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(Investigative Ophthalmology and Visual Science. 2007;48:491-499.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0765
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Persistent Hyperplastic Primary Vitreous Due to Somatic Mosaic Deletion of the Arf Tumor Suppressor

J. Derek Thornton,1 Doug J. Swanson,2 Michelle N. Mary,1 Deqing Pei,3 Amy C. Martin,1 Stanley Pounds,3 Dan Goldowitz,2 and Stephen X. Skapek1,4

1From the Departments of Oncology and 3Biostatistics, St. Jude Children’s Research Hospital and the 4Departments of Ophthalmology and Anatomy and 2Neurobiology, University of Tennessee Health Sciences Center, Memphis, Tennessee.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Mice lacking the Arf tumor-suppressor gene develop eye disease reminiscent of persistent hyperplastic primary vitreous (PHPV). The current work explores mechanisms by which Arf promotes eye development, and its absence causes a PHPV-like disease.

METHODS. Chimeric mice were made by fusing wild-type and Arf–/– morulae. In these experiments, wild-type cells are identified by transgenic expression of GFP from a constitutive promoter. PCR-based genotyping and quantitative analyses after immunofluorescence staining of tissue and cultured cells documented the relative contribution of wild-type and Arf–/– cells to different tissues in the eye and different types of cells in the vitreous.

RESULTS. The contributions of the Arf–/– lineage to the tail DNA, cornea, retina, and retina pigment epithelium (RPE) correlated with each other in wild-type{leftrightarrow}Arf–/– chimeric mice. Newborn chimeras had primary vitreous hyperplasia, evident as a retrolental mass. The mass was usually present when the proportion of Arf–/– cells was relatively high and absent when the Arf–/– proportion was low. The Pdgfrß- and Sma-expressing cells within the mass arose predominantly from the Arf–/– population. Ectopic Arf expression induced smooth muscle proteins in cultured pericyte-like cells, and Arf and Sma expression overlapped in hyaloid vessels.

CONCLUSIONS. In the mouse model, loss of Arf in only a subset of cells causes a PHPV-like disease. The data indicate that both cell autonomous and non–cell autonomous effects of Arf may contribute to its role in vitreous development.

The hyaloid vascular system (HVS), which supplies the developing vitreous and lens, undergoes dramatic involution during later stages of mammalian eye development.1 Failed HVS involution causes an eye disease known as persistent hyperplastic primary vitreous (PHPV; also known as persistent fetal vasculature).2 Hallmark features of the disease, which often presents in childhood, include microphthalmia and leukocoria, due to the presence of a fibrovascular, retrolental mass enveloping the vitreous elements of the HVS.2 3 In severe cases, the retrolental mass disrupts the posterior lens capsule and physically interacts with the inner aspect of the neuroretina causing tractional detachment of the retina. Although occasional reports of bilateral and heritable PHPV suggest a genetic basis,4 5 6 the fact that most cases are unilateral and sporadic implies that genetic or epigenetic somatic cell events during development may give rise to the disease.

Mouse models have provided some insight into the molecular and cellular mechanisms driving HVS regression. In genetically engineered mice lacking angiopoietin-2 (Ang-2), the HVS fails to regress completely, and this gene is necessary for postnatal vascular remodeling in the mouse.7 8 It is hard to assess the other "clinical" manifestations of PHPV in this model, because most Ang-2-deficient mice die in the first 2 weeks of life. The p53 tumor suppressor may also contribute to HVS regression. A PHPV-like disease occurs in p53–/– mice bred to either pure C57BL/6 or pure BALB/cOlaHsd backgrounds, whereas eyes are usually normal in p53–/– C57BL/6 x 129/Sv mice.9 10 Failed HVS regression in p53–/– BALB/c mice is associated with decreased vitreous cell apoptosis at P7 and P8.9 PHPV was also reported to develop in a single line of transgenic mice expressing IE180, which encodes a pseudorabies virus immediate early protein.11 As a transcription factor, IE180 may control other genes guiding HVS regression. Of interest, the ocular phenotype was apparently not found in several other transgenic IE180 lines; these displayed a cerebellar defect instead.12 Finally, mouse studies have revealed that macrophage-like hyalocytes represent critical effectors of HVS regression.13 How hyalocyte function may be coupled to Ang-2, p53, or IE180 is not yet clear.

