October 1999
Volume 40, Issue 11
Free
Retinal Cell Biology  |   October 1999
Cell-Specific Expression of Tubby Gene Family Members (tub, Tulp1, 2, and 3) in the Retina
Author Affiliations
  • Sakae Ikeda
    From The Jackson Laboratory, Bar Harbor, Maine; and
  • Wei He
    AXYS Pharmaceuticals, La Jolla, California.
  • Akihiro Ikeda
    From The Jackson Laboratory, Bar Harbor, Maine; and
  • Jürgen K. Naggert
    From The Jackson Laboratory, Bar Harbor, Maine; and
  • Michael A. North
    AXYS Pharmaceuticals, La Jolla, California.
  • Patsy M. Nishina
    From The Jackson Laboratory, Bar Harbor, Maine; and
Investigative Ophthalmology & Visual Science October 1999, Vol.40, 2706-2712. doi:
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      Sakae Ikeda, Wei He, Akihiro Ikeda, Jürgen K. Naggert, Michael A. North, Patsy M. Nishina; Cell-Specific Expression of Tubby Gene Family Members (tub, Tulp1, 2, and 3) in the Retina. Invest. Ophthalmol. Vis. Sci. 1999;40(11):2706-2712.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The family of tubby-like proteins (TULPs), consisting of four family members, are all expressed in the retina at varying levels. Mutations within two members, tub and TULP1, are known to lead to retinal degeneration in mouse and humans, respectively, suggesting the functional importance of this family of proteins in the retina. Despite a high degree of conservation in the carboxy-teminal region (e.g., putative functional domain of the genes) among family members, they are unable to compensate for one another. The purpose of this study was to provide a rationale for this lack of compensation by investigating the spatial distribution of tubby gene family members in the retina and to investigate the mechanism of photoreceptor cell death in tubby mice.

methods. In situ hybridization using riboprobes specific for each tubby gene family member and immunohistochemistry for TUB and TULP1 were performed to determine their expression patterns in the retina of tubby and wild-type control mice. The terminal dUTP nick-end labeling (TUNEL) assay was performed to detect apoptotic cells in the retina of tubby and wild-type control mice.

results. tub mRNA was found to be expressed throughout the retina, with highest expression in the ganglion cell layer (GCL) and photoreceptor cells. In contrast, Tulp1 expression was observed only in photoreceptor cells and Tulp3 mRNA was expressed at a moderate level only in the inner nuclear layer (INL) and GCL. The results of the immunohistochemical analysis paralleled those observed in the in situ studies. TUB immunoreactivity was most highly concentrated in the GCL, in the inner and outermost regions of the INL, in the outer plexiform layer (OPL), and in the inner segments of photoreceptor cells. Similarly, TULP1 immunoreactivity was observed in the OPL and inner segments of the photoreceptor cells. No differences in expression at the mRNA or protein level were observed for any of the molecules tested in tubby or wild-type mice. TUNEL-positive cells were detected in the ONL of tubby mice, whereas very few were seen in the same layer of age-matched control mice.

conclusions. Although all tubby gene family members are expressed in the retina, they each have different cell-specific expression patterns, which may account in part for their inability to compensate for the loss of one family member. The photoreceptor cell death in tubby mice occurs through an apoptotic mechanism, which is known to be the common final outcome of other forms of retinal degeneration.

