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(Investigative Ophthalmology and Visual Science. 2006;47:5590-5598.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0063
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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

1From the Margaret M. Dyson Vision Research Institute, and 2Department of Ophthalmology, Weill Medical College of Cornell University, New York, New York; and the 4Ophthalmic Oncology Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To define the pattern of Rb expression relative to cell cycle position and cell type in the developing human retina.

METHODS. Cryosections of fetal week 11-18 retinas were immunostained for Rb and cell cycle– or cell type–specific markers.

RESULTS. Rb was prominent in retinal progenitor cells (RPCs) expressing the cyclin D1, cyclin A, and cytoplasmic cyclin B markers of G1, S, and early to mid G2 phases, but not in RPCs expressing the phosphohistone H3 marker of late G2 and M. Rb was not detected in the earliest postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrating away from the ventricular layer, but was detected as such cells underwent further differentiation. Among photoreceptors, Rb was not detected in the earliest RXR{gamma}(+) cone precursors or in the earliest Nrl(+) rod precursors, but subsequently rose to high levels in cones and to low levels in rods. Rb was prominent at the time when Müller glia exit the cell cycle and was generally expressed in a pattern complementary to p27Kip1.

CONCLUSIONS. Rb exhibits cell cycle–specific expression in RPCs, with loss in late G2-M and restoration in G1. Rb is re-expressed after postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrate away from the ventricular layer; after the appearance of early cone and rod markers; but coinciding with Müller glia cell cycle withdrawal. The results suggest that Rb does not mediate the initial proliferative arrest of retinal neurons, but may indirectly induce arrest in RPCs or maintain an arrest in postmitotic precursors.

Retinoblastomas generally arise within the first 4 years of life and have been detected prenatally as early as fetal week (Fwk) 21.1 2 The retinoblastoma gene (RB1) and protein (Rb) are crucial to the suppression of retinoblastoma, as RB1 mutation, deletion, or silencing occurs in all such tumors, and germline mutations predispose to numerous retinoblastoma foci.3 Although these observations indicate that Rb suppresses tumorigenesis during early retinal development, the retinal cell type and developmental stage in which Rb functions have not been defined.4 5

The human retina begins its development at Fwk 5 and initially consists of proliferating retinal progenitor cells (RPCs) within a neuroblastic layer (NBL).6 RPC nuclei undergo a cell cycle–dependent migration within the NBL, in which mitotic nuclei are positioned at the ventricular (outermost) layer, S phase nuclei are located in the middle NBL, and G1 and G2 nuclei migrate between these positions.7 8 Mitotic RPCs may give rise to additional RPCs or to postmitotic precursors of each of the mature retinal cell types, with the fate influenced by cell-intrinsic competence states and by extrinsic cues.9 Retinal development proceeds in a central to peripheral direction, such that proliferation ceases in the central retina by Fwk 12 and in the far periphery by Fwk 30.10 11

Histopathological analyses have provided clues to the cell type in which Rb may suppress tumorigenesis. Retinoblastomas often contain differentiated cells that express markers of cones and, to a lesser extent, markers of rods and Müller glia, but not markers of other retinal cells.12 13 Similarly, retinoblastoma cell lines express proteins that are specific to cones and rods but not other cell types.14 15 In tumors, cells that express glial markers adjoin and may form concurrent with cells that express photoreceptor markers.12 13 These findings suggest that retinoblastomas may derive from a cell that is committed to photoreceptor and glial differentiation or from an uncommitted cell that is restricted to such differentiation in the tumor environment.

Among mammals, retinoblastoma is almost exclusively a human disease, as it has been diagnosed in only two individual animals.16 17 Moreover, in mice, loss of Rb does not predispose to retinal tumors, and combined loss of Rb and either of the related proteins p107 or p130 results in tumors with amacrine but not photoreceptor differentiation.18 19 20 21 In this study, we sought to define the spatiotemporal pattern of Rb expression in the developing human retina, as a means of identifying cell types and developmental stages in which Rb may suppress tumorigenesis.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Human fetal eyes were obtained under protocols approved by the Weill Medical College IRB and were studied in compliance with the tenets of the Declaration of Helsinki. Fetal age was determined by femur length. The cornea and lens were removed and the eyes fixed overnight at 4°C in 4% paraformaldehyde in PBS (PFA/PBS), incubated in 30% sucrose/PBS overnight at 4°C, embedded in one part 30% sucrose/PBS and two parts optimal cutting temperature compound (OCT; Miles Laboratories, Elkhart, IN), frozen, and sectioned at 10 to 15 µm.

