November 2009
Volume 50, Issue 11
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Retina  |   November 2009
CD73, a Novel Cell Surface Antigen That Characterizes Retinal Photoreceptor Precursor Cells
Author Affiliations & Notes
  • Hideto Koso
    From the Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; the
  • Chiharu Minami
    From the Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; the
    Tokyo College of Biotechnology, Tokyo, Japan; and the
  • Yoko Tabata
    From the Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; the
  • Mariko Inoue
    From the Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; the
  • Erika Sasaki
    Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan.
  • Shinya Satoh
    From the Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; the
  • Sumiko Watanabe
    From the Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; the
  • Corresponding author: Sumiko Watanabe, Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; sumiko@ims.u-tokyo.ac.jp
Investigative Ophthalmology & Visual Science November 2009, Vol.50, 5411-5418. doi:10.1167/iovs.08-3246
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      Hideto Koso, Chiharu Minami, Yoko Tabata, Mariko Inoue, Erika Sasaki, Shinya Satoh, Sumiko Watanabe; CD73, a Novel Cell Surface Antigen That Characterizes Retinal Photoreceptor Precursor Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(11):5411-5418. doi: 10.1167/iovs.08-3246.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: The authors sought to identify cell surface markers of photoreceptor and its precursor cells.

Methods.: The expression of surface CD antigens that label both temporally and spatially distinct populations of mouse retinal cells were examined. Of the antibodies that showed positive signals in retinal cells, CD73 was focused on for more detailed analyses.

Results.: Mouse retinal subpopulations that expressed CD73 first appeared around birth and subsequently increased dramatically in number, eventually representing more than 90% of the retinal cells in the adult. CD73+ cells were postmitotic and mostly rhodopsin-negative at postnatal day 1. However, in the adult retina, most of these cells expressed rhodopsin but not s-opsin. In reaggregation cultures, CD73+ cells differentiated into rhodopsin-positive cells more rapidly than CD73 cells, which supports the idea that CD73 is an early photoreceptor lineage marker. The effects of ectopic expression in retinal cells of Nrl and Crx, both of which are transcription factors known to be expressed in photoreceptor lineage, suggest that CD73 is genetically downstream of Crx in the rod cell differentiation lineage. Adult retina of the common marmoset monkey also showed correlation of the expression pattern of rhodopsin and CD73.

Conclusions.: CD73 is a cell surface marker of cone/rod common precursors and mature rod cells in mice and is genetically localized between Nrl and Crx. The expression of CD73 was conserved in primate rod cells, and CD73 provides an useful tool to purify photoreceptor cells for transplantation aimed at the regeneration of photoreceptors.

The vertebrate neural retina consists of six types of neurons and one type of glial cells, which are organized into a laminar structure. The outer nuclear layer (ONL) consists of photoreceptors, and specific loss of these cells causes several severe retinal diseases, among them retinitis pigmentosa. 1,2 Regeneration of photoreceptor cells is an important step in the regeneration of vision, and considerable effort is being invested in understanding these processes. The isolation of retinal progenitor cells or precursors of the photoreceptors lineage is one of the strategies used to achieve neural retina regeneration by transplantation. 3 However, these cell populations have not yet been adequately characterized, in part because of a lack of markers that can be used to identify the distinct stages and lineages of retinal cells. Although the patterns of expression of transcriptional factors reported to be involved in retinal development may reflect the developmental stage, these molecules are intracellular, which limits their usefulness for cell enrichment. Therefore, it is important to define surface markers that can be used to label specific retinal cell subpopulations. Surface antigens permit the isolation of a specific subset of cells from a cell mixture without damaging the cells, which facilitates the characterization of cell lineages and the identification of factors that regulate cell proliferation and differentiation. We evaluated candidate markers using flow cytometry and cell sorting in combination with retinal in vitro cultures. We screened the mouse retina at various developmental stages for reactivities with a panel of antibodies directed against cell-surface antigens and obtained unique expression patterns for more than 30 antigens. Among these, SSEA-1 (CD15) and c-kit (CD117) have been shown previously to represent the early and late immature stages of retinal progenitor cells, respectively. 4,5  
In the present study, we focused on the CD73 antigen, the expression of which was seen to increase concomitantly with retinal development. CD73, which is also known as ecto-5′-nucleotidase, is a 70-kDa glycosylphosphatidylinositol (GPI)-anchored cell surface molecule that catalyzes the extracellular conversion of 5′-adenosine monophosphate to adenosine. 6,7 We identified CD73 as a marker of the early stages of the photoreceptor lineage. CD73 is assumed to be localized genetically downstream of Crx. This is the first report describing a cell surface marker of immature photoreceptor cells. 
Materials and Methods
Mice, Common Marmoset, and Cultures
EGFP transgenic mice, which were kindly provided by Masaru Okabe (Osaka University, Japan), 8,9 were maintained in a C57BL/6J background. ICR mice were obtained from Japan SLC. Common marmoset was maintained in the Central Institute for Experimental Animals (CIEA) in accordance with CIEA guidelines. Retinal explant cultures and reaggregation cultures were prepared as described previously 4,10 and were infected with retroviruses as described previously. 10,11 All animal experiments were approved by the Animal Care Committee of the Institute of Medical Science, University of Tokyo and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Fluorescence-Activated Cell Sorting
Neural retinas were isolated and dispersed to single cells using trypsin and were stained with antibodies, as described previously. 4 The following antibodies were used: anti-CD73 (BD Bioscience, Franklin Lakes, NJ), anti-Ki67 (BD Biosciences), anti-Rho4D2 (kind gift from Robert Molday, University of British Columbia, Canada), anti-Nestin (BD Biosciences), anti-PNR (Perseus Proteomics, Tokyo, Japan), and anti-protein kinase C (anti-PKC; Oncogene Research Products, San Diego, CA). Nonlabeled antibodies were visualized with the appropriate secondary antibody conjugated with Alexa488 (Molecular Probes, Eugene, OR). At least 10,000 events for healthy cells were analyzed (FACSCalibur; BD Biosciences). Cell sorting was carried out (FACSVantage or FACSAria; BD Biosciences), as described previously, 4 and results (FACSAria; BD Biosciences) were replotted (FlowJo software; Tree Star, Ashland, OR) 
Construction and Immunostaining
The full-length cDNAs of the mouse Crx, Nrl, and CD73 genes were cloned by polymerase chain reaction (PCR) according to the sequences in the database using mouse retinal cDNA. The PCR products were cloned into a vector (pGEM-T-Easy; Promega, Madison, WI), and the inserts were subcloned into the pMX-IRES-EGFP retrovirus vector. 10 Immunostaining for sectioned or dissociated retinas was performed as described previously. 4,10 The primary antibodies used were anti-CD73, anti-rhodopsin (Rho4D2), anti-s-opsin (Chemicon, Temecula, CA), anti-glutamine synthetase (Chemicon), and anti-m-opsin (Chemicon). The primary antibodies were visualized using the appropriate secondary antibodies conjugated to Alexa-488 or Alexa-546 (Molecular Probes). DAPI was used for nuclear staining. Samples were mounted (VectaShield; Vector Laboratories, Burlingame, CA) and analyzed under a microscope (Axioplan; Zeiss, Thornwood, NY). 
RT-PCR
Total RNA was purified from CD73+ and CD73 retinal cells using the reagent (TRIZOL; Gibco BRL, Grand Island, NY), and cDNA was synthesized by reverse transcriptase (Superscript II; Gibco BRL). All the primer sets were tested for different numbers of cycles (25–35 cycles) using rTaq (Takara, Shiga, Japan), and the semiquantitative cycle number was determined for each primer set. DNA bands were visualized with ethidium bromide. 
Results
Characterization of CD73+ Cells in the Developing Retina
By screening the expression patterns of various CD antigens in the mouse retina at various developmental stages, we noted that the expression levels of some antigens changed as retinal development progressed. The expression of the CD73 antigen increased dramatically with retinal development. Initially, we examined CD73 expression in the mouse neural retina at various developmental stages using flow cytometry (Fig. 1A). CD73 expression was not detected in the retina at embryonic day (E) 14 and was first observed in approximately 10% of the cells derived from an E16 mouse (Fig. 1A). The population of CD73-expressing cells increased along with mouse development until postnatal day (P) 6, and 90% of the cells in the adult mouse retina expressed CD73. 
Figure 1.
 