We recently discovered that deficiency of the tumor-suppressor gene Arf also results in a PHPV-like disease in the mouse.14 15 Arf encodes a nuclear protein, p19Arf, which was initially shown to interact physically with and inhibit the function of Mdm2, thereby promoting p53-dependent apoptosis or cell cycle arrest as a tumor-suppressive mechanism (reviewed by Sherr16 ). More recently, several p53-independent effects have been ascribed to p19Arf.17 18 19 20 21 In contrast to the eye disease in p53–/– mice, the PHPV-like phenotype is highly penetrant in Arf–/– mice of mixed C57BL/6 x 129/Sv background,14 suggesting that p19Arf does not merely activate p53 to promote HVS involution. Arf is expressed in a subset of perivascular cells within the HVS from embryonic day (E)12.5 through postnatal day (P)5.14 15 22 In Arf–/– mice, perivascular cells expressing platelet-derived growth factor receptor ß (Pdgfrß) accumulate and completely envelop the vitreous vessels as a fibrovascular mass.22 Analysis of mouse embryos lacking both Arf and Pdgfrß shows that p19Arf checks perivascular cell accumulation by controlling signals from this receptor,22 but the mechanisms are not defined.

We have taken advantage of our mouse model to explore further the pathogenesis of the eye disease and to gain better insight into developmental functions of p19Arf. We generated and analyzed chimeric mice composed of various numbers of wild-type and Arf–/– cells to determine formally whether somatic mosaic deletion of Arf in development could produce a PHPV-like disease; to show whether p19Arf has cell autonomous or non–cell autonomous effects during development; and to begin to evaluate whether Arf expression in perivascular cells in the embryonic vitreous might guide their fate toward a specific lineage.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chimeric Mouse Production and Analysis
Mice carrying an Arf exon 1ß deletion were maintained on a mixed C57BL/6 x 129/Sv genetic background.23 24 25 Genotyping for Arf was performed using polymerase chain reaction (PCR), as previously described.14 Mice carrying a transgene for a chicken ß-actin promoter-driven green fluorescent protein (Gfp; hereafter referred to as wild-type mice), produced at the transgenic facility at the University of Tennessee Health Science Center (UTHSC; Liu et al., manuscript in preparation), were maintained on an FVB genetic background. Thirty wild-type (ß-actin-Gfp){leftrightarrow}Arf–/– chimeras were generated, essentially as previously described.26 Animal studies were approved by the St. Jude Children’s Research Hospital and the UTHSC Animal Care and Use Committees and comply with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Histology Studies
Chimeric pups were euthanatized on postnatal day 1 for routine histology studies and immunofluorescence staining. Serial sections from the midline through the vitreous cavity were examined to determine the presence or absence of a retrolental mass. The area of the retrolental mass was determined in a representative, midline photomicrograph (ImagePro Plus software; Media Cybernetics, Silver Spring, MD). Gfp expression in ß-actin-Gfp (wild-type) cells in chimeric mice and in ArfGfp/+ mice25 was assessed by direct fluorescence microscopy or antibody-based detection. Expression of Pdgfrß, CD31, and smooth muscle {alpha}-actin (Sma) were detected by immunofluorescence staining as described.15 22 Additional details are provided in Supplementary Information, online at http://www.iovs.org/cgi/content/full/48/2/491/DC1.

Quantification of the Degree of Wild-Type{leftrightarrow} Arf–/– Chimerism
The degree of wild-type{leftrightarrow}Arf–/– chimerism was quantified by analysis of PCR-amplified genomic DNA from tail biopsy samples in 30 pups and by fluorescence microscopy in 26 eyes of 18 mice. For the former, PCR products were resolved on a 2% agarose gel, detected by ethidium bromide staining, and quantified with a gel documentation system (GelDoc 2000; Bio-Rad, Hercules, CA). To assess the degree of chimerism in different ocular structures, digital photomicrographs of midline sections were taken (model BX60 light/fluorescence microscope; Olympus, Tokyo, Japan, or model 510 NLO META multiphoton microscope; Carl Zeiss Meditec, Inc., Dublin, CA). The degree of chimerism in the retina was calculated from the green fluorescence density in anatomically defined areas (ImagePro Plus software; Media Cybernetics) with fluorescence photomicrographs (neuroretina) and multiphoton photomicrographs (neuroretina and retrolental mass). The degree of chimerism in the corneal epithelium and retinal pigment epithelium (RPE) was calculated by using the software to trace a line along the entire lengths of the corneal epithelium and the RPE. The nonfluorescent areas along the line were then measured to determine the Arf –/– contribution to each structure. Midline sections from three chimeric eyes were used to quantify the fraction of cells within the retrolental mass that expressed CD31 or Sma. Additional details for quantitative analyses are provided in the Supplementary Information, http://www.iovs.org/cgi/content/full/48/2/491/DC1.