Tubby mice, also known as retinal degeneration 5 (rd5), exhibit retinal and cochlear degeneration and maturity-onset obesity. 1 2 3 4 5 The retinal degeneration in these mice is characterized by a progressive loss of photoreceptor cells starting at 3 weeks of age. 2 3 The reduction of the outer nuclear layer (ONL), along with the shortening and disorganization of the inner and outer segments, is observed through 8 months, when photoreceptor cells are diminished to a one- to two-nuclear-layer thickness. Electron microscopic studies reveal the presence of numerous membrane-bound vesicles of unknown origin in the interphotoreceptor space during the earlier stages of degeneration. 2 Pyknotic photoreceptor cell nuclei also are observed in the ONL, suggesting that the photoreceptor cells may die through an apoptotic mechanism in tubby mice. Based on nuclear morphology, cone and rod photoreceptor cells appear to die at a similar rate. Consistent with histologic observations, electroretinograms of tubby mice are abnormal, showing poorly developed a- and b-waves with progressively reduced amplitude, both of which are extinguished by 6 months of age. 2  
A mutation responsible for these phenotypes in tubby mice was identified by positional cloning within a novel gene, tub, situated on chromosome 7. 4 5 The mutation causes a splicing defect in the carboxyl-terminal intron of the tub gene, leading to a larger transcript that contains the unspliced intron. As a result, the 44 carboxyl-terminal amino acids in the wild-type protein are replaced with 24 intron-coded amino acids. The wild-type TUB protein is predominantly hydrophilic and has no signal sequence, suggesting an intracellular localization. The analysis of the amino acid sequence of TUB has not revealed the function of this protein. 
tub, however, is a member of a novel gene family with highly conserved carboxy termini and divergent amino termini. Three additional members, tubby-like protein (TULP) 1, TULP2, and TULP3, have been identified in humans, 6 7 and their mouse homologues, Tulp1, Tulp2, and Tulp3 have also been isolated 7 (Nishina et al., unpublished data, 1997). Each family member is situated on different chromosomes and exhibits a different tissue expression pattern. 6 7 Recent reports have shown that mutations within the TULP1 gene, which is expressed almost exclusively in the retina, are found in patients with retinitis pigmentosa (RP). 8 9 10 As also observed in tubby mice, these patients show a severe, progressive loss of photoreceptor cells that often leads to blindness. These retinal degeneration phenotypes indicate that at least two of the tubby family members are necessary to maintain retinal integrity and function. Interestingly, all tubby gene family members are expressed in the eye, although at varying levels. A high degree of conservation in the carboxyl-terminal region among the family members (55%–90% amino acid identity) and across multiple species from plants to humans, suggests that this region may be the functional domain that imparts a common function to the family members. 6 7 However, that retinal degeneration develops in tubby mice in spite of the normal expression of other family members suggests that they are unable to compensate for one another. One possible explanation for this lack of compensation is that although the family members share a similar function, they are not redundant because they have different cell-specific expression patterns in the retina. To investigate this possibility, we determined the expression patterns of tubby gene family members in the retina of tubby and wild-type control mice. In addition, we investigated the mechanism of photoreceptor cell death in tubby mice by examining whether fragmentation of DNA, characteristic of apoptotic cells, occurs in these mice. 
Methods
Mice
Tubby and wild-type matched control mice used in this study were obtained from our research colony at The Jackson Laboratory (Bar Harbor, ME). All mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
In Situ Hybridization
Five-week-old tubby and wild-type matched control animals were anesthetized with tribromoethanol and perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Eyes were enucleated and postfixed in the same fixative for 18 hours, dehydrated, and embedded in paraffin. A minimum of three eyes from three different animals in each group were used for this experiment. Sections of 6-μm thickness were cut and mounted on slides pretreated with poly l-lysine (Sigma, St. Louis, MO). Sections were deparaffinized in xylene and rehydrated through a graded series of alcohol and PBS. Sections were then treated with proteinase K (20μ g/ml) for 7 minutes at room temperature (RT), acetylated in 0.1 M triethenolamine (pH 8.0)-0.25% acetic anhydride solution, dehydrated, and air dried. 
[α-33P] UTP-labeled sense and antisense riboprobes (105 cpm/μl) were generated from plasmids containing cDNA fragments of tub (nt 197–599, U52433), Tulp1 (nt 81–707, AF105,711), Tulp2 (nt 40–1011, AF105,712), and Tulp3 (nt 44–623, AF045,583). To prevent cross-hybridization, all probes were prepared from the amino-terminal half of each protein, where the sequence homology between family members is low. 6 7  
