|Molecular Vision 2001;
Received 4 September 2001 | Accepted 28 November 2001 | Published 2 December 2001
Regulation of choroid development by the retinal pigment epithelium
Paul A. Overbeek
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX
Correspondence to: Shulei Zhao, Ph.D., Lexicon Genetics, Inc., 4000 Research Forest Drive, The Woodlands, TX, 77381; Phone: (281) 863-3071; FAX: (281) 863-8088; email: firstname.lastname@example.org
Purpose: The choroidal vasculature is essential for normal retinal function. However, mechanisms that control choroid development are unknown. In the present study, we provide evidence that the retinal pigment epithelium (RPE) plays an essential role in regulation of the choroid development in the mouse eye.
Methods: Transgenic mice that transiently express FGF9 in the embryonic RPE were generated. Postnatal eyes were analyzed by histology and in situ hybridizations.
Results: In the transgenic mice, most of the RPE was converted to neural retina. The choroid formed only in regions where patches of RPE were present. The choroid failed to develop in the absence of the RPE.
Conclusions: The presence of the RPE appears to be required for choroid development, suggesting that molecular interactions between RPE and periocular mesenchyme are essential for melanocyte differentiation and vascular development in the choroid.
The choroid is a layer of highly vascularized tissue surrounding the eye. Choroidal blood nourishes the retinal pigment epithelium (RPE) and the photoreceptors at the outer layer of the retina. The choroid develops from two embryonic tissues, the mesoderm and cranial neural crest cells. The endothelial cells of choroidal blood vessels are of mesodermal origin while all other cells including stromal cells, melanocytes, and pericytes are derived from neural crest cells [1-3]. During early eye development, tubes and spaces form in the periocular region next to the optic vesicle. These tubes are lined by endothelium of mesodermal origin. As the eye grows, they expand from the central axis to the caudal end of the optic vesicle and form a plexus. When the optic vesicle invaginate to form the optic cup and pigmentation appears in the presumptive RPE, primitive capillaries develop from the plexus adjacent to the pigmented RPE. Subsequently, the ophthalmic artery and the posterior ciliary arteries enter the choriocapillary layer while large capillaries fuse to form the veins. In the human embryo, pigmentation of choroidal melanocytes occurs in late gestation and is complete at birth .
The periocular mesenchyme remains close contact with the RPE during eye development, suggesting that interactions between these tissues may be important for normal ocular development. We have previously reported that transient expression of transgenic FGF9 in embryonic RPE can switch its differentiation to a neuronal pathway, resulting in a duplicate neural retina in transgenic mice [4,5]. We report here that the choroid fails to develop in these mice, indicating that RPE provides inductive signals for choroid development. In the mouse, pigmentation of choroidal melanocytes begins soon after birth and is complete by two weeks of age.
We previously described transgenic mice that express FGF9 in the presumptive RPE under the control of a tyrosinase-related protein 2 (TRP2) promoter . In these mice, the embryonic RPE is induced to differentiate into neural retina. In the present study, we further examined whether the consequent lack of an RPE affected development of periocular tissues in these mice. Since FVB/N mice from which the transgenic mice were originally derived carry an autosomal recessive mutation causing retinal degeneration [6,7], these mice were mated with wild type pigmented inbred C57BL/6 mice or albino outbred ICR mice for further analysis. Postnatal day 1 (P1) and day 7 (P7) mouse eyes were collected and fixed in 10% formalin overnight at room temperature and then rinsed in 70% ethanol for 24 h. The eyes were next dehydrated using ethanol solutions of increasing concentration, cleared in xylene, and embedded in paraffin wax. Sections (5 mm) were cut on a microtome, dewaxed with xylene, rehydrated using decreasing concentrations of ethanol, and then used for hematoxylin/eosin staining and for in situ hybridizations. Animals were handled following the guidelines provided in US Public Health Service Policy on Humane Care and Use of Laboratory Animals.