Cell Culture–Based Studies
Mouse 10T1/2 pericyte-like cells (CCL-226; ATCC) were cultivated and transduced with MSCV-based retrovirus vectors containing bicistronic cDNA encoding Gfp and p19Arf (separated by an IRES [internal ribosomal entry site] element) or Gfp alone, as previously described.22 Two or 4 days after transduction, cells were harvested for flow cytometric assessment of transduction efficiency and for immunoblotting or immunocytochemistry staining for Sma expression, as described previously.15

Statistical Analysis
A general linear model27 that accounts for intrasubject correlation was used to determine whether the chimerism percentages were equal across the five tissue types (retina, cornea, retinal pigmented layer, tail DNA, and retrolental mass). To perform pair-wise comparisons of percentages of chimerism between tissue types, we applied Wilcoxon’s signed-rank test to the within-subject differences between the chimerism percentages of the tissues. Spearman’s rank-based correlation coefficient was used to measure the association of the degree of chimerism in pairs of tissues. Among subjects developing a retrolental mass, Spearman’s correlation coefficient was used to measure the association of the size of a retrolental mass with the degree of chimerism in various tissues. The Wilcoxon rank-sum test was used to compare chimerism percentages between subjects with and without a retrolental mass. Spearman’s correlation coefficient and Wilcoxon’s signed-rank and rank-sum tests are described.28


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
We first sought to determine whether a PHPV-like eye disease would develop in animals lacking Arf in only a subset of cells. To do this, we generated chimeric mice by fusing Arf–/– morulae with Arf+/+ morulae that express Gfp under control of the chicken ß-actin promoter; therefore, Arf wild-type cells were detectable by cytoplasmic Gfp expression. Molecular analysis of tail-derived genomic DNA and direct assessment of the cornea, retina, and retina pigment epithelium (RPE) by fluorescence microscopy showed that the 30 newborn chimeras represented a spectrum of mice composed of a relatively low to a relatively high proportion of Arf–/– cells (Fig. 1) . The three different ocular tissues had slightly different mean percentages of Arf–/– composition (ranging from 20.1% to 30.9%; P < 0.048); the biological importance of this small difference is not clear. The relative representation of the Arf–/– lineage in different parts of the eye and in tail-derived DNA correlated closely (Spearman correlation coefficients of 0.78 or greater; P < 0.0002 in each case; Fig. 2 ), again implying that the presence or absence of Arf does not greatly limit or enhance the contribution of cells to these tissues.


Figure 1
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  		FIGURE 1. Quantitative analyses of the degree of chimerism in P1 wild type{leftrightarrow} Arf–/– chimeric mice. (A) Photograph of a representative ethidium bromide–stained gel (left) and quantitative analysis (right) showing relative abundance of wild-type and knockout Arf alleles. Lanes 1–6: representative of chimeric mice used to generate data for the 30 analyzed mice (A–D'). Genomic DNA from an Arf+/– mouse (+/–) and water (H2O) served as positive and negative controls. ({square}) Arf–/– contribution; ({blacksquare}) wild-type contribution. (B) Representative immunofluorescence photomicrographs (left) and quantitation (right) of degree of chimerism in the retina, RPE, and corneal epithelium. Images show regions composed of wild-type and Arf–/– cells that either do (arrows) or do not (arrowheads) express Actin-Gfp. DAPI-stained images (blue) show nuclei. The alphanumeric code for each sample in the chart represents a separate eye. Original magnification: x200. Scale bars: 85 µm (retina), 50 µm (RPE); 100 µm (cornea).

 

Figure 2
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  		FIGURE 2. The degree of chimerism correlated in the different tissues. (A–F) Representative scatterplots showing the relative contribution of Arf–/– cells (expressed as a percentage of the total) to different tissues as indicated. Each Y and N represents an eye with or without a mass, respectively. Spearman correlation coefficients as follows: (A) 0.82; (B) 0.79; (C) 0.78; (D) 0.90; (E) 0.90; and (F) 0.86 (all P ≤ 0.0002).