For Rb staining, sections were postfixed in 4% PFA/PBS for 5 minutes, washed in 0.5 M NaCl/20 mM Tris (pH 8.0; TBS), treated with 1 mM EDTA/TBS for 5 minutes, washed with TBS, incubated in 0.1% H2O2 for 15 minutes, washed in TBS, treated with ABC kit reagent A (Vector Laboratories, Burlingame, CA) in TBS for 15 minutes, washed in TBS, treated with ABC kit reagent B (Vector Laboratories) in TBS for 15 minutes, washed in TBS, blocked and permeabilized in 5% horse serum+2% human serum in TBS (block 1), with 0.1% Triton-X-100 for 20 minutes, incubated in Rb antibody G3-245 (1:200; BD-PharMingen, San Diego, CA) in block 1+0.05% Tween-20 overnight at 4°C, washed in TBS, incubated in biotinylated horse anti-mouse antibody (1:135; Vector Laboratories) in block 1 for 30 minutes, washed in TBS, washed in 0.1 M sodium bicarbonate and 0.15 M NaCl (balanced saline), incubated with FITC-conjugated streptavidin (1:100; Vector Laboratories) in balanced saline, and washed with TBS. For costaining, sections were incubated in 5% goat serum and 2% human serum in TBS (block 2) for 20 minutes, incubated overnight with primary antibody (Supplementary Information, online at http://www.iovs.org/cgi/content/full/47/12/5590/DC1) in block 2, washed in TBS, incubated for 30 minutes in block 2 with secondary antibody (Supplementary Information online), and washed in TBS. Sections were stained with (4',6'-diamino-2-phenylindole (DAPI) and analyzed by indirect immunofluorescence or confocal microscopy.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Distinct Rb Expression Patterns in Peripheral and Central Retina
We initially evaluated Rb expression at Fwk 18. At this age, there is extensive proliferation in the peripheral retina, but no proliferation in the central retina.11 In the periphery, Rb was prominent in nuclei throughout the middle NBL and in occasional nuclei in the outer NBL (Figs. 1A 1B) . A similar pattern was evident at Fwks 12 and 14, although the band of Rb(+) nuclei was positioned closer to the outer limiting membrane (OLM), due to the lack of rod precursors in the outer NBL at these ages (Supplementary Fig. S1; all Supplementary Figures are online at http://www.iovs.org/cgi/content/full/47/12/5590/DC1). In contrast, in the central retina, Rb was prominent in outer nuclear layer cells having the appearance of cones and in inner nuclear layer (INL) positions typical of Müller cell nuclei, but was detected at far lower levels elsewhere (Figs. 1C 1D) . Thus, the Rb expression pattern differed in the highly proliferating peripheral retina compared with the postmitotic central retina and exhibited cell type–specific differences among postmitotic cells.


Figure 1
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  		FIGURE 1. Rb expression in the peripheral and central retina. Fwk 18 peripheral (A, B) and central (C, D) retina stained for Rb (green) and DAPI (blue). OLM signal represents background that is independent of primary antibody. OLM, outer limiting membrane; NBL, neuroblastic layer; IPL, inner plexiform layer; GCL, ganglion cell layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer.

 
Rb Expression in RPCs
In the peripheral retina, Rb(+) nuclei were in positions that are characteristic of RPCs (Figs. 1A 1B) . To determine whether Rb is expressed in such cells, we costained for Rb and the proliferation marker Ki67. Rb was prominent in all Ki67(+) nuclei in the middle and outer NBL (Figs. 2A 2B 2C 2D , arrowheads), but was diminished or undetectable in Ki67(+) nuclei at the ventricular layer (Fig. 2 , arrows, and Supplementary Fig. S2 online). Moreover, Rb had a punctate distribution outside of the Ki67(+) region in many ventricular layer cells (Figs. 2B 2C 2D 2E 2F 2G ; thick arrows).


Figure 2
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  		FIGURE 2. Rb expression in a subset of RPCs. Fwk 18 peripheral retina costained for Rb (green), Ki67 (red), and DAPI (blue) and examined by confocal microscopy. The boxed region in (A) is shown in (B–D). A separate section is shown at higher magnification in (E–G). Arrowheads: nuclei that stain for both Rb and Ki67. Arrows: cells that stain for Ki67 but little or no Rb. Thick arrows: punctate Rb staining outside of Ki67(+) region. VL, ventricular layer; O-NBL, outer neuroblastic layer; M-NBL, middle neuroblastic layer.