To examine the possible factors that are likely to be involved in development of choroidal vasculature, in situ hybridizations were carried out with 35S-labeled antisense RNA probes specific to mouse vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 as previously described . After washing, RNase treatment, and dehydration, the slides were coated with Kodak NTB-2 emulsion for autoradiography. The slides were developed, counter-stained with hematoxylin, and then mounted with a cover slip for examination of silver grain distribution under bright-field or dark-field illumination. Both the bright- and dark-field images were collected by computer through a CCD camera and subsequently superimposed onto each other using Adobe Photoshop software. Silver grains in the dark-field images were pseudo-colored red to improve contrast in the superimposed images.
In the wild type eyes, pigmented cells appeared in the developing choroid at P7 (Figure 1A,C). Small blood vessels and blood cells were present in the developing choroid adjacent to the RPE (Figure 1C). In P7 eyes from the TRP2-FGF9 transgenic mice, the RPE was converted to a second layer of neural retina (Figure 1B). Both the original and the RPE-derived neural retinae differentiated, laminated, and expressed retina-specific markers such as rhodopsin [4,5]. The majority of the presumptive RPE was converted to neural retina except for a patch in the posterior region of the eye (Figure 1B). The failure of this RPE patch to convert to neural retina was presumably due to low levels of transgene expression in this region. Pigmented choroid tissue was found only in the region where the RPE was still present (Figure 1B,D), indicating that RPE is required for choroid development. Small blood vessels and blood cells were observed in this region (Figure 1D). In regions where the RPE had been converted to neural retina, no such vascular structures were present (Figure 1E). Occasionally, thin monolayers of melanocytes were found in regions where the RPE had been converted to the neural retina (red arrow in Figure 1B). However, these thin cell layers do not resemble the developing choroid. They possibly migrated there from adjacent regions.
To examine what factors might be involved in development of the choroidal vasculature, the expression pattern of VEGF in wild type eyes was analyzed by in situ hybridization. At P1, VEGF mRNA was expressed predominantly in the RPE and also to some extent in the differentiated cell layer of the neural retina (the ganglion cell layer; Figure 2A,B). At P7, VEGF mRNA remained expressed in the RPE and was also present at high levels in the inner nuclear layer and in astrocytes along the vitreous surface of the neural retina (Figure 2C,D). Müller glial cells in the inner nuclear layer and retinal astrocytes at the vitreous surface are known to express high levels of VEGF in postnatal rodent eyes .
In situ hybridizations were also carried out to examine expression patterns of the VEGF receptors Flt-1 (VEGFR1) and Flk-1 (VEGFR2) in wild type mouse eyes. At P1, Flt-1 mRNA was expressed in the endothelial cells of hyaloid blood vessels (Figure 3A) and in periocular mesenchyme including a layer of cells adjacent to the RPE (Figure 3A,E). Flk-1 mRNA exhibited a similar expression pattern except that it was also expressed weakly in the proliferating retinoblast layer (RBL; Figure 3B,F). At P7, Flt-1 mRNA was expressed in the vascular endothelial cells at the vitreous surface of the neural retina and in the developing choroidal tissue near the RPE (Figure 3C). It was also detected at low levels in the inner nuclear layer (Figure 3C). In P7 eyes, Flk-1 mRNA was expressed in the ganglion cell layer, the inner nuclear layer, and in the periocular mesenchymal cells adjacent to the RPE (Figure 3D). Expression of Flk-1 mRNA in the developing neural retina has been previously reported . The cells adjacent to the RPE that expressed Flt-1 and Flk-1 (Figure 3E,F) were likely to be endothelial cells of the developing choroidal vasculature.
Our previous study showed that the TRP2 promoter activated transgene expression in the developing RPE by embryonic day 9.5 (E9.5) . Our more recent study demonstrates that transgene expression directed by the TRP2 promoter was turned off by E10 as soon as RPE differentiation had been switched to the neuronal pathway . That study also showed that endogenous FGF9 is normally expressed in the developing neural retina and has no apparent effect on RPE differentiation. This might be due to the high affinity of FGFs for extracellular matrix, particularly heparan sulfate proteoglycans, which can severely limit their diffusion in interstitial spaces. As a result, FGFs often have a very limited range of action [5,10]. Therefore, the transient expression of FGF9 in the presumptive RPE between E9-E10 was not very likely to have a significant impact on choroid development in postnatal mice.
