Cell Cycle–Specific and Cell Type–Specific Expression of Rb in The

Cell Cycle–Specific and Cell Type–Specific Expression of Rb in the Developing Human Retina Thomas C. Lee,1,2,3 Dena Almeida,1,2 Nidia Claros,1,2 David H. Abramson,2,4 and David Cobrinik1,2 PURPOSE. To define the pattern of Rb expression relative to cell type and developmental stage in which Rb functions have not cycle position and cell type in the developing human retina. been defined.4,5 METHODS. Cryosections of fetal week 11-18 retinas were immu- The human retina begins its development at Fwk 5 and nostained for Rb and cell cycle– or cell type–specific markers. initially consists of proliferating retinal progenitor cells (RPCs) within a neuroblastic layer (NBL).6 RPC nuclei undergo a cell RESULTS. Rb was prominent in retinal progenitor cells (RPCs) cycle–dependent migration within the NBL, in which mitotic expressing the cyclin D1, cyclin A, and cytoplasmic cyclin B nuclei are positioned at the ventricular (outermost) layer, S markers of G1, S, and early to mid G2 phases, but not in RPCs phase nuclei are located in the middle NBL, and G1 and G2 expressing the phosphohistone H3 marker of late G2 and M. Rb nuclei migrate between these positions.7,8 Mitotic RPCs may was not detected in the earliest postmitotic ganglion, ama- give rise to additional RPCs or to postmitotic precursors of crine, horizontal, and bipolar cell precursors migrating away each of the mature retinal cell types, with the fate influenced from the ventricular layer, but was detected as such cells by cell-intrinsic competence states and by extrinsic cues.9 underwent further differentiation. Among photoreceptors, Rb Retinal development proceeds in a central to peripheral direc- was not detected in the earliest RXR␥(ϩ) cone precursors or in ϩ tion, such that proliferation ceases in the central retina by Fwk the earliest Nrl( ) rod precursors, but subsequently rose to 12 and in the far periphery by Fwk 30.10,11 high levels in cones and to low levels in rods. Rb was promi- Histopathological analyses have provided clues to the cell nent at the time when Mu¨ller glia exit the cell cycle and was Kip1 type in which Rb may suppress tumorigenesis. Retinoblasto- generally expressed in a pattern complementary to p27 . mas often contain differentiated cells that express markers of CONCLUSIONS. Rb exhibits cell cycle–specific expression in cones and, to a lesser extent, markers of rods and Mu¨ller glia, 12,13 RPCs, with loss in late G2-M and restoration in G1.Rbis but not markers of other retinal cells. Similarly, retinoblas- re-expressed after postmitotic ganglion, amacrine, horizontal, toma cell lines express proteins that are specific to cones and and bipolar cell precursors migrate away from the ventricular rods but not other cell types.14,15 In tumors, cells that express layer; after the appearance of early cone and rod markers; but glial markers adjoin and may form concurrent with cells that coinciding with Mu¨ller glia cell cycle withdrawal. The results express photoreceptor markers.12,13 These findings suggest suggest that Rb does not mediate the initial proliferative arrest that retinoblastomas may derive from a cell that is committed of retinal neurons, but may indirectly induce arrest in RPCs or to photoreceptor and glial differentiation or from an uncom- maintain an arrest in postmitotic precursors. (Invest Ophthal- mitted cell that is restricted to such differentiation in the tumor mol Vis Sci. 2006;47:5590–5598) DOI:10.1167/iovs.06-0063 environment. Among mammals, retinoblastoma is almost exclusively a etinoblastomas generally arise within the first 4 years of life human disease, as it has been diagnosed in only two individual Rand have been detected prenatally as early as fetal week animals.16,17 Moreover, in mice, loss of Rb does not predispose (Fwk) 21.1,2 The retinoblastoma gene (RB1) and protein (Rb) to retinal tumors, and combined loss of Rb and either of the are crucial to the suppression of retinoblastoma, as RB1 muta- related proteins p107 or p130 results in tumors with amacrine tion, deletion, or silencing occurs in all such tumors, and but not photoreceptor differentiation.18–21 In this study, we germline mutations predispose to numerous retinoblastoma sought to define the spatiotemporal pattern of Rb expression foci.3 Although these observations indicate that Rb suppresses in the developing human retina, as a means of identifying cell tumorigenesis during early retinal development, the retinal cell types and developmental stages in which Rb may suppress tumorigenesis. 