Characterization of CD73 expression. (A) Flow cytometric analysis of the expression of CD73 at various developmental stages of the mouse retina. The dot plot patterns of side scatter (SSC) versus CD73 are shown. (BD) Contour plot patterns of double staining of the mouse retina at P1 with anti- CD73 versus anti-Ki67 (B), and of the mouse retina at the P9 stage with antibodies against nestin, protein kinase C (PKC) (C), and at the adult stage with antibodies against PKC and islet-1 (D). (E) Coexpression of CD73 (red) and rhodopsin (green) in dissociated retinal cells was examined. Retinal cells at the indicated stages were dissociated on the plate and immunostained with anti-CD73 and anti-rhodopsin antibodies. DAPI was used to stain the nuclei. Left: views under the microscope of samples derived from P9 mice. Right: the percentages of rhodopsin/CD73 double-positive subpopulation of CD73+ cells are shown. (F) Contour plot pattern of double-staining for CD73 and rhodopsin of a retina at P6, P9, and adult. (G) Immunostaining for CD73 and rhodopsin of frozen-sectioned retina derived from mice at P1, P5, and adulthood. GCL, ganglion cell layer; NBL, neuroblastic layer; INL, inner nuclear layer; ONL, outer nuclear layer. (H) Double-staining of mouse retinal cells at the P2 stage with anti-CD73 (red) and anti-s-opsin (green) antibodies.
Figure 1.
 
Characterization of CD73 expression. (A) Flow cytometric analysis of the expression of CD73 at various developmental stages of the mouse retina. The dot plot patterns of side scatter (SSC) versus CD73 are shown. (BD) Contour plot patterns of double staining of the mouse retina at P1 with anti- CD73 versus anti-Ki67 (B), and of the mouse retina at the P9 stage with antibodies against nestin, protein kinase C (PKC) (C), and at the adult stage with antibodies against PKC and islet-1 (D). (E) Coexpression of CD73 (red) and rhodopsin (green) in dissociated retinal cells was examined. Retinal cells at the indicated stages were dissociated on the plate and immunostained with anti-CD73 and anti-rhodopsin antibodies. DAPI was used to stain the nuclei. Left: views under the microscope of samples derived from P9 mice. Right: the percentages of rhodopsin/CD73 double-positive subpopulation of CD73+ cells are shown. (F) Contour plot pattern of double-staining for CD73 and rhodopsin of a retina at P6, P9, and adult. (G) Immunostaining for CD73 and rhodopsin of frozen-sectioned retina derived from mice at P1, P5, and adulthood. GCL, ganglion cell layer; NBL, neuroblastic layer; INL, inner nuclear layer; ONL, outer nuclear layer. (H) Double-staining of mouse retinal cells at the P2 stage with anti-CD73 (red) and anti-s-opsin (green) antibodies.
Double immunostaining of CD73 and Ki67, which is a nuclear cell proliferation-associated antigen, 12 showed that CD73 expression in postmitotic cells (Fig. 1B). However, between 10% and 20% of CD73+ cells are Ki67/CD73 double positive at P1 and E17 (data not shown), suggesting that retinal progenitor cells begin to express CD73 at the end of their proliferative stage. We also examined whether CD73+ cells had the characteristics of differentiated lineages of the retinal subpopulation by costaining. Nestin is expressed in neural progenitor cells. 13 None of CD73+ cells coexpressed nestin at P9, which confirms that the CD73+ cell fraction is enriched for differentiated cells. PKC, which is a marker of bipolar cells, was expressed only in a negligible fraction (3%) of the CD73low cells at P9 (Fig. 1C). Similar results were obtained by staining adult retinal cells with antibodies against CD73 and PKC or Islet-1, which are markers of bipolar or amacrine cells, respectively (Fig. 1D). In addition to these results, our immunostaining data do not support the idea that CD73 is expressed in retinal cells other than rod photoreceptors. However, our observation that 85% to 90% of the adult retinal cells were CD73+ by FACS analysis is higher than the generally accepted value for rod photoreceptors (70%). One possible explanation for this discrepancy is the selective loss of retinal cells other than rod photoreceptors during the preparation of single cells by dissociation for FACS analysis. 
Rod photoreceptor cells express rhodopsin and represent the largest of the retinal cell subpopulations; these cells differentiate during the last stage of retinal development. 14 More than 80% of the retinal cells were rhodopsin positive at P9, and the most of these cells were double positive for rhodopsin and CD73 (Fig. 1F), which suggests that CD73 is a marker of rhodopsin-positive cells. Previous reports demonstrated the expression of 5′-nucleotidase enzyme activities in photoreceptor cells of the rat retina, 15 which is consistent with our present results. Therefore, we examined in more detail the coexpression of CD73 and rhodopsin during retinal development. Retinal cells from mice at P1, P6, and P9 were dissociated and immunostained with the anti-CD73 and anti-rhodopsin antibodies. Some of the cells were replated and used for immunohistochemistry with the same set of antibodies (Fig. 1E). Less than 30% of the CD73+ cells expressed rhodopsin in P1 mouse retinas, and more than 50% of the CD73+ cells expressed rhodopsin in P6 mouse retinas. Interestingly, although a significant number of CD73+ cells were observed at P6, not all cells were rhodopsin-positive cells at this stage (Fig. 1F). At the more advanced stage of P9, most of the cells were double-positive for CD73 and rhodopsin (Figs. 1E, 1F), and less than 10% of the cells in the P9 and adult samples were rhodopsin-positive/CD73-negative. We attempted to characterize these cells; however, we have no explanation for our observation. Taken together, these results suggest that retinal cells that commit to the rod photoreceptor cell lineage initially express CD73 and subsequently become double-positive for CD73 and rhodopsin. To examine this hypothesis at the transcriptional level, we examined the temporal transitions of mRNA expression of CD73 and rhodopsin by semiquantitative RT-PCR (see Fig. 4C). Weak expression of CD73 mRNA was observed in the retina at E16, and the strength was increased until P5, which is consistent with the transition of CD73 protein expression revealed by FACS analysis. This suggests that the expression of CD73 is regulated mainly at the transcriptional level. The onset of CD73 expression occurred earlier than that of rhodopsin, which supports the idea that CD73 is an earlier marker than rhodopsin of the photoreceptor lineage. 
We also examined the spatial localization of CD73+ cells in the developing retina by immunohistochemistry and compared this expression pattern with that of rhodopsin (Fig. 1G). In P1 retinas, CD73 was observed in the outer half of the neuroblastic layer, which corresponds to the area in which the photoreceptors start to differentiate. At P5, the ONL became visible, and CD73 expression was evident throughout the ONL. When we examined adult mouse-derived retinas, CD73 expression was strong and widespread in the ONL nucleus and in the outer region of the nucleus, and rhodopsin expression was strong in the outer region of the ONL, which corresponds to the outer segment of the photoreceptor. Although a previous study reported the expression of CD73 in Müller glial cells, 16 we did not observe the expression of CD73 in the inner nuclear layer (INL), in which the Müller glial cell body is localized, at any of the stages examined (Fig. 1G and data not shown). This notion was confirmed by the lack of expression of mRNA of glutamine synthetase (GS), which is a marker of Müller glial cells, in the purified CD73+ cell population (see Fig. 4A). Because GS antibody cannot be used for FACS analysis (data not shown), we examined its expression in dissociated CD73+ retinal cells derived from P15 mice and found that only a negligible number (1/150 CD73+ cells) of GS/CD73 double-positive cells was observed, further supporting this notion. In the ONL, in addition to the rod cells, cone cells were detected. We examined whether CD73 was expressed in cone cells by double immunostaining the adult mouse retina with the cone marker s-opsin and CD73. We found that CD73+ cells never expressed s-opsin (Fig. 1H). However, when we used semiquantitative RT-PCR to examine the expression of s-opsin mRNA in CD73+ and CD73 cells from the retinas of developing mice, s-opsin expression was observed in both cell fractions (see Fig. 4B). 
In Vitro Differentiation and Proliferation of CD73+ Cells
We next examined the differentiation and proliferation activities of isolated CD73+ cells using an in vitro culture system. We used reaggregation cultures, which have been shown to be an excellent model system for examining the intrinsic proliferation and differentiation of retinal progenitors in vitro. 4,5 In this system, the cells proliferate and differentiate into rod photoreceptor cells in a manner similar to that seen in vivo. 17 By culturing labeled donor cells with an excess number of unlabeled host retinal cells, we could evaluate the intrinsic proliferation and differentiation of the donor cells in a defined environment. 18 To distinguish transplanted cells from host cells, we used neural retinas derived from EGFP transgenic (Tg) mice 8,9 as donor cells. 4,5 CD73+ and CD73 cells of EGFP Tg mice at P1 were purified in a cell sorter (Fig. 2A) and mixed with an excess number of dissociated unfractionated host retinal cells from normal mice at P1 to prepare reaggregation cultures. When we examined the expression of rhodopsin before starting the culture, less than 5% of CD73+ cells were rhodopsin positive, but no expression of rhodopsin was observed in CD73 cells (Fig. 2B). After 3 days of culture, the number of rhodopsin-positive cells increased, but these cells were still only observed in the CD73+ cell reaggregates. On day 7 of culture, nearly 60% of the CD73+ cells were rhodopsin positive, whereas only a low percentage of the CD73 cells were rhodopsin positive. In this culture system, the efficiency of cell differentiation was lower than in the in vivo situation (using unfractionated retinal cells, the maximum percentage we observed as rhodopsin-positive cells was 60%; data not shown). On day 10 of culture, although the proportion of rhodopsin-positive cells in the CD73 cell population had increased significantly, it was far lower than the proportion of rhodopsin-positive cells in the CD73+ population. This indicates that CD73+ cells are in a more advanced stage of the rod photoreceptor cell lineage than CD73 cells. 
Figure 2.
 