It has been observed that in human patients with colobomas, failure in RPE differentiation leads to defective choroid and sclera , supporting the notion that the defective RPE but not a factor such as FGF9 causes the absence of the choroid. Early in vitro studies using chick embryos also suggest a role of the RPE in regulating differentiation of periocular mesenchyme [11,12]. These studies demonstrate that RPE induced formation of cartilage, a component of the sclera. Mutations in the microphthalmia transcription factor (Mitf) cause the colobomas phenotype in mice. Recent studies show that dorsal RPE in these mice differentiate as neural retina [13,14]. Whether there were defects in periocular mesenchymal tissues was not reported in these studies. Given the close association between the choroid and the sclera, it would not be surprising that the sclera was also defective in TRP2-FGF9 transgenic and in Mitf mutant mice although this has yet to confirmed by further studies.
We have shown that one of the signals released by RPE is VEGF. In developing rodents, VEGF expression in the RPE increases from embryonic to postnatal stages, peaks approximately a week after birth (data not shown), and then decreases with age but persists throughout adulthood [8,15]. VEGF is also present in adult human RPE . In postnatal mouse eyes, VEGF receptors Flt-1 and Flk-1 were found expressed in a subset of periocular mesenchymal cells adjacent to the RPE (Figure 3). These gene expression patterns suggest that VEGF made by RPE could play a critical role in vascular development in the choroid. Elevated levels of VEGF expression by RPE have been implicated as a cause of choroidal neovascularization in human [17-21] and in animal models [22-30].
Vascular development is a complex process and requires functional interactions of many different factors (such as VEGF, PDGF, TGF-beta, angiopoietins, etc). It is unlikely that VEGF is the only factor produced by the RPE or other tissues in the vicinity that controls choroid development. For example, since there is no evidence that melanocyte differentiation requires VEGF stimulation, migration and organization of pigmented cells in the developing choroid are likely to be regulated by other as yet unidentified factors.
This work was supported by NIH grant EY10448 (PAO), the Knights Templar Foundation and Fight For Sight, Inc. (SZ).
1. Noden DM. Periocular mesenchyme: neural crest and mesodermal interactions. In: Jakobiec FA, editor. Ocular anatomy, embryology and teratology. Philadelphia: Harper & Row; 1982. p. 79-119.
2. Torczynski E. Choroid and suprachoroid. In: Jakobiec FA, editor. Ocular anatomy, embryology and teratology. Philadelphia: Harper & Row; 1982. p. 553-85.
3. Etchevers HC, Vincent C, Le Douarin NM, Couly GF. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development 2001; 128:1059-68.
4. Zhao S, Overbeek PA. Tyrosinase-related protein 2 promoter targets transgene expression to ocular and neural crest-derived tissues. Dev Biol 1999; 216:154-63.
5. Zhao S, Hung FC, Colvin JS, White A, Dai W, Lovicu FJ, Ornitz DM, Overbeek PA. Patterning the optic neuroepithelium by FGF signaling and Ras activation. Development. In press 2001.
6. Pittler SJ, Baehr W. Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse. Proc Natl Acad Sci U S A 1991; 88:8322-6.
7. Bowes C, Li T, Frankel WN, Danciger M, Coffin JM, Applebury ML, Farber DB. Localization of a retroviral element within the rd gene coding for the beta subunit of cGMP phosphodiesterase. Proc Natl Acad Sci U S A 1993; 90:2955-9.
8. Stone J, Maslim J. Mechanisms of retinal angiogenesis. Prog Retin Eye Res 1997; 16:157-81.
9. Yang K, Cepko CL. Flk-1, a receptor for vascular endothelial growth factor (VEGF), is expressed by retinal progenitor cells. J Neurosci 1996; 16:6089-99.
