 were raised in rabbits. (A) HA-tagged Nir1, Nir2, and Nir3 expressed in HEK293 cells were immunoprecipitated with anti-HA antibodies, resolved by SDS-PAGE, and immunoblotted with anti-HA, anti-Nir1, anti-Nir2, or anti-Nir3 antibodies. As shown, all the three Nir proteins were recognized by anti-HA antibodies, but each of them was only recognized by anti-Nir1, -Nir2, or -Nir3 antibodies, respectively. (B) Rat retinal lysate was prepared, and Nir1, Nir2, or Nir3 was immunoprecipitated by anti-Nir antibodies, as indicated. Preimmune (P.I.) serum was used as the control. After immunoprecipitation the samples were washed, resolved by SDS-PAGE, and immunoblotted as; indicated.
Figure 1.
 
Anti-Nir antibodies specifically recognize the human and rat Nir proteins. Polyclonal antibodies against Nir1, Nir2, and Nir3 were raised in rabbits. (A) HA-tagged Nir1, Nir2, and Nir3 expressed in HEK293 cells were immunoprecipitated with anti-HA antibodies, resolved by SDS-PAGE, and immunoblotted with anti-HA, anti-Nir1, anti-Nir2, or anti-Nir3 antibodies. As shown, all the three Nir proteins were recognized by anti-HA antibodies, but each of them was only recognized by anti-Nir1, -Nir2, or -Nir3 antibodies, respectively. (B) Rat retinal lysate was prepared, and Nir1, Nir2, or Nir3 was immunoprecipitated by anti-Nir antibodies, as indicated. Preimmune (P.I.) serum was used as the control. After immunoprecipitation the samples were washed, resolved by SDS-PAGE, and immunoblotted as; indicated.
Figure 2.
 
Distribution profile of the three Nir proteins in the adult rat retina. Vertical cryostat sections (15 μm) of adult rat retina were immunostained with anti-Nir1, anti-Nir2, or anti-Nir3 affinity-purified polyclonal antibodies. Micrographs were taken in approximately the same region of the retina. (A) Immunofluorescence image of Nir1 immunoreactivity shows the radial fibers, branchlets, somata, and end feet of putative Müller cells. (B) Immunofluorescence image of Nir2 distribution. Nir2 immunoreactivity was detected throughout the retinal layers, preferentially localized to the IS of photoreceptor cells and the somata of various retinal neuronal cells. (C) Immunofluorescence image of Nir3 distribution. Intensive immunoreactivity of Nir3 was detected in various layers of the retina, predominantly localized in the IS of the photoreceptor cells and the OPL. Scale bar, 50 μm.
Figure 2.
 
Distribution profile of the three Nir proteins in the adult rat retina. Vertical cryostat sections (15 μm) of adult rat retina were immunostained with anti-Nir1, anti-Nir2, or anti-Nir3 affinity-purified polyclonal antibodies. Micrographs were taken in approximately the same region of the retina. (A) Immunofluorescence image of Nir1 immunoreactivity shows the radial fibers, branchlets, somata, and end feet of putative Müller cells. (B) Immunofluorescence image of Nir2 distribution. Nir2 immunoreactivity was detected throughout the retinal layers, preferentially localized to the IS of photoreceptor cells and the somata of various retinal neuronal cells. (C) Immunofluorescence image of Nir3 distribution. Intensive immunoreactivity of Nir3 was detected in various layers of the retina, predominantly localized in the IS of the photoreceptor cells and the OPL. Scale bar, 50 μm.
Figure 3.
 