Differentiation of CD73+ and CD73 cells in reaggregation cultures. (A) Dot plot pattern of EGFP (Fl-1) versus anti-CD73 antibody staining (PE [Fl-2]) of E17 neural retina derived from EGFP Tg mice. Whole cells displaying the pattern shown on the left were fractionated according to CD73 expression in the cell sorter, as shown in the middle (CD73+) and right (CD73) panels. The purities of the fraction were approximately 90% and >95%, respectively. (B) Reaggregation cultures that consisted of a mixture of donor cells and host cells were prepared to analyze the differentiation of CD73+ and CD73 cells. CD73+ or CD73 retinal cells derived from the EGFP Tg mice at P1 were mixed with a large excess of retinal cells from normal mice of the same age. The rhodopsin-expressing cells (%) in the EGFP-positive cell population are shown. Reaggregation cultures were harvested at the indicated days of culture, and the cells were replated on a chamber glass slide and immunostained with antibodies against GFP and rhodopsin. The experiments were performed at least twice, with essentially the same results.
Figure 2.
 
Differentiation of CD73+ and CD73 cells in reaggregation cultures. (A) Dot plot pattern of EGFP (Fl-1) versus anti-CD73 antibody staining (PE [Fl-2]) of E17 neural retina derived from EGFP Tg mice. Whole cells displaying the pattern shown on the left were fractionated according to CD73 expression in the cell sorter, as shown in the middle (CD73+) and right (CD73) panels. The purities of the fraction were approximately 90% and >95%, respectively. (B) Reaggregation cultures that consisted of a mixture of donor cells and host cells were prepared to analyze the differentiation of CD73+ and CD73 cells. CD73+ or CD73 retinal cells derived from the EGFP Tg mice at P1 were mixed with a large excess of retinal cells from normal mice of the same age. The rhodopsin-expressing cells (%) in the EGFP-positive cell population are shown. Reaggregation cultures were harvested at the indicated days of culture, and the cells were replated on a chamber glass slide and immunostained with antibodies against GFP and rhodopsin. The experiments were performed at least twice, with essentially the same results.
Role of CD73 in Retinal Rod Photoreceptor Cell Differentiation
CD73 is an enzyme with ecto-5′-nucleotidase activity, and the enzymatic product of CD73 is adenosine. 6,7 Of the four types of adenosine receptor (A1, A2a, A2b, A3), 19 A2a and A2b have previously been reported to be expressed in the neural retina of the rat. 20,21 We examined the temporal expression of the mRNAs for these receptors by semiquantitative RT-PCR (Fig. 3A). We found that A1 was expressed in the neural retina from an early stage (E16) of retinal development and that the A1 expression level increased as retinal development progressed (Fig. 3A). Very weak bands were observed for A2a and A2b in the embryonic retina; these receptors started to be expressed in the postnatal retina, and the expression level increased concomitantly with development. We also used semiquantitative RT-PCR of RNA samples isolated from purified CD73+ and CD73 cells at various stages to examine whether these receptors were expressed in CD73+ cells (Fig. 3B). Interestingly, all three adenosine receptor subtypes were predominantly expressed in CD73+ cells at P1 (Fig. 3B). At P2, weak expression of all three receptors was observed in the CD73 cells; at P6, significant expression of A1 and A2a was observed in both the CD73+ and CD73 cells, and A2b was detected primarily in the CD73+ fraction. 
Figure 3.
 
The roles of CD73 in retinal development. Temporal expression of mRNA for adenosine receptors in the developing retina. (A) Semiquantitative RT-PCR for adenosine receptors A1, A2a, and A2b was carried out using total RNA samples purified from mouse retinas at various developmental stages. (B) The expression of adenosine receptors A1, A2a, and A2b in CD73+ and CD73 cells at the indicated stages was examined by semiquantitative RT-PCR. (CF) Gain- and loss-of-function analyses of CD73. Retroviruses that encode IRES-EGFP or CD73-IRES-EGFP (for overexpression experiments) and CMV EGFP or shRNA against CD73-CMV EGFP (for downregulation experiments) were transduced into retinal explants at E16, followed by FACS analysis of the expression of CD73 (C) and rhodopsin (D) after 14 days of culture. (E) Quantitative results for rhodopsin-positive cells calculated from (D). The rhodopsin-positive cells in the EGFP-positive cell population (shown in D) are expressed as the relative percentage of those in the EGFP-negative cells population in each experiment. (F) Distributions of EGFP-positive cells in the ONL, INL, and GCL. Retroviruses that encode IRES-EGFP, CD73-IRES-EGFP, CMV-EGFP, or shRNA against CD73-CMV EGFP were transduced into retinal explants at E16. After 14 days of culture, frozen sections were produced, and the distribution of EGFP-positive cells in each layer was examined.
Figure 3.
 