 
We scored the chimeric eyes for the presence or absence and the size of the retrolental mass, as this is the principal abnormality uniformly detectable in Arf–/– mice at P1. A mass was often detectable in chimeric eyes, including several in which the Arf–/– contribution was quite low (e.g., cornea, 5%; retina, 1%; RPE, 17%; and tail DNA, 19%) (Fig. 3A) . Whereas the penetrance of the phenotype approaches 100% in Arf–/– eyes,14 only 14 of 26 eyes of 18 wild-type{leftrightarrow}Arf–/– chimeras had a mass. Further, it was unilateral in three mice in which the Arf–/– contribution to the retina ranged from 1% to 28%. In two of the three, Arf–/– cells contributed more to the retina in the eye with a mass (4.1% and 22.9%) than the eye without a mass (0.7% and 4.9%). Across the entire group, the presence of a retrolental mass was associated with a higher proportion of Arf–/– cells in the tail, retina, cornea, and RPE (Fig. 3B) . A threshold effect seemed evident as a mass was always present when greater than 18% of the cornea (n = 8), 28% of the retina (n = 6), 32% of the RPE (n = 7), or 50% of the tail DNA (n = 5) was Arf–/–, whereas it was always absent when less than 5% of the cornea (n = 6), 1% of the retina (n = 2), 17% of the RPE (n = 5), and 19% of the tail DNA (n = 2) was derived from cells lacking Arf. Among samples with a retrolental mass, the mass size (a marker of disease severity) was not significantly associated with the relative amount of Arf–/– cells in the cornea (P = 0.2799), retina (P = 0.4593), RPE (P = 0.3249), or tail DNA (P = 0.6415). However, due to the relatively small sample size, the statistical power for detecting a difference was somewhat limited. We conclude that, although a PHPV-like eye disease can develop in eyes lacking Arf in only a subset of cells, the abnormal phenotype can sometimes be suppressed in chimeric eyes containing relatively more wild-type cells.


Figure 3
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  		FIGURE 3. Retrolental mass is often absent in eyes composed of mostly wild-type cells. (A) Representative photomicrographs of eye sections of hematoxylin and eosin-stained (Aa), DAPI-stained (Ab) images and direct green fluorescence (Ac) from eyes with low (Aa–c) and high degrees (Aa'–c') of Arf–/– cell contribution. The retrolental mass (arrow) was visible in the eye of a high percentage of Arf–/– chimeric mice. Original magnification: x40, insets x200. (B) Graphic representation showing the degree of chimerism in the retina (Ba), tail-derived DNA (PCR) (Bb), cornea (Bc), and RPE (Bd) in eyes in which a mass was absent or present as indicated. In each case, the percentage of Arf–/– contribution differed in the two groups. P = (A) 0.0100; (B) 0.0068; (C) 0.0031; and (D) 0.0068.

 
We focused similar quantitative analyses on the retrolental mass to gain a better understanding of certain aspects of its pathogenesis. Although it contains endothelial cells and vascular smooth muscle cells, most of the mass is composed of perivascular cells expressing Pdgfrß.22 We determined the relative contribution of the Arf–/– lineage to the specific types of cells in the mass to gain insight into whether it had cell autonomous effects and whether it played a role in specification or differentiation of any of these cells in the retrolental mass.

We first documented the validity of the ß-actin-Gfp reporter by showing that it was uniformly expressed in the retrolental mass in nonchimeric Arf–/– mice (Supplementary Fig. S1, http://www.iovs.org/cgi/content/full/48/2/491/DC1). In chimeric mice, however, the reporter was expressed in only a subset of cells in the mass (Fig. 4A) . As in other pair-wise comparisons, the contribution of the Arf–/– cells to the mass correlated with their contribution to other areas of the eye (Spearman correlation coefficients 0.64 or greater; corresponding P = 0.01–0.08). Despite the correlation, the relative contribution of the Arf–/– lineage to the mass was consistently greater than its contribution to other areas of the eye. This was apparent when one compared the mean values of the contribution of Arf–/– to the retrolental mass (65.28%) versus the other tissues (25.69%–46.50%; Fig. 4B ). The selective contribution was even more pronounced in the retrolental Pdgfrß expressing cells—nearly all (95.9% ± 3.0%) (n = 642 cells from three separate eyes) were Arf–/–, even though that lineage contributed little to the retina (14.4% ± 12.9%) in the examined eyes (Fig. 4C) . In contrast to the Pdgfrß-positive cells, relatively few (~10%–20%) of the endothelial cells within the retrolental mass were Arf–/– (Fig. 5) . These findings suggest that Arf loss causes a cell intrinsic defect promoting the accumulation of Pdgfrß-positive cells in the vitreous. Because many of the endothelial cells in the mass were wild-type, additional non–cell autonomous effects of Arf loss in Pdgfrß-positive perivascular cells may selectively recruit wild-type endothelial cells to the mass.