1 2 From the Margaret M. Dyson Vision Research Institute, and De- METHODS partment of Ophthalmology, Weill Medical College of Cornell Univer- 4 sity, New York, New York; and the Ophthalmic Oncology Service, Human fetal eyes were obtained under protocols approved by the Department of Surgery, Memorial Sloan-Kettering Cancer Center, New Weill Medical College IRB and were studied in compliance with the York, New York. 3Current address: Division of Ophthalmology, Department of Sur- tenets of the Declaration of Helsinki. Fetal age was determined by gery, Childrens Hospital Los Angeles, Los Angeles, California. femur length. The cornea and lens were removed and the eyes fixed Supported by The C.V. Starr Foundation, The Fred Gluck Founda- overnight at 4°C in 4% paraformaldehyde in PBS (PFA/PBS), incubated tion, The New York Community Trust, and The Fund for Ophthalmic in 30% sucrose/PBS overnight at 4°C, embedded in one part 30% Knowledge. sucrose/PBS and two parts optimal cutting temperature compound Submitted for publication January 20, 2006; revised August 1, (OCT; Miles Laboratories, Elkhart, IN), frozen, and sectioned at 10 to 2006; accepted October 11, 2006. 15 ␮m. Disclosure: T.C. Lee, None; D. Almeida, None; N. Claros, None; For Rb staining, sections were postfixed in 4% PFA/PBS for 5 D.H. Abramson, None; D. Cobrinik, None minutes, washed in 0.5 M NaCl/20 mM Tris (pH 8.0; TBS), treated with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- 1 mM EDTA/TBS for 5 minutes, washed with TBS, incubated in 0.1% ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. H2O2 for 15 minutes, washed in TBS, treated with ABC kit reagent A Corresponding author: David Cobrinik, Margaret M. Dyson Vision (Vector Laboratories, Burlingame, CA) in TBS for 15 minutes, washed Research Institute, Weill Medical College of Cornell University, 1300 in TBS, treated with ABC kit reagent B (Vector Laboratories) in TBS for York Ave, LC303, New York, NY 10021; [email protected]. 15 minutes, washed in TBS, blocked and permeabilized in 5% horse Investigative Ophthalmology & Visual Science, December 2006, Vol. 47, No. 12 5590 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/24/2021 IOVS, December 2006, Vol. 47, No. 12 Expression of Rb in the Developing Human Retina 5591 serumϩ2% human serum in TBS (block 1), with 0.1% Triton-X-100 for line), apart from rare cells at the interface of the outer and 20 minutes, incubated in Rb antibody G3-245 (1:200; BD-PharMingen, middle NBL (Figs. 3A–C, arrows). Notably, cyclin D1(ϩ) cells San Diego, CA) in block 1ϩ0.05% Tween-20 overnight at 4°C, washed often had weak Rb signals relative to the surrounding cells (Figs. in TBS, incubated in biotinylated horse anti-mouse antibody (1:135; 3A–C, compare cells marked by arrowheads with those marked Vector Laboratories) in block 1 for 30 minutes, washed in TBS, washed by asterisks). in 0.1 M sodium bicarbonate and 0.15 M NaCl (balanced saline), Cells expressing cyclin A and cytoplasmic cyclin B were incubated with FITC-conjugated streptavidin (1:100; Vector Laborato- detected in both the middle and outer NBL (Figs. 3E, 3H). In ries) in balanced saline, and washed with TBS. For costaining, sections the middle NBL, nearly all cyclin A(ϩ) and cytoplasmic cyclin were incubated in 5% goat serum and 2% human serum in TBS (block B(ϩ) cells had strong Rb signals (Figs. 3D–I, arrowheads; Sup- 2) for 20 minutes, incubated overnight with primary antibody (Sup- plementary Fig. S2 online). The higher Rb signal in cyclin A(ϩ) plementary Information, online at http://www.iovs.org/cgi/content/ and cytoplasmic cyclin B(ϩ) cells, relative to cyclin D1(ϩ) full/47/12/5590/DC1) in block 2, washed in TBS, incubated for 30 cells, suggests that Rb expression increases as RPCs progress ϩ minutes in block 2 with secondary antibody (Supplementary Informa- from G1 into S and G2. In contrast, many cyclin A( ) and cyclin tion online), and washed in TBS. Sections were stained with (4Ј,6Ј- B(ϩ) cells at the ventricular layer had little or no Rb (Figs. diamino-2-phenylindole (DAPI) and analyzed by indirect immunofluo- 3D–I, arrows), although some cells had punctate Rb signals rescence or confocal microscopy. (Figs. 3D–I, thick arrows) similar to those in Ki67(ϩ) cells in similar positions (Fig. 2). To define further the cell-cycle position in which Rb RESULTS signal declines, we costained for Rb and phosphorylated Distinct Rb Expression Patterns in Peripheral and histone H3 (PH3), which is specific to late G2 and mitotic 27 ϩ Central Retina prophase, metaphase, and anaphase. PH3( ) cells had either a low, barely detectable nuclear Rb signal (not We initially evaluated Rb expression at Fwk 18. At this age, shown), a punctate Rb signal outside the PH-3(ϩ) region there is extensive proliferation in the peripheral retina, but no (Figs. 3J–L, thick arrows), or no detectable Rb in the case of 11 proliferation in the central retina.

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