To confirm the specificity of these probes, northern hybridization was performed as described in Nishina et al. 7 Membranes with 5μ g poly(A)+ RNA from mouse eyes and testes were hybridized with 32P random-labeled DNA probes for tub, Tulp1, and Tulp2 obtained from the clones described above. The specificity for the Tulp3 probe has previously been confirmed. 7  
Hybridization solution (50% formamide, 0.3 M NaCl, 20 mM Tris-HCl [pH 8.0], 5 mM EDTA, 10 mM NaPO4, [pH 8.0], 10% dextran sulfate, 1× Denhardt’s, and 0.5 mg/ml yeast tRNA) containing 50,000 cpm/μl labeled probe was applied to each slide. Sections were covered with coverslips and incubated at 60°C in a humidified chamber overnight. The slides were washed in a solution of 50% formamide-2× SSC at 65°C for 30 minutes, rinsed twice in NTE (0.5 M NaCl, 10 mM Tris-HCl [pH 7.5], 5 mM EDTA) at 37°C for 15 minutes, treated with RNaseA (1 μg/ml) at 37°C for 15 minutes, and rinsed in NTE for another 15 minutes. The slides were then washed in a solution of 50% formamide-2× SSC at 65°C for 20 minutes, in 2× SSC at RT for 15 minutes, and in 0.1× SSC at RT for 15 minutes and dehydrated. The slides were coated with NTB-2 emulsion (Eastman Kodak, Rochester, NY) and, after exposure for 5 days, 10 days, and 3 weeks (tub and Tulp1: 5 days, Tulp2: 3 weeks, Tulp3: 10 days) at 4°C, they were developed (D19; Eastman Kodak) and counterstained with hematoxylin. 
Generation of Polyclonal Antibodies for TUB and TULP1
Polyclonal antibodies were generated against the amino- and carboxyl-terminal half of human TUB and against the amino-terminal half of human TULP1. 
Tub-N2.
An 852-bp DNA fragment (nt 242-1093, HSU82467) was cloned into pET16b (Novagen, Madison, WI). The resultant 6X-His–tagged Tub-N fusion protein was purified using Ni-NTA agarose resin (Qiagen, Santa Clara, CA). Approximately 0.6 mg purified protein in Tris buffer was sent to Pocono Rabbit Farm and Laboratory (Candensis, PA) for injection into a rabbit. The rabbit antiserum was collected and purified against Tub-N coupled to Affi-Gel 10 (Bio-Rad, Hercules, CA). 
Tub-C3.
An 834-bp DNA fragment (nt 1094–1927, HSU82467) was cloned into pET16b (Novagen). 6X-His–tagged Tub-C fusion protein was purified using Ni-NTA agarose resin (Qiagen). Approximately 0.4 mg purified protein in Tris buffer and TiterMax Gold (CytRX, Norcross, GA) were used for injection into a rat. The rat antiserum was collected and purified against the antigen coupled to Affi-Gel 10 (Bio-Rad). 
TULP1-N.
A 601-bp DNA fragment (nt 37–637, HSU82468) was cloned into pET16b (Novagen). 6X-His–tagged TULP1-N fusion protein was purified using Ni-NTA agarose resin (Qiagen). The semipurified protein was run on a 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel. Approximately 0.2 mg protein from one single band was cut out, diced, and mixed with TiterMax Gold (CytRX) and injected into a rat. The rat antiserum was collected and used directly for immunostaining. 
Immunohistochemistry
Five-week-old tubby and wild-type matched control mice were anesthetized and perfused as described earlier, except that Bouin’s fixative was used. A minimum of three eyes from three different animals in each group were used. Tissues were postfixed, embedded in paraffin, sectioned, and rehydrated as described. Sections were heated in the microwave for 8 minutes in sodium citrate buffer (pH 6.5) for antigen retrieval 11 and then incubated overnight with either rabbit polyclonal antiserum against the amino-terminal half of the tubby protein (Tub-N2), or rat polyclonal antiserum against the carboxyl-terminal half of the tubby protein (Tub-C3) or the amino-terminal half of TULP1 (TULP1-N). Antibody binding was detected using biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) and an ABC kit (Vectastain Elite; Vector) using 3,3-diaminobenzidine tetrahydrocholoride as enzyme substrate (Vector). 
Terminal dUTP Nick-End Labeling Assay
Tubby and wild-type control mice at 4, 6, and 8 weeks of age were anesthetized and perfused with 10% neutral-buffered formalin. Three eyes from three different animals in each group were assayed per time point. After postfixation and embedding in paraffin, sections were prepared in the manner described earlier. Slides were treated with proteinase K (20 μg/ml) in PBS for 15 minutes at RT and washed four times for 2 minutes in distilled water. The terminal dUTP nick-end labeling (TUNEL) assay 12 was performed using a kit (Apop Tag In Situ Apoptosis Detection; Oncor, Gaithersburg, MD). For a negative control, water was substituted for the terminal deoxynucleotidyl transferase (TdT) enzyme in the reaction buffer of the kit. For each mouse, three to four sections spanning the optic nerve were used for quantitative analysis. Positively labeled cells were counted in the ONL. The images of the ONL were visualized by light microscopy and captured by camera (Megaplus 1.4; Eastman Kodak). Areas were measured using a quantification system (Quantimet Q600HR; Leica, Deerfield, IL) that was directly connected to a video camera. The data are expressed as numbers of positively labeled nuclei per square micrometer of the ONL. 