 
The diminished Rb signal in ventricular layer RPCs suggests that Rb may be decreased in the G2- or M-phase nuclei that are typically located in this position. To examine Rb in different cell cycle phases, retinas were costained for Rb and a series of cyclin proteins. Cyclin D1 was used as a marker of cells in G1, as it is expressed in G1 and degraded in S,22 and was not detected in outer NBL positions typical of G2 and M.23 24 Cyclin A was used as a marker of S, G2, and mitotic prophase,25 and cyclin B1 as a marker that accumulates in the cytoplasm in G2, enters the nucleus on entry into prophase, and is degraded in anaphase.26 Consistent with these assignments, cyclins D1 and A were detected in distinct cell populations (Supplementary Fig. S3 online).

As in other species, cyclin D1 was detected in the middle NBL (Fig. 3B) . Rb was detected in nearly all cyclin D1(+) cells (Figs. 3A 3B 3C , arrowheads; Supplementary Fig. S2 online), apart from rare cells at the interface of the outer and middle NBL (Figs. 3A 3B 3C , arrows). Notably, cyclin D1(+) cells often had weak Rb signals relative to the surrounding cells (Figs. 3A 3B 3C , compare cells marked by arrowheads with those marked by asterisks).


Figure 3
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  		FIGURE 3. Cell cycle–specific Rb expression in RPCs. (A–L) Fwk 18 peripheral retina costained for Rb (green) and either cyclin D1 (red, A–C), cyclin A (red, D–F), cyclin B1 (red, G–I), or phosphohistone H3 (red, J–L), and examined by confocal microscopy. Arrowheads: cells that costain for Rb and the cell cycle marker. Arrows: cells that strongly stain for the cell cycle marker but not Rb. Thick arrows: cells with punctate Rb staining. (A–C, *) Cells that strongly stain for Rb but not cyclin D1. (M) Rb expression relative to cell cycle phase and nuclear position in the NBL. Green shading: subcellular localization and intensity of Rb signal.

 
Cells expressing cyclin A and cytoplasmic cyclin B were detected in both the middle and outer NBL (Figs. 3E 3H) . In the middle NBL, nearly all cyclin A(+) and cytoplasmic cyclin B(+) cells had strong Rb signals (Figs. 3D 3E 3F 3G 3H 3I , arrowheads; Supplementary Fig. S2 online). The higher Rb signal in cyclin A(+) and cytoplasmic cyclin B(+) cells, relative to cyclin D1(+) cells, suggests that Rb expression increases as RPCs progress from G1 into S and G2. In contrast, many cyclin A(+) and cyclin B(+) cells at the ventricular layer had little or no Rb (Figs. 3D 3E 3F 3G 3H 3I , arrows), although some cells had punctate Rb signals (Figs. 3D 3E 3F 3G 3H 3I , thick arrows) similar to those in Ki67(+) cells in similar positions (Fig. 2) .

To define further the cell-cycle position in which Rb signal declines, we costained for Rb and phosphorylated histone H3 (PH3), which is specific to late G2 and mitotic prophase, metaphase, and anaphase.27 PH3(+) cells had either a low, barely detectable nuclear Rb signal (not shown), a punctate Rb signal outside the PH-3(+) region (Figs. 3J 3K 3L , thick arrows), or no detectable Rb in the case of anaphase cells (Figs. 3J 3K 3L , thin arrow). This observation suggests that Rb levels decline at or before the time when histone H3 is phosphorylated in late G2.

After undergoing mitosis at the ventricular layer, early G1-phase RPC nuclei and postmitotic retinal precursor nuclei migrate through the outer NBL in the inward (vitread) direction. However, all Rb(+) cells in the outer NBL expressed cyclin A (Figs. 3D 3E 3F and data not shown), which is specific to S, G2, and early M.25 The lack of Rb in cyclin A(–) cells implies that Rb was not appreciably expressed in early G0 or G1 nuclei during their vitread migration.

In sum, Rb was present in cyclin D1(+) cells in the middle NBL, was detected at higher levels in cyclin A(+) and cytoplasmic cyclin B(+) cells in the middle and outer NBL, and was diminished in cyclin A(+), cyclin B(+), and PH3(+) cells at the ventricular layer (Fig. 3M) . The results imply that Rb expression increases during progression from G1 into S and G2, declines in late G2 and M coinciding with a punctate distribution pattern, and does not reaccumulate until RPC nuclei return to the middle NBL.

