768 X 512 pixels were opened in Adobe Photoshop. (either v. 2.5.Ib5 or 3.0; Adobe Systems Mountain. View CA). Higher resolution images were used for.
thymocytes.2 Thy-1 is found in the nervous systems of a wide range of species and is present on neurons at concentrations of as much as 500 molecules per Catherine Jui-ling Liu,* Neena Chaturvedi,* mm2—representing up to as much as 7.5% of the surface protein.3 Evolutionarily, Thy-1 is highly conColin J. Barnstable,-f and Evan B. Dreyer* served.3 Recent work in our laboratory4 has suggested Purpose. To evaluate the developmental expression of that during development, retinal ganglion cell process Thy-1 in the retina. Thy-1, the most abundant mammaoutgrowth is modulated by an interaction between lian neuronal surface glycoprotein, is likely to play a Thy-1 and a glial Thy-1 receptor. significant role in retinal development. In the mammaPrevious immunohistochemistry studies5 in adult lian retina, it is found predominantly, if not exclusively, rat retinas have localized Thy-1 predominantly to the on retinal ganglion cells. ganglion cell layer, with some staining also seen in the Methods. Rat retinae of various ages were stained immunerve fiber and inner plexiform layers. These data nohistochemically for Thy-1 with 2G12, a monoclonal suggest a significant role for Thy-1 in the retina. We Thy-1 antibody. Sections were analyzed digitally to have therefore chosen to explore the expression of quantify bound antibody. Using semiquantitative reThy-1 in the developing rat retina. verse transcription-polymerase chain reaction (RTAlthough Thy-1 is a surface component of most PCR), the expression of Thy-1 protein was compared neurons in the adult nervous system, it is present only with the levels of mRNA detected. on certain neurons during early development. MoreResults. Thy-1-dependent fluorescence was detected in over, detectable antigen can be lost from cells that, rat retinae from birth, albeit at low levels. Thy-1 labeling at earlier development, were Thy-1 positive, whereas was localized predominantly to the ganglion cell layer. formerly Thy-1 negative cells can acquire high levels Minimal, fine patterns of linear and reticular fluoresof the antigen during later stages of development.3 cence were noted in the inner nuclear layer. Thy-1 levBarnstable and coworkers6 identified Thy-1 in retinal els reached a maximal level at approximately postnatal sections at postnatal day 2, although Thy-1 positive day 14. RT-PCR measurements showed a similar time course for the increase in Thy-1 expression. ganglion cells could be identified in high-density culConclusions. The Thy-1 antigen is present in the inner tures from newborn rats. To quantify Thy-1 expression during rat retinal development, the decision was made retina at birth. Its level increases steadily after birth and to use quantitative immunohistochemistry for two reapeaks during the second week of life. Thy-1 expression sons. In situ hybridization studies7 of Thy-1 in Purkinje is approximately coterminous with synaptogenesis of the inner plexiform layer and may play a role in synapcells have demonstrated a temporal incongruity betogenesis of the inner retina or in other developmental tween mRNA appearance and glycoprotein expresmilestones in the formation of the visual system. Invest sion. Furthermore, anti-Thy-1 antibodies have not Ophthalmol Vis Sci. 1996;37:1469-1473. been effective in identifying Thy-1 on Western blots.
Retinal Thy-1 Expression During Development
Adhesion molecules in the immunoglobulin superfamily, for example, neuronal cell adhesion molecule, LI, fasciclins, and myelin-associated glycoprotein, play a crucial role in the development of the nervous system.1 Thy-1, the smallest member of the immunoglobulin superfamily, was originally identified in brain and
From the *Defmrtmenl of Ophthalmology, Massachusetts Eye 6f Ear Infirmary, Harvard Medical School, Boston, Massachusetts, and the f Department of Of>hthalmology and Visual Science, Yale University, New Haven, Connecticut. Supported in part liy giants from the Potts and Glaucoma Foundations, the National Glaucoma Trust, the Massachusetts and Connecticut Lions Eye Research Funds, and the National Institutes of Health (R01-EY10009). Submitted for publication October 5, 1995; revised January 2, 1996; accepted February 5, 1996. ftoprietary interest category: N Ref/rint requests: Evan B. Dreyer, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114.
Investigative Ophthalmology & Visual Science, June 1996, Vol. 37, No. 7 Copyright © Association for Research in Vision and Ophthalmology
METHODS. Tissue. These experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Long-Evans rats were used. Pups from a single litter were killed at different ages, ranging from birth to the 30th day of life. Five litters were studied by immunofluorescence and two by reverse transcription-polymerase chain reaction (RT-PCR). For immunocytochemistry, all eyes were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4 to 7.45) for 2 hours at 4°C and were transferred to 30% sucrose solution overnight at 4°C. Eight-micron transverse cryostat sections were cut, mounted on gelatinized slides, air dried, and stored at -20°C. For RT-PCR, retinas were dissected rapidly and flash frozen in liquid nitrogen until use.