Results
In Situ Hybridization for tub and Tulp Genes
The spatial mRNA expression patterns of tub, Tulp1, Tulp2, and Tulp3 in the retina of normal and tubby mice were determined by in situ hybridization. 
The specificity of the probes used for in situ hybridization was tested by northern analysis (Fig. 1) . tub and Tulp1 probes detected major transcripts at 6.3 and 2.4 kb in the eye, respectively. Because of the low abundance in the eye, 6 Tulp2 probe was tested on a membrane with testis RNA, where it detected a major 2.0-kb transcript. 
In both normal and tubby mice, tub mRNA was highly expressed in the ganglion cell layer (GCL) and photoreceptor cells and moderately expressed in the inner nuclear layer (INL; Figs. 2 C, 2D). Tulp1 expression, which was observed in the photoreceptor layer (Figs. 2E 2F) , was more highly expressed than tub. Tulp2 mRNA expression was not detected above background level (data not shown). Tulp3 mRNA was moderately expressed in the INL and also in the GCL (Figs. 2G 2H) . No specific signal was detected in the tissues hybridized with sense probes (data not shown). No significant differences in the pattern of expression of the tubby gene family members were observed between tubby and control mice. 
Immunohistochemistry for TUB and TULP1
Immunohistochemical analyses were performed for TUB and TULP1 proteins, for which polyclonal antibodies were generated. We confirmed that the antibodies recognized each fusion protein by incubating them with western blot analysis containing lysate from Escherichia coli that were transfected with the expression vectors (data not shown). 
With the Tub-N2 antibody directed against the amino-terminal half of TUB, strong immunoreactivity was detected in the GCL and the inner segments of photoreceptor cells in both normal and tubby mice (Figs. 3 A, 3B, 3C). Moderate immunoreactivity was also detected in cells located in the innermost and outermost regions of the INL (Fig. 3B) . Staining patterns for Tub-N2 were the same in control and tubby mice, except that the staining of the inner segments in tubby mice appeared more diffuse, presumably because of the disorganization of the inner segments. With the Tub-C3 antibody, strong staining was seen in the outer half of the outer plexiform layer (OPL) and in the inner segments of photoreceptor cells of wild-type control mice (Fig. 3D) . Cells in the GCL were weakly stained (Fig. 3D) . In tubby mice, Tub-C3 immunoreactivity was not observed (Fig. 3E) , suggesting that this antibody recognized the carboxyl-terminal portion of the protein that is modified by the tubby mutation. Strong TULP1-N immunoreactivity was detected in the outer half of the OPL and inner segments of the photoreceptor cells. Staining patterns were the same in control and tubby mice for the TULP1-N antibody (Figs. 3F 3G) . No staining was seen in the control tissues incubated with water in place of primary antibodies (data not shown). 
TUNEL Assay
To determine whether photoreceptor cell loss in tubby mice occurs through an apoptotic mechanism, detection of 3′-OH DNA ends was performed by TUNEL assay. Representative light photomicrographs of these data are shown in Figure 4 . At all ages examined, the TUNEL-positive nuclei were confined to the ONL of tubby mice. These apoptotic nuclei were scattered throughout the layer without aggregation, surrounded by normal-appearing nuclei. Among these positively stained nuclei were cells that were stained peripherally or darkly throughout, presumably indicating different stages of chromatin condensation during apoptosis. 
As shown in Figure 5 , TUNEL-positive cells were detected in the ONL of tubby mice at 4, 6, and 8 weeks of age, whereas very few positive cells were seen in the same layer of age-matched control mice. TUNEL-positive nuclei were most abundant at 4 weeks. At this age, the thickness of ONL of tubby mice is approximately two thirds that of control mice, and by 8 weeks it is further reduced to approximately one third to one half of normal. 
Discussion
In this study, we performed in situ hybridization to compare the expression patterns of tubby gene family members in the retina of tubby and wild-type control mice. In both control and tubby mice, tub mRNA is highly expressed in the GCL and photoreceptor cells and moderately in the INL. In contrast to the broad distribution of tub throughout the retina, other family members exhibited a more restricted layer-specific localization. mRNA for Tulp1, which is expressed almost exclusively in the eye at very high levels, 6 was detected only in the photoreceptor cells. As expected from cDNA screening of a human retinal library in which only one in 10−6 plaques was TULP2 positive, 6 the expression level of Tulp2 by in situ hybridization was very low in the retina. We did not detect a signal above background level. Tulp3, which shows a rather broad pattern of tissue expression similar to that of tub, 7 was detected in the INL and GCL but not in the photoreceptor cells. To determine in which cells in the INL TULP3 protein is expressed, immunohistochemical analysis for TULP3 and double labeling with cell markers for different cell types are needed. 