Rb Expression in Postmitotic Retinal Cells
After undergoing mitosis at the ventricular layer, RPCs give rise to additional RPCs or to postmitotic retinal precursors. To define the Rb expression pattern during the genesis of postmitotic cells, we stained for Rb and early markers of the different retinal cell types.

Rb in Ganglion Cell Precursors.
Ganglion cells are produced in the retinal far periphery at Fwk 12.11 Islet-1 is an early marker of such cells and is first expressed at the ventricular layer soon after mitosis.28 29 Islet-1 was highly expressed in precursors migrating through the NBL as well as in the incipient ganglion cell layer (GCL; Figs. 4A 4B ). Rb was not detected in migrating Islet-1(+) cells in the outer NBL (Figs. 4A 4B 4C , arrows), but was prominent in Islet-1(+) cells at the interface of the NBL and GCL (Figs. 4A 4B 4C , arrowheads). Rb was also detected at low levels in a subset of cells in the incipient GCL and in the more mature GCL at later ages (Figs. 1 4) , suggesting that Rb levels may decline during ganglion cell maturation. To define further the timing of Rb expression, we costained for Rb and Brn-3b, which is specific to postmitotic ganglion cells.30 31 Rb was prominent in Brn-3b(+) cells at the interface between the NBL and GCL (Figs. 4D 4E 4F , arrowheads), suggesting that Rb accumulates at or before the time when Brn-3b is first expressed.


Figure 4
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  		FIGURE 4. Rb expression in ganglion cell precursors. Fwk 12 peripheral retina costained for Rb (green) and either Islet-1 (red, A–C) or Brn-3b (red, D–F). Arrowheads: nuclei that costain for Rb and either Islet-1 or Brn-3b. Arrows: nuclei that stain for Islet-1 but not Rb. iGCL, incipient ganglion cell layer.

 
Rb in Horizontal and Amacrine Cell Precursors.
Horizontal and amacrine cell precursors initially migrate from the ventricular layer to the inner NBL.7 32 However, as Rb was not detected in postmitotic nuclei in the outer NBL, Rb evidently did not accumulate during the early stages of this process.

After migrating to the inner NBL, horizontal cell precursors migrate in the outward direction and express the Prox1 and Lim1 proteins.33 34 35 At Fwk 11, Prox1 was detected at low levels in Ki67(+) RPCs, but at far higher levels in distinctively shaped, Ki67(–) nuclei in the central and midperipheral retina (data not shown). The time of appearance and position of the strongly Prox1(+) nuclei was consistent with that of horizontal cell precursors.35 At this age, Rb was not detected in the most peripheral, and thus most recently formed, Prox1(+) nuclei (Figs. 5A 5B 5C) , but was detected at low levels in occasional Prox1(+) cells in more central positions (Figs. 5D 5E 5F , arrowheads). Similarly, at Fwk 18, Rb was not detected in the most peripheral Prox1(+) horizontal cell precursors (Figs. 5G 5H 5I) , but was highly expressed in the more mature horizontal cells in the central retina (Figs. 5J 5K 5L) . Rb expression in horizontal cell precursors was confirmed by costaining with Lim1 (Supplementary Fig. S4 online). The expression of Rb in the more central, but not the peripheral, Prox1(+) nuclei implies that Rb is expressed after the initial expression of Prox1 during horizontal cell maturation.


Figure 5
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  		FIGURE 5. Rb expression in horizontal cell precursors. Fwk 11 peripheral (A–C) and central (D–F) retina or Fwk 18 peripheral (G–I) and central (J–L) retina costained for Prox1 (red) and Rb (green). Arrows: nuclei that stain for Prox1 but not Rb. Arrowheads: nuclei that costain for Prox1 and Rb. The positions of strongly Prox1(+) cells are consistent with that of horizontal cell precursors at all ages and with that of a second, uncharacterized population in the Fwk 18 INL.

 
To date, we have not identified a marker that is specific to postmitotic amacrine precursors and compatible with Rb staining (see Supplementary Information, http://www.iovs.org/cgi/content/full/47/12/5590/DC1). However, Rb was detected at low levels in the inner INL where amacrine precursors accumulate (Fig. 1B) , consistent with Rb’s having minimal expression during amacrine cell maturation.