The roles of CD73 in retinal development. Temporal expression of mRNA for adenosine receptors in the developing retina. (A) Semiquantitative RT-PCR for adenosine receptors A1, A2a, and A2b was carried out using total RNA samples purified from mouse retinas at various developmental stages. (B) The expression of adenosine receptors A1, A2a, and A2b in CD73+ and CD73 cells at the indicated stages was examined by semiquantitative RT-PCR. (CF) Gain- and loss-of-function analyses of CD73. Retroviruses that encode IRES-EGFP or CD73-IRES-EGFP (for overexpression experiments) and CMV EGFP or shRNA against CD73-CMV EGFP (for downregulation experiments) were transduced into retinal explants at E16, followed by FACS analysis of the expression of CD73 (C) and rhodopsin (D) after 14 days of culture. (E) Quantitative results for rhodopsin-positive cells calculated from (D). The rhodopsin-positive cells in the EGFP-positive cell population (shown in D) are expressed as the relative percentage of those in the EGFP-negative cells population in each experiment. (F) Distributions of EGFP-positive cells in the ONL, INL, and GCL. Retroviruses that encode IRES-EGFP, CD73-IRES-EGFP, CMV-EGFP, or shRNA against CD73-CMV EGFP were transduced into retinal explants at E16. After 14 days of culture, frozen sections were produced, and the distribution of EGFP-positive cells in each layer was examined.
To examine the role of CD73 in retinal development, we performed gain- and loss-of-function analyses of CD73 using retinal explant culture. For the gain-of-function analysis, CD73 was expressed by retrovirus-mediated gene expression in the retinal explant. A retrovirus that encoded the wild-type CD73-IRES-EGFP was transduced into retinal explants prepared from an E16 mouse. After 14 days of culture, CD73 expression was examined by FACS analysis (Fig. 3C). The control EGFP virus-expressing population contained both CD73+ and CD73 cells. In contrast, the CD73-IRES-EGFP virus-transduced cell population contained only CD73+ cells (Fig. 3C). In addition, the expression level of CD73 in this cell population was higher than that of the control samples. After 14 days of retinal explant culture, the expression of rhodopsin was examined by FACS analysis (Figs. 3D, 3E). In the CD73-overexpressed fraction, the proportion of rhodopsin-positive cells was similar to that observed in the control EGFP virus-infected population (Fig. 3E). The downregulation of CD73 was achieved using shRNA, which was introduced into the retinal explant cultures by retroviruses. The suppression of CD73 expression was confirmed by FACS analysis (Fig. 3C). The levels of rhodopsin expression were similar in the EGFP-positive and EGFP-negative cells after 14 days of culture (Fig. 3D). Once again, the levels of rhodopsin expression (Fig. 3E) and distribution of cells into subretinal layers (Fig. 3F) were similar in the EGFP-positive, CD73-downregulated cells and the control EGFP virus-infected cells. Taken together, these results suggest that CD73 does not mediate autonomous cellular signals to promote rhodopsin lineage differentiation. We also examined whether the overexpression or downregulation of CD73 affects other cell lineages, such as Müller glia, ganglion, and amacrine cells by immunostaining with specific markers such as glutamine synthetase and HuC/D. We did not detect significant differences between the two fractions and the controls (data not shown). 
CD73 Expressed Downstream of Crx
To analyze the detailed molecular mechanisms, we examined the gene expression patterns of CD73+ and CD73 cells by semiquantitative RT-PCR. As expected, rhodopsin was expressed exclusively in CD73+ fractions (Fig. 4B). Crx and Nrl are transcription factors that play important roles in photoreceptor cell development. 22,23 Nrl was observed exclusively in CD73+ cells, whereas Crx was observed in both CD73+ and negative cells, which suggests that Crx is expressed earlier than CD73. We also examined the expression of early retinal progenitor markers, such as Chx10 24 and Hes1, and found that they were expressed exclusively in CD73 cells, as expected (Fig. 4A). 
Figure 4.
 
Mapping of CD73 in the hierarchy of genes involved in photoreceptor cell differentiation. (A, B) Semiquantitative RT-PCR of CD73+ and CD73 populations from the P1 (A) and P2 and P6 (B) mouse retina. The RT-PCR products were separated in a 1% agarose gel and visualized with ethidium bromide. G3PDH was used as a control. The experiments were performed at least twice, for all the primers, using independently prepared samples, with essentially the same results. (C) Kinetics of gene expression in the mouse retina. Semiquantitative RT-PCR was performed using RNA from mouse retinas at various developmental stages. G3PDH was used as a control. (D, E) Effects of retrovirus-mediated expression of Crx and Nrl transcription factors on CD73 expression. Retinal explant cultures prepared from E16 mouse retinas were infected with retroviruses that contained EGFP, Crx-IRES-EGFP, or Nrl-IRES-EGFP, and the cells were cultured for 4 days and then dissociated. The expression of CD73 in the EGFP-positive and EGFP-negative cells was examined by flow cytometry. The flow cytometric patterns (D) and the calculated values for the CD73+ cells in the EGFP-positive or EGFP-negative populations are shown (E). The experiments were performed at least three times with essentially the same results.
Figure 4.
 
