The official journal of INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH

PIGMENT CELL & MELANOMA Research Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation Bhushan Sarode, Craig S. Nowell, JongEun Ihm, Corinne Kostic, Yvan Arsenijevic, Alexandre P. Moulin, Daniel F. Schorderet, Friedrich Beermann and Freddy Radtke

DOI: 10.1111/pcmr.12236 Volume 27, Issue 4, Pages 580–589 If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here

Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr

Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org

To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org

ORIGINAL ARTICLE

Pigment Cell Melanoma Res. 27; 580–589

Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation Bhushan Sarode1,2, Craig S. Nowell1,2, JongEun Ihm1, Corinne Kostic3,4, Yvan Arsenijevic3,4, Alexandre P. Moulin4, Daniel F. Schorderet1,4,5, Friedrich Beermann1,2 and Freddy Radtke1,2 1 School of Life Science, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland 2 Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland 3 Unit of Gene Therapy and Stem Cell Biology, Jules-Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland 4 Eye Pathology Laboratory, Jules-Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland 5 IRO – Institute for Research in Ophthalmology, Sion, Switzerland

KEYWORDS Notch/ciliary body/ocular hypertension/phthisis bulbi/glaucoma/pigmented epithelium/ iris

CORRESPONDENCE Freddy Radtke, e-mail: [email protected]

PUBLICATION DATA Received 16 July 2013, revised and accepted for publication 12 March 2014, Published online 15 March 2014

Sarode is the sole first author, and Nowell is the second author.

doi: 10.1111/pcmr.12236

Summary The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intra-ocular pressure and regulate exposure of the internal eye structures to light, respectively. The cellular and molecular factors that mediate the development of the ciliary body and iris from the ocular pigmented epithelium remain to be fully elucidated. Here, we have investigated the role of Notch signaling during the development of the anterior pigmented epithelium by using genetic loss- and gain-of-function approaches. Loss of canonical Notch signaling results in normal iris development but absence of the ciliary body. This causes progressive hypotony and over time leads to phthisis bulbi, a condition characterized by shrinkage of the eye and loss of structure/function. Conversely, Notch gain-of-function results in aniridia and profound ciliary body hyperplasia, which causes ocular hypertension and glaucoma-like disease. Collectively, these data indicate that Notch signaling promotes ciliary body development at the expense of iris formation and reveals novel animal models of human ocular pathologies.

Introduction The pigmented epithelium of the eye consists of the retinal pigment epithelium (RPE), iris, and ciliary body (CB), which develop during embryogenesis and early postnatal stages from structures at the margin of the optic cup. The RPE controls proper lamination of the retina and differentiation of the photoreceptors (Martinez-

Morales et al., 2004). The main function of the iris is to regulate the amount of light entering the eye. Congenital aniridia, in which the iris is absent, results in impaired vision and is also associated with ocular pathologies such as cataracts and certain types of glaucoma. Loss of function mutations in the Pax6 gene, a master regulator of eye development, is currently the only genetic lesion known to be causative for aniridia (Lee et al., 2008).

Significance The iris and ciliary body are pigmented structures of the anterior eye segment and are each essential for normal ocular function. Perturbed development and/or function of either structure results in serious pathological conditions such as glaucoma and phthisis bulbi. Despite their importance, little is known about the mechanisms that regulate their development. Here, we demonstrate that canonical Notch signaling promotes ciliary body development and opposes iris formation. In addition, we demonstrate that genetic manipulation of Notch signaling in ocular pigment cells facilitates robust modeling of human pathologies such as glaucoma and phthisis bulbi.

580

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Notch and ocular pigmented epithelium