Rb in Cone Precursors.
Rb was highly expressed in foveal cone precursors at Fwk 18 (Fig. 1C) . To define the timing of Rb expression in this lineage, we stained for Rb and several cone precursor markers. One of the earliest cone markers in chicks and mice is the retinoid X receptor {gamma} (RXR{gamma}).36 37 38 At Fwk 12, Rb was detected in only a subset of the RXR{gamma}(+) nuclei in the fovea (Figs. 6A 6B 6C , arrowheads), and in none of the younger RXR{gamma}(+) nuclei at more peripheral positions (data not shown). At Fwk 15, Rb was detected in all the RXR{gamma}(+) nuclei in the central retina, but not in those in the periphery (Figs. 6D 6E 6F 6G 6H 6I) . Similarly, Rb was not detected in peripheral RXR{gamma}(+) nuclei at Fwk 18 (data not shown). The increased prevalence of Rb in RXR{gamma}(+) cells in more developed retinal regions implies that Rb is expressed after RXR{gamma} appears.


Figure 6
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  		FIGURE 6. Rb expression in cone precursors. Fwk 12 (A–C) or Fwk 15 (D–I) central (A–F) or peripheral (G–I) retina costained for Rb (green), RXR{gamma} (red), and DAPI (blue). Arrows: cells that stain for RXR{gamma} but not Rb. Arrowheads: cells that costain for both RXR{gamma} and Rb.

 
Two other early cone markers are thyroid hormone receptor ß2 (TRß2)39 40 and interphotoreceptor retinoid binding protein (IRBP), which is specific to cone precursors in the early fovea.10 At Fwk 12, TRß2 and IRBP were detected only in the fovea, and Rb was detected in all TRß2(+) and all IRBP(+) cells (Supplementary Fig. S5 online).

Rb in Rod Precursors.
Rod precursors are initially positioned below the outer limiting membrane. As additional rod nuclei accumulate, the earlier born rod nuclei are displaced to progressively deeper layers.7 32 To evaluate Rb expression during rod development, we costained for Rb and Nrl, which is the earliest known rod-specific protein.10 41 At Fwk 14, the most peripheral (and hence, least mature) Nrl(+) nuclei were positioned below the outer limiting membrane (Figs. 7A 7B 7C , arrows). Rb was not detected in these peripheral Nrl(+) nuclei, but was detected in more mature Nrl(+) nuclei at more central and basal positions (Figs. 7A 7B 7C , arrowhead). This phenomenon was more clear in the Fwk 18 periphery, where Rb was not evident in Nrl(+) nuclei near the outer limiting membrane, but was detected in more mature Nrl(+) nuclei at more basal positions (Figs. 7D 7E 7F 7G , arrowheads). Similarly, Rb was detected in Nrl(+) nuclei in the parafoveal region, albeit at far lower levels than in Nrl(–) cones (Supplementary Fig. S6 online). Notably, Nrl was expressed at higher levels in older, basally positioned Rb(+) nuclei than in the younger, apically positioned nuclei that lacked Rb expression (Figs. 7D 7E 7F 7G) .


Figure 7
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  		FIGURE 7. Rb expression in rod precursors. Fwk 14 midperipheral retina (A–C) and Fwk 18 peripheral retina (D–G) costained for Rb (green) and Nrl (red). The boxed region in (D) is shown at higher magnification in (E–G). Arrows: cells that stain for Nrl but not Rb. Arrowheads: cells that costain for Rb and Nrl. The directions toward the retinal periphery or center are indicated for (A–C).

 
Rb in Bipolar Cell Precursors.
Bipolar cells comprise much of the outer aspect of the INL and can be identified by their prominent nuclear expression of Chx10.42 Chx10 is also expressed in RPCs, but at far lower levels and without the central-to-peripheral distribution of bipolar cell precursors.

At Fwk 15, the most peripheral, and thus youngest, strongly Chx10(+) bipolar cell nuclei were interspersed within the NBL and did not have detectable Rb (Figs. 8A 8B 8C) . However, in the central retina, Chx10(+) nuclei formed a distinct layer, and in most cases had moderate, above background Rb expression (Figs. 8D 8E 8F) , indicating that Rb was first detected in bipolar cells after Chx10 accumulation.


Figure 8
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  		FIGURE 8. Rb expression in bipolar cell precursors. Fwk 15 peripheral (A–C) or central (D–F) retina costained for Chx10 (red) and Rb (green). Arrows: nuclei that stain for Chx10 but not Rb. Arrowheads: nuclei that costain for Rb and Chx10.