Localization of Nir1 and Nir2 in different retinal cells. (A) Vertical retinal sections were double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. The merged confocal image shows the red immunostaining for Nir1 and the overlap with GFAP appears in yellow. Although Nir1 immunoreactivity extensively overlapped with GFAP (yellow), Nir1 was also expressed in other cell types (red) that were not labeled with anti-GFAP antibody. Nir2 was expressed in amacrine cells. (BD) Vertical retinal sections were double immunostained with anti-Nir2 polyclonal antibody and anti-parvalbumin monoclonal antibody. The distribution of Nir2 (B; red) and parvalbumin (C; green) are shown, along with the merged confocal image demonstrating the overlapping immunostaining (D; yellow). (EG) Serial retinal sections were immunostained with anti-calbindin (E), anti-Nir2 (F), or anti-PKCα (G) polyclonal antibodies. Both Nir2 and calbindin immunoreactivities appeared in horizontal cells (E, F, arrows), whereas Nir2 immunoreactivity outlined cells with morphology similar to that obtained by PKCα immunoreactivity (G). Scale bar, 25 μm.
Figure 3.
 
Localization of Nir1 and Nir2 in different retinal cells. (A) Vertical retinal sections were double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. The merged confocal image shows the red immunostaining for Nir1 and the overlap with GFAP appears in yellow. Although Nir1 immunoreactivity extensively overlapped with GFAP (yellow), Nir1 was also expressed in other cell types (red) that were not labeled with anti-GFAP antibody. Nir2 was expressed in amacrine cells. (BD) Vertical retinal sections were double immunostained with anti-Nir2 polyclonal antibody and anti-parvalbumin monoclonal antibody. The distribution of Nir2 (B; red) and parvalbumin (C; green) are shown, along with the merged confocal image demonstrating the overlapping immunostaining (D; yellow). (EG) Serial retinal sections were immunostained with anti-calbindin (E), anti-Nir2 (F), or anti-PKCα (G) polyclonal antibodies. Both Nir2 and calbindin immunoreactivities appeared in horizontal cells (E, F, arrows), whereas Nir2 immunoreactivity outlined cells with morphology similar to that obtained by PKCα immunoreactivity (G). Scale bar, 25 μm.
Figure 4.
 
Nir3 is highly expressed in synaptic terminals. Vertical cryostat sections (15 μm) were taken in adult rat retina and double immunostained with anti-Nir2 or -Nir3 polyclonal antibodies and anti-SNAP-25 monoclonal antibody or biotin-PNA. Intensive immunoreactivity of Nir3 was detected in the OPL, where it colocalized with PNA and SNAP-25. In contrast, weak immunoreactivity of Nir2 was observed in the OPL and colocalization with either PNA or SNAP-25 was hardly detectable. Scale bar, 25 μm.
Figure 4.
 
Nir3 is highly expressed in synaptic terminals. Vertical cryostat sections (15 μm) were taken in adult rat retina and double immunostained with anti-Nir2 or -Nir3 polyclonal antibodies and anti-SNAP-25 monoclonal antibody or biotin-PNA. Intensive immunoreactivity of Nir3 was detected in the OPL, where it colocalized with PNA and SNAP-25. In contrast, weak immunoreactivity of Nir2 was observed in the OPL and colocalization with either PNA or SNAP-25 was hardly detectable. Scale bar, 25 μm.
Figure 5.
 
Expression of Nir1 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir1 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18, diffuse Nir1 immunoreactivity was detected throughout the retina (A). From P0 to P2, Nir1 immunoreactivity was enhanced in the GCL (B, C). At P4, the premature OPL was visualized with Nir1 immunoreactivity (D). From P6 to P8, Nir1 immunoreactivity gradually displayed the distinct distribution pattern of Müller cells (E, F). From P12 to P16, Nir1 immunoreactivity was significantly enhanced in Müller cells (G, H). Scale bar, 30 μm.
Figure 5.
 
Expression of Nir1 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir1 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18, diffuse Nir1 immunoreactivity was detected throughout the retina (A). From P0 to P2, Nir1 immunoreactivity was enhanced in the GCL (B, C). At P4, the premature OPL was visualized with Nir1 immunoreactivity (D). From P6 to P8, Nir1 immunoreactivity gradually displayed the distinct distribution pattern of Müller cells (E, F). From P12 to P16, Nir1 immunoreactivity was significantly enhanced in Müller cells (G, H). Scale bar, 30 μm.
Figure 6.
 