Figure 4
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  		FIGURE 4. Cells derived from the Arf–/– lineage selectively contribute to the Pdgfrß-expressing cells in the retrolental mass. (A) Representative phase contrast (Aa) and confocal photomicrographs showing DAPI (Ab) and green fluorescence (Ac) of the retrolental mass (arrowheads, circumscribed) adjacent to lens (L) in a chimeric mouse. Most cells in the retrolental mass are from the Arf–/– lineage. Note that the retrolental mass adheres to the neuroretina (Figure 4), which is largely Gfp-positive. Original magnification: x200. (B) Graphic representation of a subset of eyes showing a percentage of Arf–/– contribution to the retrolental mass (RLM) and other tissues, as indicated. An ANOVA model indicates that the average percentage in each group is significantly different from that in the other groups (P = 0.001). Pair-wise comparisons show RLM (*) is significantly different from retina (P = 0.0156), cornea (P = 0.0078), RPE (P = 0.0078), and tail-derived DNA (PCR) (P = 0.0156). (C) Representative confocal immunofluorescence photomicrograph of the retrolental mass in chimeric mouse showing Pdgfrß (Ca) and Gfp to mark cells of wild-type lineage (Cb). Multicolor overlay (Cc). Pdgfrß-expressing cells in the wild-type lineage were rarely observed (arrows).

 

Figure 5
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  		FIGURE 5. Different contribution of Arf–/– cells to smooth muscle (Sma positive) and endothelial (CD31 positive) cells in the retrolental mass. (A) Representative confocal immunofluorescence photomicrographs of chimeric eyes stained for CD31 (Aa), Sma (Ad), and Gfp, to mark wild-type cells (Ab, Ae). Multicolor overlays include DAPI (blue) (Ac, Af). Most CD31-expressing endothelial cells were wild-type (arrowheads), whereas Sma-positive cells were also Arf–/– (arrows). Original magnification: x200. (B) Graphic representation of a fraction of Arf–/–-derived cells in the entire retrolental mass (black) versus CD31-positive (red) and Sma-positive (white) cells in the retrolental mass from eyes from three separate chimeric mice. Approximately 60 and 120 cells were counted in each eye, to obtain the quantitative data for CD31 and Sma lineage tracing, respectively.

 
It has been proposed that Pdgfrß-positive perivascular cells represent progenitors that can give rise to vascular smooth muscle cells.29 If true, we expected that most of the Sma-expressing cells would be derived from the Arf–/– lineage like the Pdgfrß-positive cells. Instead, only approximately 50% of the Sma-positive cells were Arf–/– (Fig. 5) . Because the Arf promoter is predominantly expressed in Pdgfrß-positive perivascular cells in the developing vitreous,22 we considered that p19Arf might facilitate smooth muscle maturation, even though it is not formally necessary for the process. To explore this notion, we determined whether Arf and Sma expression overlapped in nonchimeric ArfGfp/+ mice in which Arf expression can be followed by Gfp immunostaining.25 Dual immunofluorescence staining showed that Gfp and Sma were expressed together in a subset of mural cells at P1 (Fig. 6A) . To directly test whether Arf could promote smooth muscle cell maturation, we took advantage of the mouse 10T1/2 pericyte-like cell line. These cells localize to perivascular sites when implanted with tumor cells in vivo,30 and they expressed several genes relevant to mural cell biology, including Pdgfrß, Ang-1, and Vegf (Fig. 6B) . 10T1/2 cells lack Arf, but they can be efficiently transduced by retrovirus encoding p19Arf (Supplementary Fig. S2, http://www.iovs.org/cgi/content/full/48/2/491/DC1). Arf–/– 10T1/2 cells expressed low levels of Sma at baseline (Fig. 6C) ; 2 to 4 days after retrovirus transduction, the Arf-expressing cells assumed a flattened morphology and expressed high levels of Sma (Figs. 6C 6D) and smooth muscle myosin (Supplementary Fig. S3). Taken together, these data are consistent with a model in which Arf expression in Pdgfrß-positive perivascular cells enveloping the HVS facilitates their maturation into smooth muscle cells.