Mapping of CD73 in the hierarchy of genes involved in photoreceptor cell differentiation. (A, B) Semiquantitative RT-PCR of CD73+ and CD73 populations from the P1 (A) and P2 and P6 (B) mouse retina. The RT-PCR products were separated in a 1% agarose gel and visualized with ethidium bromide. G3PDH was used as a control. The experiments were performed at least twice, for all the primers, using independently prepared samples, with essentially the same results. (C) Kinetics of gene expression in the mouse retina. Semiquantitative RT-PCR was performed using RNA from mouse retinas at various developmental stages. G3PDH was used as a control. (D, E) Effects of retrovirus-mediated expression of Crx and Nrl transcription factors on CD73 expression. Retinal explant cultures prepared from E16 mouse retinas were infected with retroviruses that contained EGFP, Crx-IRES-EGFP, or Nrl-IRES-EGFP, and the cells were cultured for 4 days and then dissociated. The expression of CD73 in the EGFP-positive and EGFP-negative cells was examined by flow cytometry. The flow cytometric patterns (D) and the calculated values for the CD73+ cells in the EGFP-positive or EGFP-negative populations are shown (E). The experiments were performed at least three times with essentially the same results.
We examined in more detail the time course of gene expression (Fig. 4C). We first examined the temporal transitions of mRNA expression of CD73 and rhodopsin by semiquantitative RT-PCR (Fig. 4C). Weak expression of CD73 mRNA was observed in the retina at E16, and the strength was increased until P5, which is consistent with the transition of CD73 protein expression revealed by FACS analysis. This suggests that the expression of CD73 is regulated mainly at the transcriptional level. The onset of CD73 expression occurred earlier than that of rhodopsin, which supports the idea that CD73 is an earlier marker than rhodopsin of the photoreceptor lineage. Crx was found to be expressed even at E14, and CD73 expression started around E16 to E18. A transition in Nrl expression in the developing mouse retina was previously reported. Bibb et al. 25 reported that Nrl was expressed after birth, whereas Akimoto et al. 26 reported that Nrl was first expressed during early embryogenesis around E12. In our study, Nrl and rhodopsin expression began around P1. The difference in Nrl expression between these previous studies and our own may be attributed to a difference in primer sensitivity. Our results confirm that the onset of CD73 expression occurs between Crx and Nrl expression. Therefore, we examined the role of Crx in CD73 expression. To test the effects of Crx, we used Nrl for comparison purposes and incorrectly expressed these genes in retinal explants derived from E16 mice by retroviral-mediated gene transfer. 11 Retinal explants were infected with retrovirus that encoded Crx- or Nrl-IRES-EGFP; they were cultured for 4 days, harvested, and dissociated. CD73 expression was analyzed by flow cytometry (Figs. 4D, 4E). In the control EGFP virus-transfected cells, approximately 30% of the cells expressed CD73 in both the EGFP-positive and EGFP-negative fractions. However, in the Crx retrovirus-infected explant samples, more than 40% of the EGFP-expressing cells also expressed CD73, which was significantly higher than that of the control cells (Fig. 4E). In the Nrl-expressing cell population, the proportion of CD73+ cells in the EGFP-positive fraction was almost the same as in the uninfected parental cells or control EGFP virus-infected cells (Figs. 4D, 4E). Taken together, these results suggest that the activation of Crx positively regulates CD73-expressing cells and that Crx (but not Nrl) is upstream of CD73 in the photoreceptor development hierarchy. 
CD73, a Marker for Rod Photoreceptor Cells in Retina of Common Marmoset (Callithrix jacchus)
Finally, we asked whether our finding that CD73 is a marker for rod photoreceptor is also applicable for primates. Retinas from adult common marmoset were isolated, and we first examined the expression of rhodopsin and M-opsin in the frozen sectioned retina, including the fovea region. The fovea pit is depicted at the central region of Figure 5A, identified by its unique bended structure of nuclear layers (Fig. 5B). As previously reported, 27 M-opsin was predominantly expressed in the fovea region, and, in contrast, rhodopsin was distributed in regions other than the fovea (Figs. 5A, 5B). Although anti-human CD73 antibody did not give reliable signals of frozen sectioned retina by immunohistochemistry, FACS analysis using whole retina showed strong expression of CD73 (Fig. 5C). Therefore, under a microscope, we mechanically divided the fovea and other regions with the use of a scalpel and used FACS to examine the CD73 expression of these two fractions (Fig. 5D). Strong CD73 expression was observed with retina except for fovea, and only a small population of cells at the foveal region expressed CD73. We purified CD73+ and CD73 cells of the adult marmoset retina by cell sorter, and the cells were seeded on the slide glass and immunostained of the purified cells with anti-rhodopsin and -PNR, which is rod photoreceptor specific marker, antibodies (Fig. 5E). Almost all cells in CD73+ fractions were rhodopsin or PNR positive; in contrast, only a few positive cells were observed in CD73 cells. Taken together, we concluded that CD73 is a marker of rod photoreceptor cells in the mature retina of the common marmoset. 
Figure 5.
 
Expression of CD73 in marmoset retina. (A, B) Expression of M-opsin and rhodopsin in adult common marmoset retina. Immunostaining was performed using frozen-sectioned retina, and nuclei were visualized by DAPI. (C) FACS pattern of CD73 staining of whole retina of adult common marmoset. Green line: CD73 staining. Black line: control IgG staining. (D) Adult common marmoset retina was mechanically divided into fovea and other regions, and FACS analysis of CD73 expression was performed for both fractions. Violet region, green line: staining patterns of fovea and other regions, respectively. (E) Whole retina of adult common marmoset was dissociated and sorted to CD73+ and CD73 populations by cell sorter, and the cells were replated on a chamber glass slide and immunostained with antibodies against rhodopsin (upper four panels) and PNR (lower four panels). Nuclei were stained with DAPI.
Figure 5.
 
Expression of CD73 in marmoset retina. (A, B) Expression of M-opsin and rhodopsin in adult common marmoset retina. Immunostaining was performed using frozen-sectioned retina, and nuclei were visualized by DAPI. (C) FACS pattern of CD73 staining of whole retina of adult common marmoset. Green line: CD73 staining. Black line: control IgG staining. (D) Adult common marmoset retina was mechanically divided into fovea and other regions, and FACS analysis of CD73 expression was performed for both fractions. Violet region, green line: staining patterns of fovea and other regions, respectively. (E) Whole retina of adult common marmoset was dissociated and sorted to CD73+ and CD73 populations by cell sorter, and the cells were replated on a chamber glass slide and immunostained with antibodies against rhodopsin (upper four panels) and PNR (lower four panels). Nuclei were stained with DAPI.
Discussion
In the present study, we show that CD73 labels precursor and mature populations of photoreceptor cells. The present report is the first to show specific expression of a cell surface molecule in a photoreceptor cell lineage and the application of this molecule for the enrichment of photoreceptor precursor cells. This is important in terms of applications related to transplantation for the treatment of retinal diseases. Using reaggregation cultures of retinal cells, we show that the purification of CD73 is an efficient way to achieve photoreceptor cell generation by transplantation. This is an in vitro model system, and we are in the process of developing an in vivo transplantation system. 
Recently, in vivo retinal repair by transplantation of photoreceptor precursors has been reported. 3 Successful regeneration of functional rod photoreceptors in the mouse is encouraging for researchers working on regenerative medicine of the neural retina. However, in the previous report, the isolated retinas were from transgenic mice that expressed EGFP under the control of the Nrl promoter, which limits the use of this protocol for human applications. CD73 is a cell surface molecule and an anti-CD73 antibody is commercially available, allowing for transplantation and regeneration using CD73-expressing cells without risk for gene transfer. Furthermore, it is important that we found that CD73 is also a marker for the common marmoset photoreceptor. Thus, we can apply this knowledge easily and immediately to human systems using an anti-human CD73 antigen antibody. 
Our observation of exclusive expression of CD73 in a photoreceptor lineage is supported by recent DNA microarray analyses of knockout mice. In Nrl-knockout and rhodopsin-knockout mice, both of which lack rod photoreceptors, DNA microarray analyses comparing the retinas of wild-type mice and the knockout mice have revealed that the expression of CD73 is downregulated in these knockout mice. 28,29 However, previous reports have suggested that the 5′-nucleotidase activity is localized in photoreceptor cells and in Müller cells. 30,31 More recently, immunostaining of the 5′-nucleotidase with polyclonal antibody in the developing retina of the mouse has concluded that this enzyme is distributed in Müller cells. 16 In contrast, we did not observe the expression of CD73 in the INL, where the nuclei of Müller glial cells are localized, and CD73 expression never overlapped with glutamine synthetase expression, which is a marker of Müller glial cells, thereby suggesting that CD73 is not expressed in Müller glial cells. Because the previous report did not describe double immunostaining for the 5′-nucleotidase and markers of retinal cells, 16 it is not possible to compare directly the previous results with our present results. However, given that the polyclonal antibody used in the previous paper was raised against the soluble form of bovine 5′-nucleotidase, 16 it is possible that the antibody recognizes a molecule different from that recognized by the anti-CD73 monoclonal antibody used in our present study. This notion is supported by their observation that the expression of the antigen during embryonic development was also associated with proliferating cellular elements, 16 which contrasts with our finding that CD73 is expressed exclusively in nonproliferating cells. Nevertheless, we cannot exclude the possibility that the tertiary structure of CD73 is different in different cell types and that the antibodies only recognize a certain structure of CD73. 
Initially, we expected that cone-specific genes would not be expressed in CD73+ cells. However, cone s-opsin mRNA was detected in CD73 cells derived from neonatal mice. In contrast, in mature retinas, we did not observe CD73 expression in s-opsin-expressing cone cells. We speculate that the transition of CD73 expression during photoreceptor cell differentiation occurs as shown schematically in Figure 6. Thus, CD73 is expressed in the common progenitors of cone and rod cells. After terminal differentiation, CD73 continues to be expressed in rod cells until adulthood, whereas it is downregulated in the cone cell lineage. 
Figure 6.
 