For TUB and TULP1, immunohistochemical analysis was also performed to determine further their expression at the protein level. The Tub-N2 antibody, generated against the amino-terminal half of TUB, gave a result consistent with mRNA distribution revealed by in situ hybridization. Strong immunoreactivity was detected in the cells in the GCL and the inner segments of photoreceptor cells. Also, some cells in the INL, possibly amacrine cells and horizontal cells, judging from their location, were moderately stained. There were some differences in staining patterns using the Tub-C3 antibody raised against the carboxyl-terminal half of TUB. The GCL showed strong immunoreactivity with Tub-N2 but only weak immunoreactivity with Tub-C3. Cells in the INL that were stained with Tub-N2 were not stained with Tub-C3. A potential explanation for these differences is that, when only Tub-N2 staining is detected, TUB may be bound by another protein or to a cellular membrane, so that the Tub-C3 antibody cannot recognize its epitope. Computer-assisted analysis of the TUB primary sequence suggests a potential transmembrane helix at the carboxyl-terminal end of the protein, 7 which is removed by the mutation in tubby mice. Although Tub-C3 was generated against the entire carboxyl-terminal half of TUB where the sequence is highly conserved among all tubby gene family members, we exclude the possibility that it recognizes other members, because no staining was detected in tubby animals in which TULPs are expressed normally. 
Consistent with the in situ hybridization results, the immunoreactivity for TULP1 was detected only in the photoreceptor cells. TULP1 protein was found in the outer half of the OPL, where photoreceptor cells form synapses with the horizontal and bipolar cells in the INL, and in the inner segments of photoreceptor cells. 
Because carboxyl-terminal sequences show high conservation among tubby gene family members and across species, we speculate that the family members share a similar function that is imparted by this conserved region. It has been shown that another family of proteins, retinoic acid receptors (RARs), whose DNA- and ligand-binding regions are highly conserved among family members (RAR-α, -β, and -γ and their isoforms), show a high degree of functional redundancy in the eye. 13 Congenital eye malformations were observed in RAR compound null mutants, but not in mice deficient for only one type of RAR. However, in case of the tubby gene family, a mutation in one of the family members (tub or TULP1) is enough to cause retinal degeneration, suggesting that they do not compensate for each other. The results here show that each tubby gene family member is expressed in specific cell types in the retina. This cell-specific expression may account in part for their inability to compensate for the loss of one family member. For example, because Tulp3 is not expressed in the photoreceptor cells, it is clear that it cannot compensate for the loss of tub or Tulp1 in these cells, even if the family members share a conserved function. However, each family member must also have acquired some unique functions that cannot be compensated for by other family members, inasmuch as tub and Tulp1 are both expressed in the photoreceptor cells and are still not redundant. Generation of mice deficient in Tulp genes is currently under way. By characterizing their eye phenotype and by crossing them with tubby mice and to each other, we may be able to clarify their functional relationship further. 
It has been reported that apoptosis is the underlying mechanism of photoreceptor cell death in several mouse models of retinal degeneration. 14 15 16 17 In this study, we examined whether photoreceptor cell death in tubby mice also occurs by this mechanism. In tubby mice, the loss of photoreceptor cells starts at approximately 3 weeks of age and proceeds until 8 months. 2 3 Our results show that at 4 to 8 weeks of age tubby mice have TUNEL-labeled photoreceptor cell nuclei that are rarely present in normal control mice. Together with the previous observation that pyknotic nuclei are present in the ONL of tubby mice, 2 our observation strongly suggests that photoreceptor cells die by apoptosis in this model. In other mouse models of retinal degeneration, 14 15 16 17 as well as in tubby mice, the exact mechanism by which each primary mutation triggers apoptosis is unknown. By revealing the function of tubby gene family members in the retina and the relationship between the mutations in these genes and the retinal phenotypes they induce, we may be able to obtain new information about the mechanisms underlying apoptotic photoreceptor cell death. 
 