 
Rb in Müller Glia.
Rb was highly expressed in INL positions that are characteristic of Müller glia nuclei at Fwk 18 (Figs. 1C 9F) . This distribution was also evident at Fwk 15, although the pattern was less obvious due to higher Rb expression in surrounding INL cells (Fig. 9C) . Nonetheless, at Fwk 15 each of the strongly Rb(+) INL nuclei was surrounded by the Müller cell–specific marker, cellular retinal–binding protein (CRALBP)43 (Figs. 9A 9B 9C) , confirming their Müller cell identity.


Figure 9
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  		FIGURE 9. Rb expression in Müller glia. Fwk 15 (A–C) or Fwk 18 (D–F) fovea costained for Rb (green), and either CRALBP (red, A–C) or cyclin D1 (red, D–F). Arrowheads: cells that costain for Rb and either CRALBP or cyclin D1.

 
Müller glia resemble RPCs in their morphology, gene expression profile, and ability to re-enter the cell cycle.44 45 46 In keeping with this relationship, Müller glia express cyclin D1 for a limited time while they undergo cell cycle exit.46 We confirmed that cyclin D1 persisted in the Fwk 18 central retina, after proliferation had ceased (Fig. 9E and data not shown). Rb was prominent in all cyclin D1(+) nuclei (Figs. 9D 9E 9F) , indicating that it is highly expressed as Müller glia exit the cell cycle.

Complementary Expression of Rb and p27Kip1
The lack of Rb in early postmitotic retinal neurons suggested that other cell cycle regulators mediate the proliferative arrest of such cells. Because p27Kip1 has an important antiproliferative role in murine retina,23 24 we examined its expression relative to that of Rb.

In the Fwk 18 peripheral retina, p27 and Rb were generally expressed in a complementary pattern (Figs. 10A 10B 10C) . A similar complementarity was evident at Fwk 14 (data not shown). RPCs comprising most of the the NBL prominently expressed Rb but not p27, whereas rod precursors in the outer NBL and amacrine and ganglion cell precursors in the inner retina expressed p27 but little or no Rb. Rare cells expressed both Rb and p27 (Figs. 10A 10B 10C , arrowheads), particularly in outer NBL positions where Rb and Nrl were coexpressed (see Figs. 7D 7E 7F ), and in middle and inner NBL positions where Rb and Prox1 were coexpressed (see Fig. 5 ). Thus, Rb may be superimposed over p27 expression during the differentiation of these cells.


Figure 10
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  		FIGURE 10. Complementary expression of Rb and p27Kip1. Fwk 18 retina costained for Rb (green) and p27Kip1 (red). Images of peripheral (A–C), transition zone (D–F), and central (G–I) retina are from the same section and were imaged in identical fashion. Arrowheads: nuclei that costain for Rb and p27. Arrows: nuclei that stain strongly for Rb but not p27.

 
Rb and p27 were also expressed in a complementary manner in the central retina. p27 was highly expressed and Rb had minimal expression in positions typical of ganglion cells, amacrine cells, horizontal cells, bipolar cells, and rods, whereas little or no p27 was detected in the strongly Rb(+) Müller glia and cones (Figs. 10G 10H 10I , arrows).

To assess the relationship between Rb and p27 expression in earlier cones and Müller cells, we examined a transition zone having the most peripheral (and hence least mature) Rb(+) cone precursors and only rare Ki67(+) cells. In contrast to the central retina, Rb(+) cone precursors in this region had high levels of p27 (Figs. 10D 10E 10F , arrowheads). This observation implies that cone precursors transition from a state in which they express both Rb and p27 to a state in which they express predominantly Rb. However, we found no evidence of such a transition in Müller glia. Rather, the less mature glial cells in the transition zone generally had no detectable p27 (Figs. 10D 10E 10F) , whereas those in the central retina had low but detectable p27 (Figs. 10G 10H 10I) .


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
This study aimed to define the pattern of Rb expression in the human fetal retina, from gestational weeks 11 to 18. This interval is characterized by RPC proliferation, production of each of the retinal cell types, and transition from a proliferative to a postmitotic state. The data showed that Rb is expressed in a cell cycle–specific manner in RPCs and accumulates at various times and levels in the different types of postmitotic retinal precursors.