10. Ornitz DM. FGFs, heparan sultate and FGFRs: complex interactions essential for development. BioEssays 2000; 22:108-12.
11. Newsome DA. Cartilage induction by retinal pigmented epithelium of chick embryo. Dev Biol 1972; 27:575-9.
12. Newsome DA. In vitro stimulation of cartilage in embryonic chick neural crest cells by products of retinal pigmented epithelium. Dev Biol 1976; 49:496-507.
13. Bumsted KM, Barnstable CJ. Dorsal retinal pigment epithelium differentiates as neural retina in the microphthalmia (mi/mi) mouse. Invest Ophthalmol Vis Sci 2000; 41:903-8.
14. Nguyen M, Arnheiter H. Signaling and transcriptional regulation in early mammalian eye development: a link between FGF and MITF. Development 2000; 127:3581-91.
15. Yi X, Mai LC, Uyama M, Yew DT. Time-course expression of vascular endothelial growth factor as related to the development of the retinochoroidal vasculature in rats. Exp Brain Res 1998; 118:155-60.
16. Kvanta A. Expression and regulation of vascular endothelial growth factor in choroidal fibroblasts. Curr Eye Res 1995; 14:1015-20.
17. Reddy VM, Zamora RL, Kaplan HJ. Distribution of growth factors in subfoveal neovascular membranes in age-related macular degeneration and presumed ocular histoplasmosis syndrome. Am J Ophthalmol 1995; 120:291-301.
18. Lopez PF, Sippy BD, Lambert HM, Thach AB, Hinton DR. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1996; 37:855-68.
19. Frank RN, Amin RH, Eliott D, Puklin JE, Abrams GW. Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthalmol 1996; 122:393-403.
20. Kvanta A, Algvere PV, Berglin L, Seregard S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci 1996; 37:1929-34.
21. Kliffen M, Sharma HS, Mooy CM, Kerkvliet S, de Jong PT. Increased expression of angiogenic growth factors in age-related maculopathy. Br J Ophthalmol 1997; 81:154-62.
22. Yi X, Ogata N, Komada M, Yamamoto C, Takahashi K, Omori K, Uyama M. Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefes Arch Clin Exp Ophthalmol 1997; 235:313-9.
23. Ishibashi T, Hata Y, Yoshikawa H, Nakagawa K, Sueishi K, Inomata H. Expression of vascular endothelial growth factor in experimental choroidal neovascularization. Graefes Arch Clin Exp Ophthalmol 1997; 235:159-67.
24. Wada M, Ogata N, Otsuji T, Uyama M. Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization. Curr Eye Res 1999; 18:203-13.
25. Spilsbury K, Garrett KL, Shen WY, Constable IJ, Rakoczy PE. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol 2000; 157:135-44.
26. Baffi J, Byrnes G, Chan CC, Csaky KG. Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor. Invest Ophthalmol Vis Sci 2000; 41:3582-9.
27. Yu MJ, Shen WY, Lai MC, Constable IJ, Papadimitriou JM, Rakoczy PE. The role of vascular endothelial growth factor (VEGF) in abnormal vascular changes in the adult rat eye. Growth Factors 2000; 17:301-12.
28. Schwesinger C, Yee C, Rohan RM, Joussen AM, Fernandez A, Meyer TN, Poulaki V, Ma JJ, Redmond TM, Liu S, Adamis AP, D'Amato RJ. Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium. Am J Pathol 2001; 158:1161-72.
29. Seo MS, Kwak N, Ozaki H, Yamada H, Okamoto N, Yamada E, Fabbro D, Hofmann F, Wood JM, Campochiaro PA. Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor. Am J Pathol 1999; 154:1743-53.
30. Kwak N, Okamoto N, Wood JM, Campochiaro PA. VEGF is major stimulator in model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2000; 41:3158-64.