Schematic representation of photoreceptor cell differentiation and CD73. CD 73 is first expressed after Crx in the common precursors of the rod and cone cells and continues to be expressed in the rod cell lineage. However, CD73 appears to be downregulated once the cells commit to differentiation into the cone cell lineage.
Figure 6.
 
Schematic representation of photoreceptor cell differentiation and CD73. CD 73 is first expressed after Crx in the common precursors of the rod and cone cells and continues to be expressed in the rod cell lineage. However, CD73 appears to be downregulated once the cells commit to differentiation into the cone cell lineage.
Nrl and Crx are key transcriptional factors that control photoreceptor differentiation. 23 The homeodomain protein Crx is required for both rod and cone differentiation and regulates the transcription of many photoreceptor-specific genes. 32 The Maf-family bZIP transcription factor NRL is essential for rod differentiation and controls the expression of most of the rod-specific genes. 23 Based on our observations of differential expression of Crx and Nrl in CD73+ and CD73 cells, we hypothesize that CD73 is downstream of Crx and upstream or parallel to Nrl in rod cell differentiation. Furthermore, detailed examination of the transition of expression of the mRNAs for Crx, CD73, and Nrl also support the idea of ordered expression of these genes in the developing retina. Previous studies of the promoter for CD73 in humans have revealed the involvement of various immune-related transcription factors such as NFAT, hypoxia-related factor 1, and the Wnt signaling pathway. 33,34 We looked for the Crx consensus-binding sequence (C/T TAATCC 35 ) in approximately 1 kb of the 5′ upstream region of the mouse CD73 gene and found one matching sequence at −870 nt from the initiation codon. However, this sequence was not found within the region covering 1 kb of human CD73 5′ upstream. We do not know whether the enhancement of CD73-expressing cells by Crx is due to the direct effects of Crx on CD73 transcription or simply results from an expanded population of rod photoreceptors. The most important outcome of the present study is that CD73 is a marker for cells at defined stages of the photoreceptor lineage and can be used in the isolation and transplantation of these cell populations. 
Footnotes
 Supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Footnotes
 Disclosure: H. Koso, None; C. Minami, None; Y. Tabata, None; M. Inoue, None; E. Sasaki, None; S. Satoh, None; S. Watanabe, None
Footnotes
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The authors thank Yasuo Ouchi for helpful discussions, and Takashi Shibata and Nobukazu Watanabe of the FACS Core Laboratory (Institute of Medical Science, University of Tokyo, Tokyo, Japan) for technical support with the sorting. 
References
Rattner A Nathans J . Macular degeneration: recent advances and therapeutic opportunities. Nat Rev Neurosci. 2006;7:860–872. [CrossRef] [PubMed]
Hartong DT Berson E Dryja TP . Retinitis pigmentosa. Lancet. 2006;368:1795–1809. [CrossRef] [PubMed]
MacLaren RE Pearson RA MacNeil A . Retinal repair by transplantation of photoreceptor precursors. Nature. 2006;444.
Koso H Ouchi Y Tabata Y . SSEA-1 marks regionally restricted immature subpopulations of embryonic retinal progenitor cells. Dev Biol. 2006;292:265–276. [CrossRef] [PubMed]
Koso H Satoh S Watanabe S . c-Kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina. Dev Biol. 2007;301.
Zimmermann H . 5′-Nucleotidase: molecular structure and functional aspects. Biochem J. 1992;285:345–365. [PubMed]
Deussen A Bading B Kelm M Schrader J . Formation and salvage of adenosine by macrovascular endothelial cells. Am J Physiol. 1993;264:692–700.
Okabe M Ikawa M Kominami K Nakanishi T Nishimune Y . ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 1997;407:313–319. [CrossRef] [PubMed]
Ikawa M Yamada S Nakanishi T Okabe M . Green fluorescent protein (GFP) as a vital marker in mammals. Curr Top Dev Biol. 1999;44:1–20. [PubMed]
Tabata Y Ouchi Y Kamiya H Manabe T Arai K Watanabe S . Retinal fate specification of mouse embryonic stem cells by ectopic expression of Rx/rax, a homeobox gene. Mol Cell Biol. 2004;24:4513–4521. [CrossRef] [PubMed]
Ouchi Y Tabata Y Arai K Watanabe S . Negative regulation of retinal-neurite extension by β-catenin signalling pathway. J Cell Sci. 2005;118:4473–4483. [CrossRef] [PubMed]
Gerdes J Schwab U Lemke H Stein H . Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 1983;31:13–20. [CrossRef] [PubMed]
Lendahl U Zimmerman LB McKay RD . CNS stem cells express a new class of intermediate filament protein. Cell. 1990;60:585–595. [CrossRef] [PubMed]
Marquardt T Gruss P . Generating neuronal diversity in the retina: one for nearly all. Trends Neurosci. 2002;25:32–38. [CrossRef] [PubMed]
Takizawa T . 5′-Nucleotidase in rat photoreceptor cells and pigment epithelial cells processed by rapid-freezing enzyme. J Histochem Cytochem. 1998;46:1091–1095. [CrossRef] [PubMed]
Braun N Brendel P Zimmerman H . Distribution of 5′-nucleotidase in the developing mouse retina. Dev Brain Res. 1995;88:79–86. [CrossRef]
Watanabe T Raff MC . Rod photoreceptor development in vitro: intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina. Neuron. 1990;4:461–467. [CrossRef] [PubMed]
Belliveau MJ Cepko CL . Extrinsic and intrinsic factors control the genesis of amacrine and cone cells in the rat retina. Development. 1999;126:555–566. [PubMed]
Yaar R Jones MR Chen J-F Ravid K . Animal models for the study of adenosine receptor function. J Cell Physiol. 2005;202:9–20. [CrossRef] [PubMed]
Yang D Zhang Y Nguyen HG . The A2B adenosine receptor protects against inflammation and excessive vascular adhesion. J Clin Invest. 2006;116:1913–1923. [CrossRef] [PubMed]
Kvanta A Seregard S Sejersen S Kull B Fredholm BB . Localization of adenosine receptor messenger RNAs in the rat eye. Exp Eye Res. 1997;65:595–602. [CrossRef] [PubMed]
Furukawa T Morrow EM Li T Davis FC Cepko CL . Retinopathy and attenuated circadian entrainment in Crx-deficient mice. Nat Genet. 1999;23:466–470. [CrossRef] [PubMed]
Mears AJ Kondo M Swain PK . Nrl is required for rod photoreceptor development. Nat Genet. 2001;29:447–452. [CrossRef] [PubMed]
Burmeister M Novak J Liang M-Y . Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nature. 1996;12:376–384.
Bibb LC Holt JK Tarttelin EE . Temporal and spatial expression patterns of the Crx transcription factor and its downstream target: critical differences during human and muse eye development. Hum Mol Genet. 2001;15:1571–1579. [CrossRef]
Akimoto M Cheng H Zhu D . Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors. Proc Natl Acad Sci U S A. 2006;103:3890–3895. [CrossRef] [PubMed]
Martin PR Grunert U . Analysis of the short wavelength-sensitive (“Blue”) cone mosaic in the primate retina: comparison of new world and old world monkeys. J Comp Neurol. 1999;406:1–14. [CrossRef] [PubMed]
Yoshida S Mears AJ Fiedman JS . Expression profiling of the developing and mature Nrl−/− mouse retina: identification of retinal disease candidates and transcriptional regulatory targets of Nrl. Hum Mol Genet. 2004;13:1487–1503. [CrossRef] [PubMed]
Kennan A Aherne A Palfi A . Identification of an IMPDH1 mutation in autosomal dominant retinitis pigmentosa (RP10) revealed following comparative microarray analysis of transcripts derived from retinas of wild-type and Rho (−/−) mice. Hum Mol Genet. 2002;11:547–557. [CrossRef] [PubMed]
Kreutzberg GW Hussain ST . Cytochemical heterogeneity of the glial plasma membrane: 5′-nucleotidase in retinal Muller cells. J Neurocytol. 1982;11:53–64. [CrossRef] [PubMed]
Kreutzberg GW Hussain ST . Cytochemical localization of 5′-nucleotidase activity in retinal photoreceptor cells. Neuroscience. 1984;11:857–866. [CrossRef] [PubMed]
Furukawa T Morrow EM Cepko CL . Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell. 1997;91:531–541. [CrossRef] [PubMed]
Spychala J Kitajewski J . Wnt and beta-catenin signaling target the expression of ecto-5′-nucleotidase and increase extracellular adenosine generation. Exp Cell Res. 2004;296:99–108. [CrossRef] [PubMed]
Synnestvedt K Furuta GT Comerford KM . Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-a mediates permeability changes in intestinal epithelia. J Clin Invest. 2002;110:993–1002. [CrossRef] [PubMed]
Chen S Wang QL Nie Z . Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron. 1997;19:1017–1030. [CrossRef] [PubMed]
Figure 1.
 