Localization of Nir1 in the Müller cells. Vertical cryostat sections (15 μm) were taken from rat retina at P12 and double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. Both Nir1 (A; red) and GFAP (B; green) immunoreactivity displayed typical Müller cell morphology, and were well colocalized in Müller cells (C, yellow). Scale bar, 30 μm.
Figure 6.
 
Localization of Nir1 in the Müller cells. Vertical cryostat sections (15 μm) were taken from rat retina at P12 and double immunostained with anti-Nir1 polyclonal antibody and anti-GFAP monoclonal antibody. Both Nir1 (A; red) and GFAP (B; green) immunoreactivity displayed typical Müller cell morphology, and were well colocalized in Müller cells (C, yellow). Scale bar, 30 μm.
Figure 7.
 
Expression of Nir2 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir2 polyclonal antibody. Immunofluorescence images were taken from approximately the same region of the retina. From E18 to P0, Nir2 immunoreactivity was present throughout the retinal layers (A, B). At P2, newly differentiated photoreceptor, amacrine, and ganglion cells were observed (C). At P4, the OPL and horizontal cells (arrows) were detected (D). From P6 (E) to P8 (F), the immunoreactivity of Nir2 in horizontal and amacrine cells was enhanced. At P12, Nir2 immunoreactivity displayed distinct horizontal cell morphology (arrows) and Nir2-positive bipolar cells were observed (G). At P16, the expression pattern of Nir2 was virtually the same as the adult profile (H). Scale bar, 30 μm.
Figure 7.
 
Expression of Nir2 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir2 polyclonal antibody. Immunofluorescence images were taken from approximately the same region of the retina. From E18 to P0, Nir2 immunoreactivity was present throughout the retinal layers (A, B). At P2, newly differentiated photoreceptor, amacrine, and ganglion cells were observed (C). At P4, the OPL and horizontal cells (arrows) were detected (D). From P6 (E) to P8 (F), the immunoreactivity of Nir2 in horizontal and amacrine cells was enhanced. At P12, Nir2 immunoreactivity displayed distinct horizontal cell morphology (arrows) and Nir2-positive bipolar cells were observed (G). At P16, the expression pattern of Nir2 was virtually the same as the adult profile (H). Scale bar, 30 μm.
Figure 8.
 
Expression of Nir3 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir3 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18 (A), Nir3 immunoreactivity was observed throughout the retina, with a stronger intensity over the GCL and the outermost portion of the NBL. At P0 (B) to P2 (C), Nir3 immunoreactivity was enhanced in the GCL. At P4 (D), similar to the profile of Nir1 and Nir2, the premature OPL was detected. Sections of retinas at P6 (E), P8 (F), P12 (G), and P16 (H) show that Nir3 immunoreactivity in the IS, the OPL, and the outer portion of the IPL was significantly enhanced. Nir3 was also detected to a lesser extent in retinal neuronal cells localized in the ONL and INL. Scale bar, 30 μm.
Figure 8.
 
Expression of Nir3 in the developing rat retina. Vertical cryostat sections (15 μm) of rat retina were taken at the indicated developmental stages and immunostained with anti-Nir3 polyclonal antibody. Immunofluorescence images were taken in approximately the same region of the retina. At E18 (A), Nir3 immunoreactivity was observed throughout the retina, with a stronger intensity over the GCL and the outermost portion of the NBL. At P0 (B) to P2 (C), Nir3 immunoreactivity was enhanced in the GCL. At P4 (D), similar to the profile of Nir1 and Nir2, the premature OPL was detected. Sections of retinas at P6 (E), P8 (F), P12 (G), and P16 (H) show that Nir3 immunoreactivity in the IS, the OPL, and the outer portion of the IPL was significantly enhanced. Nir3 was also detected to a lesser extent in retinal neuronal cells localized in the ONL and INL. Scale bar, 30 μm.
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