Figure 1.
 
Specificity of probes for tub, Tulp1, and Tulp2 used for in situ studies, as assessed by northern hybridization analysis. Five micrograms poly(A)+ RNA from eyes (lanes a and b) or testes (lane c) was analyzed with tub (lane a), Tulp1 (lane b), or Tulp2 (lane c) probes. A major band of expected size was detected in each lane. The positions of RNA size markers are shown on the left. Lane a was exposed for 3 hours, and lanes b and c were exposed for 7 minutes.
Figure 1.
 
Specificity of probes for tub, Tulp1, and Tulp2 used for in situ studies, as assessed by northern hybridization analysis. Five micrograms poly(A)+ RNA from eyes (lanes a and b) or testes (lane c) was analyzed with tub (lane a), Tulp1 (lane b), or Tulp2 (lane c) probes. A major band of expected size was detected in each lane. The positions of RNA size markers are shown on the left. Lane a was exposed for 3 hours, and lanes b and c were exposed for 7 minutes.
Figure 2.
 
The expression of tubby gene family members in the retina of wild-type control and tubby mice at 5 weeks of age. Bright-field (A, B) and corresponding dark-field sections (C through H) are shown. tub was detected throughout the retina of control (C) and tubby (D) mice and was especially prominent in the GCL and in the inner segments (ISs) of photoreceptor (PR) cells (arrow). A strong signal for Tulp1 (E, F) was detected exclusively in PR cells, most prominently in the inner segments of PR cells. Tulp3 (G, H) was detected in the INL and GCL, but not in PR cells. Note: The separation of retina from the retinal pigment epithelium and choroid is an artifact of fixation and was observed in both tubby and wild-type control sections. Scale bar, 40 μm.
Figure 2.
 
The expression of tubby gene family members in the retina of wild-type control and tubby mice at 5 weeks of age. Bright-field (A, B) and corresponding dark-field sections (C through H) are shown. tub was detected throughout the retina of control (C) and tubby (D) mice and was especially prominent in the GCL and in the inner segments (ISs) of photoreceptor (PR) cells (arrow). A strong signal for Tulp1 (E, F) was detected exclusively in PR cells, most prominently in the inner segments of PR cells. Tulp3 (G, H) was detected in the INL and GCL, but not in PR cells. Note: The separation of retina from the retinal pigment epithelium and choroid is an artifact of fixation and was observed in both tubby and wild-type control sections. Scale bar, 40 μm.
Figure 3.
 
Immunohistochemical localization of TUB and TULP1 in the retina of wild-type control and tubby mice at 5 weeks of age. Control (A, B) and tubby (C) retina stained with Tub-N2 antibody. Strong immunoreactivity was detected in the GCL of both control and tubby mice. Immunoreactivity was also observed in the inner segments (IS) of photoreceptor (PR) cells (A, C) and in the cells at the innermost and outermost regions of the INL (B, arrowheads). Control (D) and tubby (E) retina stained with the Tub-C3 antibody. In the control section (D), there was strong immunoreactivity in the OPL and in the ISs of PR cells. Weak immunoreactivity was also detected in cells in the GCL. Note that no staining was observed in the tubby retina (E). Control (F) and tubby (G) retina stained with TULP1-N antibody, counterstained with hematoxylin. Strong immunoreactivity was observed in the OPL and in the IS of PR cells. Scale bar (A, C, D, E) 18 μm; (B) 7μ m.
Figure 3.
 