Characterization of CD73 expression. (A) Flow cytometric analysis of the expression of CD73 at various developmental stages of the mouse retina. The dot plot patterns of side scatter (SSC) versus CD73 are shown. (BD) Contour plot patterns of double staining of the mouse retina at P1 with anti- CD73 versus anti-Ki67 (B), and of the mouse retina at the P9 stage with antibodies against nestin, protein kinase C (PKC) (C), and at the adult stage with antibodies against PKC and islet-1 (D). (E) Coexpression of CD73 (red) and rhodopsin (green) in dissociated retinal cells was examined. Retinal cells at the indicated stages were dissociated on the plate and immunostained with anti-CD73 and anti-rhodopsin antibodies. DAPI was used to stain the nuclei. Left: views under the microscope of samples derived from P9 mice. Right: the percentages of rhodopsin/CD73 double-positive subpopulation of CD73+ cells are shown. (F) Contour plot pattern of double-staining for CD73 and rhodopsin of a retina at P6, P9, and adult. (G) Immunostaining for CD73 and rhodopsin of frozen-sectioned retina derived from mice at P1, P5, and adulthood. GCL, ganglion cell layer; NBL, neuroblastic layer; INL, inner nuclear layer; ONL, outer nuclear layer. (H) Double-staining of mouse retinal cells at the P2 stage with anti-CD73 (red) and anti-s-opsin (green) antibodies.
Figure 1.
 
Characterization of CD73 expression. (A) Flow cytometric analysis of the expression of CD73 at various developmental stages of the mouse retina. The dot plot patterns of side scatter (SSC) versus CD73 are shown. (BD) Contour plot patterns of double staining of the mouse retina at P1 with anti- CD73 versus anti-Ki67 (B), and of the mouse retina at the P9 stage with antibodies against nestin, protein kinase C (PKC) (C), and at the adult stage with antibodies against PKC and islet-1 (D). (E) Coexpression of CD73 (red) and rhodopsin (green) in dissociated retinal cells was examined. Retinal cells at the indicated stages were dissociated on the plate and immunostained with anti-CD73 and anti-rhodopsin antibodies. DAPI was used to stain the nuclei. Left: views under the microscope of samples derived from P9 mice. Right: the percentages of rhodopsin/CD73 double-positive subpopulation of CD73+ cells are shown. (F) Contour plot pattern of double-staining for CD73 and rhodopsin of a retina at P6, P9, and adult. (G) Immunostaining for CD73 and rhodopsin of frozen-sectioned retina derived from mice at P1, P5, and adulthood. GCL, ganglion cell layer; NBL, neuroblastic layer; INL, inner nuclear layer; ONL, outer nuclear layer. (H) Double-staining of mouse retinal cells at the P2 stage with anti-CD73 (red) and anti-s-opsin (green) antibodies.
Figure 2.
 
Differentiation of CD73+ and CD73 cells in reaggregation cultures. (A) Dot plot pattern of EGFP (Fl-1) versus anti-CD73 antibody staining (PE [Fl-2]) of E17 neural retina derived from EGFP Tg mice. Whole cells displaying the pattern shown on the left were fractionated according to CD73 expression in the cell sorter, as shown in the middle (CD73+) and right (CD73) panels. The purities of the fraction were approximately 90% and >95%, respectively. (B) Reaggregation cultures that consisted of a mixture of donor cells and host cells were prepared to analyze the differentiation of CD73+ and CD73 cells. CD73+ or CD73 retinal cells derived from the EGFP Tg mice at P1 were mixed with a large excess of retinal cells from normal mice of the same age. The rhodopsin-expressing cells (%) in the EGFP-positive cell population are shown. Reaggregation cultures were harvested at the indicated days of culture, and the cells were replated on a chamber glass slide and immunostained with antibodies against GFP and rhodopsin. The experiments were performed at least twice, with essentially the same results.
Figure 2.
 
Differentiation of CD73+ and CD73 cells in reaggregation cultures. (A) Dot plot pattern of EGFP (Fl-1) versus anti-CD73 antibody staining (PE [Fl-2]) of E17 neural retina derived from EGFP Tg mice. Whole cells displaying the pattern shown on the left were fractionated according to CD73 expression in the cell sorter, as shown in the middle (CD73+) and right (CD73) panels. The purities of the fraction were approximately 90% and >95%, respectively. (B) Reaggregation cultures that consisted of a mixture of donor cells and host cells were prepared to analyze the differentiation of CD73+ and CD73 cells. CD73+ or CD73 retinal cells derived from the EGFP Tg mice at P1 were mixed with a large excess of retinal cells from normal mice of the same age. The rhodopsin-expressing cells (%) in the EGFP-positive cell population are shown. Reaggregation cultures were harvested at the indicated days of culture, and the cells were replated on a chamber glass slide and immunostained with antibodies against GFP and rhodopsin. The experiments were performed at least twice, with essentially the same results.
Figure 3.
 