Cell Cycle–Specific Expression of Rb in RPCs
Others had previously shown that Rb is expressed in RPCs in the developing mouse and human retina.47 48 49 By using cyclins and phosphorylated histone H3 (PH3) as markers, we deduced that Rb is expressed in G1 nuclei in the middle NBL, increases expression in S and early G2 nuclei traversing the NBL, declines to undetectable levels from late G2 to anaphase at the ventricular layer, and does not reaccumulate until G1 nuclei return to the middle NBL (Fig. 3M) . This cell cycle-specific pattern in human RPCs is consistent with the general decline in Rb expression in M- and G1-phase RPCs in mice,47 and further indicates that Rb is both dramatically and consistently diminished during RPC mitosis.

The cyclical expression of Rb in RPCs contrasts with Rb’s constitutive expression throughout the cell cycle in diverse cell types that were previously analyzed with the same antibody.50 In those other cell types, Rb associated with condensing chromatin in prophase, redistributed to the cytoplasm on nuclear envelope breakdown in metaphase, and returned to daughter nuclei on nuclear envelope restoration in telophase.50 However, in RPCs, Rb declined before prophase (when PH3 becomes prominent27 ) and was not restored to telophase, or G1, nuclei until such nuclei had migrated to the middle NBL.

The mechanism by which Rb expression declines in late G2 and M is unknown. However, it was notable that some cells near the ventricular layer had a punctate Rb signal immediately outside of their Ki67(+) or PH3(+) regions (Figs. 2B 2C 2D 2E 2F 2G) . As Rb was not detected in metaphase or later M-phase nuclei, the punctate Rb signal may designate sites to which Rb translocates before its degradation. Of interest, an earlier study of osteosarcoma cells showed that high Cdk activity can induce phosphorylation of Rb residue 567 and may thereby induce Rb cytoplasmic translocation and degradation.51 Thus, the dramatically increased Cdk activity at the G2–M transition may elicit cytoplasmic translocation and degradation of most of the Rb in RPCs, even though it apparently does not do so in other cell types.

It seems plausible that Rb’s cell cycle–specific expression serves a developmental role. However, the cyclical loss of Rb in the M phase seems unlikely to regulate RPC fate, as Rb was lacking in M-phase cells both in the periphery (where RPCs mainly produce more RPCs) and in more central positions (where RPCs mainly produce postmitotic precursors). Alternatively, the decline in Rb expression before mitosis may preclude Rb from inhibiting proliferation early in the subsequent cell cycle. Our detection of occasional cyclin D1(+), Rb(–) cells in the outer NBL (Figs. 3A 3B 3C) suggests that Rb may be re-expressed only after RPCs acquire cyclin D1-dependent kinase activity that can suppress Rb function.

Delayed Expression of Rb in Postmitotic Retinal Precursors
Our analyses show that Rb is expressed in all cell types in the developing human retina. This result is unsurprising in light of Rb’s widespread expression and diverse functions in many developmental contexts.52 Moreover, Rb has been detected in cell types other than amacrine cells in the early mouse retina.48 However, the present study also defined the timing of Rb expression relative to that of cell type–specific markers and p27 and thereby provides novel insights into the potential role of Rb in retinal development.

Unexpectedly, Rb was not detected at the time when postmitotic retinal neurons were born, but accumulated well after the terminal mitosis. For example, Rb was not detected in postmitotic ganglion, horizontal, amacrine, and bipolar cell precursors migrating away from the ventricular layer, but was detected at the time when ganglion cells first expressed Brn-3b and after horizontal cells first expressed Prox1. Similarly, Rb was not detected in nascent Chx10(+) bipolar precursors, but was detected in more mature Chx10(+) cells. This evidence suggests that Rb does not directly mediate cell cycle withdrawal or early differentiation events in postmitotic retinal precursors.

The timing of Rb expression in developing photoreceptors was of particular interest, given that retinoblastoma may derive from a photoreceptor-directed cell. Surprisingly, Rb was not detected at the time when rod precursors first expressed Nrl in the outer retinal layers, but was detected in more mature rod nuclei at more basal positions. This suggests that in rod precursors, Rb mediates neither cell cycle withdrawal nor the initial expression of Nrl. That Rb does not mediate a rod precursor arrest is consistent with the lack of rod precursor proliferation in Rb-deficient mouse retinas.49 Nevertheless, the lack of Rb in early Nrl(+) precursors was unexpected, given that Rb-deficient mouse retinas had impaired rod differentiation and decreased Nrl mRNA.49 Our finding that Nrl was more highly expressed in Rb(+) versus Rb(–) cells (Figs. 7E 7F 7G) suggests that Rb may promote Nrl expression during late but not early rod differentiation.