Figure 6
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  		FIGURE 6. Arf expression in pericyte-like cells can induce smooth muscle proteins. (A) Representative photomicrograph of vitreous hyaloid vessel in P1 Arf+/Gfp mouse shows most Arf-expressing cells (green) are flanked by Sma-positive cells (red, top), but some cells (3 of 11 Arf-expressing cells in two longitudinal sections through vitreous vessels) expressed Arf and Sma (bottom). Original magnification: x400. (B) Photograph of ethidium bromide-stained gel after electrophoresis of RT-PCR products shows the indicated genes to be expressed in 10T1/2 pericyte-like cells. (C) Representative immunoblots for the indicated proteins in 10T1/2 cells 48 hours after transduction with Gfp or Arf retrovirus. (D) Photomicrographs of 10T1/2 cells 48 or 96 hours after Gfp- and Arf-transduction and immunohistochemical staining for Sma. Original magnification: x40.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We can draw the following conclusions from our studies of wild-type{leftrightarrow} Arf–/– chimeric mice: First, consistent with our previous finding that Arf expression in the eye is limited to the vitreous, the presence or absence of Arf does not dramatically influence the contribution of cells to the retina, cornea, or RPE. Second, a PHPV-like eye disease can occur in mice lacking Arf in only a subset of cells; but in some chimeric eyes composed of relatively more wild-type cells, the developmental defect can be suppressed. Third, our data suggest that both cell autonomous and non–cell autonomous effects of Arf may contribute to vitreous development. Specifically, the fact that nearly all the Pdgfrß-expressing cells in the retrolental mass stemmed from the Arf–/– lineage implies that p19Arf uses cell autonomous mechanisms to block their accumulation. Additional non–cell autonomous effects of Arf loss may be relevant because some wild-type cells also accumulated in the mass. Fourth, the Arf–/– lineage appears to contribute more to the Pdgfrß expressing cells than to smooth muscle and endothelial cells in the retrolental mass. Finally, Arf expression overlaps with Sma in the vitreous, and ectopic p19Arf can promote smooth muscle protein expression in pericyte-like cells in vitro.

Although cell autonomous activity is implicit in current models of Arf-dependent tumor suppression, this activity has not been formally demonstrated during development. The retinoblastoma tumor suppressor gene provides an important precedent, in that it has both cell autonomous mechanisms to control cell proliferation (reviewed by Classon and Harlow31 ) and non–cell autonomous effects during embryonic development.32 33 34 Our chimera studies shed some light on the relative importance of cell autonomous and non–cell autonomous effects of p19Arf.

The earliest detectable abnormality in Arf–/– mice is the accumulation of vitreous cells expressing Pdgfrß.22 That approximately 95% of these cells arose from the Arf–/– lineage supports the concept that p19Arf primarily uses cell intrinsic mechanisms either to block their accumulation or to repress the expression of Pdgfrß. This conclusion is supported by an earlier finding that nearly all the Pdgfrß-expressing cells in ArfGfp/Gfp (effectively Arf–/–) mice coexpress the Arf promoter.22 It should be emphasized, though, that not all the cells in the retrolental mass were Arf–/–. In particular, many smooth muscle cells and most of the endothelial cells within the mass were wild type (see Figs. 4A 5A ). As such, it is formally possible that additional non–cell autonomous effects of Arf loss in Pdgfrß expressing cells allow wild-type cells to accumulate within the mass.

If p19Arf had predominantly non–cell autonomous effects, we expected two things. First, the Pdgfrß-positive cells would not selectively arise from the Arf–/– pool. As just discussed, most of them are Arf–/–. Second, we expected that non–cell autonomous effects, such as the repression of an antimitogenic factor in the vitreous, would suppress the developmental defect in chimeras. Of interest, the eyes appeared normal in some bona fide chimeras, especially those composed of a relatively small fraction of Arf–/– cells. It is formally possible that non–cell autonomous actions contributed to the suppression of the phenotype in these mice. But, the suppression may also be explained without invoking non–cell autonomous effects: In some animals with relatively low Arf–/– composition, the Pdgfrß expressing cells in the embryonic vitreous may have all originated from the wild-type lineage. We cannot discriminate between these two possibilities. Although a previous, unbiased search for genes induced or repressed by p19Arf in cultured fibroblasts did not reveal obvious candidate signaling proteins to mediate cell extrinsic effects,35 the developing vitreous seems to represent an ideal system to search further for such proteins.