The roles of CD73 in retinal development. Temporal expression of mRNA for adenosine receptors in the developing retina. (A) Semiquantitative RT-PCR for adenosine receptors A1, A2a, and A2b was carried out using total RNA samples purified from mouse retinas at various developmental stages. (B) The expression of adenosine receptors A1, A2a, and A2b in CD73+ and CD73 cells at the indicated stages was examined by semiquantitative RT-PCR. (CF) Gain- and loss-of-function analyses of CD73. Retroviruses that encode IRES-EGFP or CD73-IRES-EGFP (for overexpression experiments) and CMV EGFP or shRNA against CD73-CMV EGFP (for downregulation experiments) were transduced into retinal explants at E16, followed by FACS analysis of the expression of CD73 (C) and rhodopsin (D) after 14 days of culture. (E) Quantitative results for rhodopsin-positive cells calculated from (D). The rhodopsin-positive cells in the EGFP-positive cell population (shown in D) are expressed as the relative percentage of those in the EGFP-negative cells population in each experiment. (F) Distributions of EGFP-positive cells in the ONL, INL, and GCL. Retroviruses that encode IRES-EGFP, CD73-IRES-EGFP, CMV-EGFP, or shRNA against CD73-CMV EGFP were transduced into retinal explants at E16. After 14 days of culture, frozen sections were produced, and the distribution of EGFP-positive cells in each layer was examined.
Figure 3.
 
The roles of CD73 in retinal development. Temporal expression of mRNA for adenosine receptors in the developing retina. (A) Semiquantitative RT-PCR for adenosine receptors A1, A2a, and A2b was carried out using total RNA samples purified from mouse retinas at various developmental stages. (B) The expression of adenosine receptors A1, A2a, and A2b in CD73+ and CD73 cells at the indicated stages was examined by semiquantitative RT-PCR. (CF) Gain- and loss-of-function analyses of CD73. Retroviruses that encode IRES-EGFP or CD73-IRES-EGFP (for overexpression experiments) and CMV EGFP or shRNA against CD73-CMV EGFP (for downregulation experiments) were transduced into retinal explants at E16, followed by FACS analysis of the expression of CD73 (C) and rhodopsin (D) after 14 days of culture. (E) Quantitative results for rhodopsin-positive cells calculated from (D). The rhodopsin-positive cells in the EGFP-positive cell population (shown in D) are expressed as the relative percentage of those in the EGFP-negative cells population in each experiment. (F) Distributions of EGFP-positive cells in the ONL, INL, and GCL. Retroviruses that encode IRES-EGFP, CD73-IRES-EGFP, CMV-EGFP, or shRNA against CD73-CMV EGFP were transduced into retinal explants at E16. After 14 days of culture, frozen sections were produced, and the distribution of EGFP-positive cells in each layer was examined.
Figure 4.
 
Mapping of CD73 in the hierarchy of genes involved in photoreceptor cell differentiation. (A, B) Semiquantitative RT-PCR of CD73+ and CD73 populations from the P1 (A) and P2 and P6 (B) mouse retina. The RT-PCR products were separated in a 1% agarose gel and visualized with ethidium bromide. G3PDH was used as a control. The experiments were performed at least twice, for all the primers, using independently prepared samples, with essentially the same results. (C) Kinetics of gene expression in the mouse retina. Semiquantitative RT-PCR was performed using RNA from mouse retinas at various developmental stages. G3PDH was used as a control. (D, E) Effects of retrovirus-mediated expression of Crx and Nrl transcription factors on CD73 expression. Retinal explant cultures prepared from E16 mouse retinas were infected with retroviruses that contained EGFP, Crx-IRES-EGFP, or Nrl-IRES-EGFP, and the cells were cultured for 4 days and then dissociated. The expression of CD73 in the EGFP-positive and EGFP-negative cells was examined by flow cytometry. The flow cytometric patterns (D) and the calculated values for the CD73+ cells in the EGFP-positive or EGFP-negative populations are shown (E). The experiments were performed at least three times with essentially the same results.
Figure 4.
 
Mapping of CD73 in the hierarchy of genes involved in photoreceptor cell differentiation. (A, B) Semiquantitative RT-PCR of CD73+ and CD73 populations from the P1 (A) and P2 and P6 (B) mouse retina. The RT-PCR products were separated in a 1% agarose gel and visualized with ethidium bromide. G3PDH was used as a control. The experiments were performed at least twice, for all the primers, using independently prepared samples, with essentially the same results. (C) Kinetics of gene expression in the mouse retina. Semiquantitative RT-PCR was performed using RNA from mouse retinas at various developmental stages. G3PDH was used as a control. (D, E) Effects of retrovirus-mediated expression of Crx and Nrl transcription factors on CD73 expression. Retinal explant cultures prepared from E16 mouse retinas were infected with retroviruses that contained EGFP, Crx-IRES-EGFP, or Nrl-IRES-EGFP, and the cells were cultured for 4 days and then dissociated. The expression of CD73 in the EGFP-positive and EGFP-negative cells was examined by flow cytometry. The flow cytometric patterns (D) and the calculated values for the CD73+ cells in the EGFP-positive or EGFP-negative populations are shown (E). The experiments were performed at least three times with essentially the same results.
Figure 5.
 
Expression of CD73 in marmoset retina. (A, B) Expression of M-opsin and rhodopsin in adult common marmoset retina. Immunostaining was performed using frozen-sectioned retina, and nuclei were visualized by DAPI. (C) FACS pattern of CD73 staining of whole retina of adult common marmoset. Green line: CD73 staining. Black line: control IgG staining. (D) Adult common marmoset retina was mechanically divided into fovea and other regions, and FACS analysis of CD73 expression was performed for both fractions. Violet region, green line: staining patterns of fovea and other regions, respectively. (E) Whole retina of adult common marmoset was dissociated and sorted to CD73+ and CD73 populations by cell sorter, and the cells were replated on a chamber glass slide and immunostained with antibodies against rhodopsin (upper four panels) and PNR (lower four panels). Nuclei were stained with DAPI.
Figure 5.
 
Expression of CD73 in marmoset retina. (A, B) Expression of M-opsin and rhodopsin in adult common marmoset retina. Immunostaining was performed using frozen-sectioned retina, and nuclei were visualized by DAPI. (C) FACS pattern of CD73 staining of whole retina of adult common marmoset. Green line: CD73 staining. Black line: control IgG staining. (D) Adult common marmoset retina was mechanically divided into fovea and other regions, and FACS analysis of CD73 expression was performed for both fractions. Violet region, green line: staining patterns of fovea and other regions, respectively. (E) Whole retina of adult common marmoset was dissociated and sorted to CD73+ and CD73 populations by cell sorter, and the cells were replated on a chamber glass slide and immunostained with antibodies against rhodopsin (upper four panels) and PNR (lower four panels). Nuclei were stained with DAPI.
Figure 6.
 
Schematic representation of photoreceptor cell differentiation and CD73. CD 73 is first expressed after Crx in the common precursors of the rod and cone cells and continues to be expressed in the rod cell lineage. However, CD73 appears to be downregulated once the cells commit to differentiation into the cone cell lineage.
Figure 6.
 
Schematic representation of photoreceptor cell differentiation and CD73. CD 73 is first expressed after Crx in the common precursors of the rod and cone cells and continues to be expressed in the rod cell lineage. However, CD73 appears to be downregulated once the cells commit to differentiation into the cone cell lineage.
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