As in rods, Rb was not detected in the earliest cones. Whereas cone precursors are evident at Fwk 9,10 Rb was detected in only a subset of such precursors in the central retina at Fwk 12 and was detected in central but not midperipheral cones at Fwks 15 and 18. Although we cannot determine the precise length of the delay, the evidence suggests that foveal cones may arrest for 1 to 3 weeks before having detectable Rb expression. In contrast to rods, Rb rose to high levels in early cones. Spencer et al.48 similarly detected high Rb expression in adult cones. We extend their finding by showing that cone precursors prominently express Rb in the fetal period when retinoblastoma tumorigenesis begins.

In contrast to the delayed Rb expression in postmitotic retinal neurons, Rb was highly expressed in Müller glia at the time of cell cycle exit. As Müller glia resemble quiescent RPCs,44 45 46 Rb’s expression may be governed by the same process in the two cell types. Notably, p27 was lacking at the time when Müller glia exit the cell cycle. This is consistent with the lack of p27 in RPCs, but contrasts with the prominent expression of p27 in postmitotic retinal neurons.

Relationship of Rb Expression to Tumor Suppression
In mice, Rb was found to be highly expressed in each of the tissues in which it has crucial developmental roles.53 54 Thus, although a protein’s expression level does not necessarily reflect its function, it is of interest to consider whether Rb might suppress retinoblastoma in the retinal cell types in which it is expressed at high levels.

Rb was most highly expressed in RPCs. However, Rb seems unlikely to directly induce a proliferative arrest in such cells, as RPCs are not known to exit the cell cycle without differentiating into one of the postmitotic retinal cell types. Moreover, our data argue that Rb does not accumulate in RPCs in preparation for a role after such cells divide, because Rb expression declined in late G2/M and was not rapidly restored to postmitotic cells. Thus, if Rb were to suppress tumorigenesis through its expression in RPCs, it would appear to do so by indirectly eliciting cell cycle exit and differentiation after a subsequent mitosis. Notably, in cortical neurogenesis, progenitor cells undergoing neurogenic divisions have longer cell cycles than those undergoing proliferative divisions, and artificially lengthening G1 suffices to induce neurogenic differentiation after an intervening mitosis.55 56 Thus, Rb might indirectly promote neurogenic differentiation by lengthening G1 or through other effects that precede Rb’s degradation in late G2 and M.

Rb was also highly expressed at the time when Müller glia exit the cell cycle. Because p27 was not concurrently expressed, it is tempting to infer that Rb may be needed to suppress Müller cell proliferation. However, arguing against this possibility are the lack of Müller cell markers in proliferating retinoblastoma cells12 13 and evidence that adult Müller glia express high levels of the Rb-related p107 and p130.48

In cone precursors, Rb rose to high levels concurrent with a decline in p27. This reciprocal increase in Rb and loss of p27 was unique to cone precursors at the ages that we examined. Notably, Rb is prominent in cone precursors and mature cones in the human retina, but in neither developing nor adult cones in mice (this study and Refs. 21 , 48 ). Moreover, Rb’s expression in human cone precursors correlates with its role as a retinoblastoma suppressor in humans and with the predominant cone phenotype of differentiated retinoblastoma cells.12 13 These observations suggest that Rb may have a human-specific function in cone precursors, that coincides with the decline in p27. Nevertheless, additional studies are needed to address whether Rb suppresses retinoblastoma in cone precursors, in other cells that have high Rb levels such as RPCs or Müller glia, or in still other cell types that had less prominent Rb expression.


   Acknowledgements
 
The authors thank John Saari, Thomas Jessel, Barbara Wiggert, Hemant Khanna, Anand Swaroop, Lily Ng, and Douglas Forrest for gifts of antibodies used in this study.


   Footnotes
 
3 Current address: Division of Ophthalmology, Department of Surgery, Childrens Hospital Los Angeles, Los Angeles, California. Back

Supported by The C.V. Starr Foundation, The Fred Gluck Foundation, The New York Community Trust, and The Fund for Ophthalmic Knowledge.

Submitted for publication January 20, 2006; revised August 1, 2006; accepted October 11, 2006.

Disclosure: T.C. Lee, None; D. Almeida, None; N. Claros, None; D.H. Abramson, None; D. Cobrinik, None

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.

Corresponding author: David Cobrinik, Margaret M. Dyson Vision Research Institute, Weill Medical College of Cornell University, 1300 York Ave, LC303, New York, NY 10021; dec2014{at}med.cornell.edu.


   References
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 Abstract
 Methods
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