Immunohistochemical localization of TUB and TULP1 in the retina of wild-type control and tubby mice at 5 weeks of age. Control (A, B) and tubby (C) retina stained with Tub-N2 antibody. Strong immunoreactivity was detected in the GCL of both control and tubby mice. Immunoreactivity was also observed in the inner segments (IS) of photoreceptor (PR) cells (A, C) and in the cells at the innermost and outermost regions of the INL (B, arrowheads). Control (D) and tubby (E) retina stained with the Tub-C3 antibody. In the control section (D), there was strong immunoreactivity in the OPL and in the ISs of PR cells. Weak immunoreactivity was also detected in cells in the GCL. Note that no staining was observed in the tubby retina (E). Control (F) and tubby (G) retina stained with TULP1-N antibody, counterstained with hematoxylin. Strong immunoreactivity was observed in the OPL and in the IS of PR cells. Scale bar (A, C, D, E) 18 μm; (B) 7μ m.
Figure 4.
 
TUNEL staining of tubby and wild-type control retina at 4 weeks of age. Sections are counterstained with methyl green. (A) Control retina. No TUNEL-positive nuclei are observed in this field. (B) Tubby retina. TUNEL-positive nuclei are observed only in the ONL. Scale bar, 15 μm.
Figure 4.
 
TUNEL staining of tubby and wild-type control retina at 4 weeks of age. Sections are counterstained with methyl green. (A) Control retina. No TUNEL-positive nuclei are observed in this field. (B) Tubby retina. TUNEL-positive nuclei are observed only in the ONL. Scale bar, 15 μm.
Figure 5.
 
The number of TUNEL-positive photoreceptor cell nuclei in tubby and wild-type control mice at 4, 6, and 8 weeks of age. For each time point, the left column represents the number of TUNEL-positive nuclei per square micrometer of the outer nuclear layer in tubby mice, and the right column represents that of wild-type control mice. Error bars represent the SE.
Figure 5.
 
The number of TUNEL-positive photoreceptor cell nuclei in tubby and wild-type control mice at 4, 6, and 8 weeks of age. For each time point, the left column represents the number of TUNEL-positive nuclei per square micrometer of the outer nuclear layer in tubby mice, and the right column represents that of wild-type control mice. Error bars represent the SE.
The authors thank Cindy S. Avery for mouse colony management; Irina Sorokina for assistance in generation of antibodies; Stefan A. Przyborski for excellent advice; and Richard S. Smith, Verity A. Letts ,and Barbara B. Knowles for careful review of the manuscript. 
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Figure 1.
 
Specificity of probes for tub, Tulp1, and Tulp2 used for in situ studies, as assessed by northern hybridization analysis. Five micrograms poly(A)+ RNA from eyes (lanes a and b) or testes (lane c) was analyzed with tub (lane a), Tulp1 (lane b), or Tulp2 (lane c) probes. A major band of expected size was detected in each lane. The positions of RNA size markers are shown on the left. Lane a was exposed for 3 hours, and lanes b and c were exposed for 7 minutes.
Figure 1.
 
Specificity of probes for tub, Tulp1, and Tulp2 used for in situ studies, as assessed by northern hybridization analysis. Five micrograms poly(A)+ RNA from eyes (lanes a and b) or testes (lane c) was analyzed with tub (lane a), Tulp1 (lane b), or Tulp2 (lane c) probes. A major band of expected size was detected in each lane. The positions of RNA size markers are shown on the left. Lane a was exposed for 3 hours, and lanes b and c were exposed for 7 minutes.
Figure 2.
 
The expression of tubby gene family members in the retina of wild-type control and tubby mice at 5 weeks of age. Bright-field (A, B) and corresponding dark-field sections (C through H) are shown. tub was detected throughout the retina of control (C) and tubby (D) mice and was especially prominent in the GCL and in the inner segments (ISs) of photoreceptor (PR) cells (arrow). A strong signal for Tulp1 (E, F) was detected exclusively in PR cells, most prominently in the inner segments of PR cells. Tulp3 (G, H) was detected in the INL and GCL, but not in PR cells. Note: The separation of retina from the retinal pigment epithelium and choroid is an artifact of fixation and was observed in both tubby and wild-type control sections. Scale bar, 40 μm.
Figure 2.
 