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Immunohistochemistry. A monoclonal antibody
against Thy-1, 2G12, was used as the primary probe for Thy-1. Its specificity for Thy-1 has been well documented.50 Rhodamine-conjugated goat anti-mouse immunoglobulin G was used to detect 2G12 binding (Boehringer Mannheim, Indianapolis, IN). Retinal sections were first incubated widi blocking solution (10% calf serum combined with 15% nonfat dry milk in phosphate-buffered saline) for 90 minutes, washed with PBS, and incubated widi primary antibody overnight at 4°C at a constant dilution of 1:100 in PBS. Sections were washed with PBS, and incubated with secondary antibody for 1 hour at room temperature. Finally, sections were mounted in a nonglycerolbased mounting medium (Gel/Mount; Biomeda, Foster City, CA) and viewed with epifluorescence optics. Sections incubated without primary antibody were used as controls. All sections from a given litter were prepared simultaneously, using a single set of reagents. Incubation periods and antiserum concentrations (in each case, across a 0.5- to 2-fold range from that noted above) were varied with litde effect on Thy1 staining (not shown). Reverse Transcription-Polymerase Chain Reaction. RNA was isolated using RNAzol B as previously described.8 cDNA synthesis was performed using MuMLV reverse transcriptase (Gibco, Bediesda, MD) and random hexamer primers (Boehringer Mannheim) and was carried out for 2 hours at 37°C. Polymerase chain reaction primers used were 5'-TGCCTGGTGAACCAGAACCTT-3' and reverse 5'-TCACAGAGAAATGAAGTCCGTGGC-3'. These primers correspond to sequences in exons 2 and 3 of the Thy1 gene and give an amplified fragment of 393 bp from mature Thy-1 RNA. As controls, cDNA samples also were also amplified with primers corresponding to a 548-bp fragment of /3-actin.8 Polymerase chain reactions were carried out in a 100 1 volume in a PerkinElmer (Norwalk, CT) thermal cycler using conditions previously described8; PCR products were analyzed on 1% agarose gels. Control experiments have established tfiat under the conditions used, die amount of PCR product obtained is proportional to the amount of RNA present in die original sample (not shown). Quantitative Analysis. To permit simple quantification of immunofluorescence and PCR amplification, the following technique was derived from previously published mediods.9 Photographs of retinal sections were taken under identical illumination conditions using a single exposure time (generally 8 seconds) per litter. The ganglion cell layer was aligned to the upper edge of the eyepiece reticule. This allowed for establishment of a "standard fluorescence photograph." The field extended through the full retinal thickness. Forty randomly selected fields were
photographed of both control and Thy-1 labeled sections. These images were then transferred to a Kodak Photo-CD (Eastman Kodak, Rochester, NY) for subsequent analysis on a Macintosh 6100/60 computer (Apple, Cupertino, CA). Resolution images measuring 768 X 512 pixels were opened in Adobe Photoshop (either v. 2.5.Ib5 or 3.0; Adobe Systems Mountain View CA). Higher resolution images were used for comparison but did not yield any further information (data not shown). Each image was then converted to gray scale. Using die Histogram command, two measurements were made. First, die mean intensity for the black channel was measured for the entire field. A second set of measurements was made of the ganglion cell layer only. For these, die image was cropped along the x-axis to the entire width of the field and along the y-axis to the diameter of the widest retinal ganglion cell in the image. These measurements were performed only for those sections stained with antiThy-1 antibody because it was not possible to identify the ganglion cell layer unambiguously in control sections. Measurements were made masked to animal age and litter, but it was not possible to mask for control versus Thy-1. For quantification of PCR data, photographs of the ethidium bromide-stained gels were scanned into Adobe Photoshop, and the intensity of the pixels summed in a constant window for each band. The intensity of bands obtained by amplification of a /?actin fragment from each cDNA sample was used to control for variations in RNA or cDNA yield. RESULTS. Thy-1 labeling in die retina at all ages was localized predominandy to the ganglion cell layer (Fig. 1). There was good correlation in both localization and fluorescent intensity at all ages between litters (not shown). Thy-1 labeling could be detected in die inner retina of rats from the date of birdi (Fig. 2). However, the staining intensity was very low for the first few days of postnatal development. There was a sharp increase in Thy-1 intensity at day 5, and Thy1-dependent fluorescence reached maximal intensity toward the end of the second postnatal week (Fig. 2). Thy-1 staining declined minimally from this point on and remained at relatively constant levels in adulthood. The inner plexiform and nuclear layers were also faindy Thy-1 positive. Though the staining intensity in diese layers was relatively low, it was significantly higher than that found in control sections and may represent displaced ganglion cells or ganglion cell dendrites. Polymerase chain reaction amplification of cDNA produced from retinal RNA detected mature Thy-1 RNA at day 4 (Fig. 3). The intensity of the amplified
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FIGURE l. Thy-1 immunoflu-
orescence of the retina. Retinal section of a 2- (A) and a 14- (B) day-old rat pup stained with the anti-Thy-1 antibody, 2G12. Prominent staining can be seen of the ganglion cell layer and ganglion cell processes, particularly in the retina from die older animal.