Beyond helping to identify cell extrinsic and intrinsic effects of p19Arf, we used our chimera studies to evaluate whether it may also control aspects of cellular differentiation. We considered this because the strikingly specific expression of p19Arf in only a subset of vitreous cells15 22 suggested that it might do more than merely control excessive mitogenic or oncogenic signals, as is the current dogma.16 Cells migrating into the vitreous at ~E12.5 have been proposed to represent pluripotent progenitors of the hyaloid vasculature.36 Arf expression is detectable in the vitreous from ~E12.5 as well.22 Several lines of evidence indicate that Pdgfrß-positive cells give rise to different mural cell subtypes.29 37 38 Although Pdgfrß is essential for smooth muscle cell localization to many vessels,37 39 Pdgf-B can also impede smooth muscle differentiation in cultured cells.40 41 Last, p19Arf blocks Pdgf-B-dependent mitogenic signals and dampens Pdgfrß expression in fibroblasts.22 Therefore, it seemed reasonable to hypothesize that Arf expression in Pdgfrß-positive mural progenitors in the vitreous might promote smooth muscle cell maturation.

Several of our findings support this concept. First, the Arf–/– lineage seemed to contribute less to the Sma expressing cells than to the Pdgfrß-positive cells within the retrolental mass. Second, Arf and Sma expression overlapped in a subset of mural cells in vivo. Finally, ectopic expression of p19Arf in pericyte-like cells induced smooth muscle proteins in vitro. Conceptually, Arf-mediated inhibition of Pdgfrß-positive cell accumulation and its ability to promote their maturation to smooth muscle cells may be particularly important in a "transient" vasculature like the hyaloid vessels. For example, limiting coverage of the HVS by mature smooth muscle cells may render the vessels more accessible to hyalocytes which are direct effectors of HVS regression.13 The notion that cell cycle arrest and smooth muscle differentiation may be coupled is not without precedent. Expression of the adenovirus E1A oncoprotein prevents cell cycle exit and represses Sma expression in smooth muscle cells in vitro.42 Although downregulation of Pdgfrß by p19Arf can occur without p53,22 Arf-dependent induction of Sma may be p53-dependent.43

We were somewhat surprised to find that Arf–/– endothelial cells seemed underrepresented in the retrolental mass. As discussed earlier, the Arf–/– cells filling the vitreous might selectively attract wild-type endothelial cells by non–cell autonomous mechanisms. Alternatively, Arf may play a more direct role in endothelial cell lineage commitment or differentiation. It should be noted, though, that we did not observe Arf expression in endothelial cells in the developing eye at or after E14.5.15 22 Perhaps earlier developmental studies including analyses of wild type{leftrightarrow}Arf–/– mouse chimeras will shed additional light on this possibility.


   Acknowledgements
 
The authors thank the St. Jude Children’s Research Hospital Scientific Imaging Shared Resource for technical assistance; Michael B. Kastan, Martine F. Roussel, and Charles J. Sherr, as well as members of the Skapek Laboratory for helpful discussions; and particularly Charles Sherr for encouraging us to undertake the investigation.


   Footnotes
 
Supported by National Eye Institute Grant R01 EY014368 (SXS), National Mental Health Institute Grants U01 MH61971 and R25 MH066890 (DG), and National Cancer Institute Grant CA021765 (DP, SP), and by the American Lebanese Syrian Associated Charities (ALSAC).

Submitted for publication July 6, 2006; revised August 22, 2006; accepted November 30, 2006.

Disclosure: J.D. Thornton, None; D.J. Swanson, None; M.N. Mary, None; D. Pei, None; A.C. Martin, None; S. Pounds, None; D. Goldowitz, None; S.X. Skapek, 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: Stephen X. Skapek, Department of Oncology, St. Jude Children’s Research Hospital, 332 N. Lauderdale Street, Memphis, TN 38105; steve.skapek{at}stjude.org.


   References
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 Abstract
 Materials and Methods
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 Discussion
 References
 

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