The expression of tubby gene family members in the retina of wild-type control and tubby mice at 5 weeks of age. Bright-field (A, B) and corresponding dark-field sections (C through H) are shown. tub was detected throughout the retina of control (C) and tubby (D) mice and was especially prominent in the GCL and in the inner segments (ISs) of photoreceptor (PR) cells (arrow). A strong signal for Tulp1 (E, F) was detected exclusively in PR cells, most prominently in the inner segments of PR cells. Tulp3 (G, H) was detected in the INL and GCL, but not in PR cells. Note: The separation of retina from the retinal pigment epithelium and choroid is an artifact of fixation and was observed in both tubby and wild-type control sections. Scale bar, 40 μm.
Figure 3.
 
Immunohistochemical localization of TUB and TULP1 in the retina of wild-type control and tubby mice at 5 weeks of age. Control (A, B) and tubby (C) retina stained with Tub-N2 antibody. Strong immunoreactivity was detected in the GCL of both control and tubby mice. Immunoreactivity was also observed in the inner segments (IS) of photoreceptor (PR) cells (A, C) and in the cells at the innermost and outermost regions of the INL (B, arrowheads). Control (D) and tubby (E) retina stained with the Tub-C3 antibody. In the control section (D), there was strong immunoreactivity in the OPL and in the ISs of PR cells. Weak immunoreactivity was also detected in cells in the GCL. Note that no staining was observed in the tubby retina (E). Control (F) and tubby (G) retina stained with TULP1-N antibody, counterstained with hematoxylin. Strong immunoreactivity was observed in the OPL and in the IS of PR cells. Scale bar (A, C, D, E) 18 μm; (B) 7μ m.
Figure 3.
 
Immunohistochemical localization of TUB and TULP1 in the retina of wild-type control and tubby mice at 5 weeks of age. Control (A, B) and tubby (C) retina stained with Tub-N2 antibody. Strong immunoreactivity was detected in the GCL of both control and tubby mice. Immunoreactivity was also observed in the inner segments (IS) of photoreceptor (PR) cells (A, C) and in the cells at the innermost and outermost regions of the INL (B, arrowheads). Control (D) and tubby (E) retina stained with the Tub-C3 antibody. In the control section (D), there was strong immunoreactivity in the OPL and in the ISs of PR cells. Weak immunoreactivity was also detected in cells in the GCL. Note that no staining was observed in the tubby retina (E). Control (F) and tubby (G) retina stained with TULP1-N antibody, counterstained with hematoxylin. Strong immunoreactivity was observed in the OPL and in the IS of PR cells. Scale bar (A, C, D, E) 18 μm; (B) 7μ m.
Figure 4.
 
TUNEL staining of tubby and wild-type control retina at 4 weeks of age. Sections are counterstained with methyl green. (A) Control retina. No TUNEL-positive nuclei are observed in this field. (B) Tubby retina. TUNEL-positive nuclei are observed only in the ONL. Scale bar, 15 μm.
Figure 4.
 
TUNEL staining of tubby and wild-type control retina at 4 weeks of age. Sections are counterstained with methyl green. (A) Control retina. No TUNEL-positive nuclei are observed in this field. (B) Tubby retina. TUNEL-positive nuclei are observed only in the ONL. Scale bar, 15 μm.
Figure 5.
 
The number of TUNEL-positive photoreceptor cell nuclei in tubby and wild-type control mice at 4, 6, and 8 weeks of age. For each time point, the left column represents the number of TUNEL-positive nuclei per square micrometer of the outer nuclear layer in tubby mice, and the right column represents that of wild-type control mice. Error bars represent the SE.
Figure 5.
 
The number of TUNEL-positive photoreceptor cell nuclei in tubby and wild-type control mice at 4, 6, and 8 weeks of age. For each time point, the left column represents the number of TUNEL-positive nuclei per square micrometer of the outer nuclear layer in tubby mice, and the right column represents that of wild-type control mice. Error bars represent the SE.
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