band increased with retinal age. At embryonic day 15 and postnatal day 2, a larger band was detected in the position expected for either genomic DNA or unprocessed RNA. As shown in Figure 4, the intensities of
Ganglion cell Thy-1 Whole retinalThy-1 Retinal Control
Age (days)
FIGURE 2. Quantification of Thy-1 immunofluorescence of the retina. Bound Thy-1 was quantified as described in the Methods for both the ganglion cell layer and whole retinal sections. Background (control) fluorescence was quantified for the whole retina only. Each value is the mean of 40 determinations for the black channel (based on the Histogram command in Photoshop; see Methods) for each of five animals. Error bars are standard deviations.
the Thy-1 bands amplified from retinal cDNAs were quantified and normalized to an amplified /3-a.ctin fragment. Between postnatal day 2 and adult, the level of Thy-1 cDNA increased more than 15-fold (Fig. 4).
DISCUSSION. Our data indicate that Thy-1 labeling was located predominantly in the ganglion cell layer, with faint labeling found in the inner plexiform and nuclear layers, confirming several earlier results.5()1(> This pattern of Thy-1 fluorescence suggests that Thy-1 is expressed on retinal ganglion cell surfaces and processes. Our study shows that Thy-1 dependent fluorescence can be detected in the retina from the day of birth, though the labeling intensity is very low during the first few days. The staining increased steadily to its maximal intensity at the end of the second postnatal week. This progressive increase in Thy-1 expression also was seen at the RNA level, suggesting that Thy-1 is regulated primarily at the transcriptional level. The larger band seen after PCR amplification of embryonic and early postnatal cDNA could have been caused by the accumulation of eiuher unprocessed RNA or to residual genomic DNA contamination. Because of the developmental change in this band and the lack of any evidence of DNA contamination in the controls, we think the latter is unlikely. Our results suggest a possible dissociation between transcription and ap-
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pearance of mature Thy-1 RNA at early developmental stages. The peak level of Thy-1 in the developing retina coincides with synapse formation in the superior colliculus, dorsal lateral geniculate, and other central visual centers.3'" This suggests a central role for Thy-1 in retinal ganglion cell development in the retina. Other investigators have postulated a similar role for Thy-1 in postnatal rat cerebellum, where dendritic growth and synaptogenesis are accompanied by high levels of Thy-1.3 The persistence of Thy-1 in adult retina—albeit at lower levels than that seen during the second week— indicates a role for this molecule beyond development alone. Morris and Raisman11 also found a 20% to 30% decrease in Thy-1 density between 4 and 34 weeks of age. The decrease in Thy-1 density in the adult may reflect diminished Thy-1 expression on each ganglion cell body and its processes, or it may be the result of decreased ganglion cell density as the eyes enlarge with increasing age.
Pixel Density
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P4
P7
P10
Adult
Age
FIGURE 4.
Quantitation of the data shown in Figure 3. The intensity of the amplified bands was plotted after normalization to a constant level of /?-actin and subtraction of background pixel densities. The results show an increase beginning at postnatal day 2. The level seen in adult samples was approximately 15-fold higher than that seen in the postnatal day 2 retina. Although the biologic significance of Thy-1 remains obscure, the current study indicates a temporal course that may reflect a significant role for this ubiquitous molecule in retinal development. Further studies may help to define a role for Thy-1 in the retina more accurately. Key Words immunohistochemistry, neural development, retinal ganglion cells, reverse transcription -polymerase chain reaction (RT-PCR), Thy-1 References
FIGURE 3. Amplification of Thy-1 cDNA from rat retina. RNA was extracted from retina of the ages shown and converted into cDNA. Samples were amplified using Thy-1 primers and /3-actin (not shown). Controls used were embryonic day 15 cortical cDNA, a negative control reaction without cDNA (Thy-1 (—)), and a positive control reaction of amplification of adult retinal cDNA using primers specific for the 61 -kDa isoform of a CaCaM-dependent phosphodiesterase (61(+)). Lane M shows X.HindUl and