The function of the CB is more complex and includes aqueous humor (AH) production, accommodation, and formation of the blood aqueous barrier. The CB is composed of smooth muscle cells and the ciliary epithelium, which consists of two layers, one of which is pigmented. The non-pigmented cell layer represents the inner part of the epithelium, which is continuous with the neural retina and the iris. It generates fibrillins, the major component of the suspensory ligaments of the lens (Hanssen et al., 2001). In contrast, the pigmented outer epithelium lies between the retinal pigmented epithelium (RPE) and the root of the iris (Beebe, 1986). One of the main functions of the CB is the production and secretion of AH, which is a transparent fluid consisting of a mixture of growth factors, organic solutes, and other proteins that nourish the non-vascular tissue of the anterior chamber (Coca-Prados and Escribano, 2007). The AH inflates the eye and establishes an intra-ocular pressure (IOP) assuring proper alignment of the optical structures. Appropriate IOP is the result of a dynamic balance between secretion and drainage of the AH. Drainage of the AH occurs through the trabecular meshwork (TM) and the Schlemm’s canal (Do and Civan, 2009), which are anterior to the ciliary body. Damage or dysfunction of the CB can lead to ocular hypotony due to impaired AH production (Coleman, 1995; Yu et al., 2007). This is characterized by reduced visual function and in severe cases can lead to a shrunken, non-functional eye, a pathology known as phthisis bulbi (Coleman, 1995; Fine et al., 2007). In contrast, overproduction of AH or defective drainage is associated with ocular hypertension, which is one of the main risk factors for developing glaucoma, an optic neuropathy leading to visual field loss and eventually to irreversible blindness (Quigley, 2011). Given the importance of the RPE, iris, and CB with respect to ocular function, elucidation of the cellular and molecular mechanisms that regulate their development from the anterior pigmented epithelium is of considerable value and may facilitate the generation of novel genetic models, which can serve as tools to study the etiology and treatment of human ocular diseases. In addition, determination of the mechanisms that control ocular pigment cell development will provide important insight with respect to the site-specific regulatory factors that control the mammalian pigmentary system. For example, while both cutaneous and extracutaneous pigment cells produce melanin, it is not clear whether the development of these cells or the production of pigment is controlled by the same signaling pathways. In fact, there is evidence that pigment-cell-specific genes are regulated differently between pigment cells of the skin and the eye due to independent evolution of regulatory DNA sequences in pigment-specific promoters (Camacho-Hubner and Beermann, 2001; Murisier and Beermann, 2006; Murisier et al., 2007). The Notch pathway is an evolutionarily conserved cellto-cell communication cascade involved in many differª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

entiation processes in fetal and postnatal development as well as in homeostasis of adult self-renewing organs (Koch et al., 2013). In the pigmentary system of the skin, Notch signaling is required for melanocyte development. Genetic inactivation of Notch1 and Notch2 or RBP-J results in progressive hair graying caused by depletion of melanocyte precursor and stem cells (Aubin-Houzelstein et al., 2008; Kumano et al., 2008; Moriyama et al., 2006; Schouwey and Beermann, 2008; Schouwey et al., 2007). Although the Notch cascade has been shown to be instrumental in multiple aspects of eye development (Lee et al., 2005; Papayannopoulos et al., 1998; Rowan et al., 2008; Schouwey et al., 2011; Singh et al., 2012; Zheng et al., 2009), its putative role in pigmented eye structures including the RPE, CB, and iris is less clear. In this study, we have functionally assessed the role of Notch in pigmented structures of the eye using genetic loss and gain-of-function approaches.

Results and discussion Loss of Notch signaling causes ciliary body aplasia and phthisis bulbi To investigate how loss of Notch signaling in pigmented eye structures affects the physiology and function of the eye, we used a conditional gene-targeting approach to circumvent the early embryonic lethality of conventional Notch mutant mice (Besseyrias et al., 2007; Han et al., 2002). Mice carrying floxed alleles for Notch1, Notch2, or RBP-Jj were crossed with pigment-cell-specific Mart1::Cre transgenic mice, thus generating Notch1lox/loxMart1::Cre, Notch2lox/loxMart1::Cre, and RBP-Jjlox/loxMat1::Cre mice (herein referred to as Notch1Δ/Δ, Notch2Δ/Δ, and RBP-JjΔ/Δ). The activity of Mart1::Cre in the eye is restricted to the pigmented epithelial cells of the iris, CB, and RPE (Figure S1), thus allowing deletion of Notch1, Notch2, and RBP-Jj specifically in these structures. Consistent with a previous report, loss of Notch2 in pigmented epithelial cells resulted in CB aplasia (Zhou et al., 2013), a phenotype that was recapitulated in RBP-JjΔ/Δ mice (Figure 1A). Further histological analysis indicated that other pigmented eye structures such as iris and RPE were normal in RBP-JjΔ/Δ mutants (Figure 1A and Figure S2), indicating that the CB is the only pigmented structure that requires Notch signaling. Notch1Δ/Δ mice exhibited normal development of all eye structures derived from the pigmented epithelium (Figure S3). Interestingly, Notch2 expression in the CB is conserved between human and mouse (Figure S4), suggesting a similar requirement for Notch2 in the human CB. These data indicate that Notch2 is the essential non-redundant receptor required for CB development and that it executes this function via canonical RBP-Jj-mediated signaling. To determine the pathophysiological effects of CB aplasia due to loss of Notch signaling in the ocular pigmented epithelium, we characterized the ocular structure and function throughout the lifetime of Notch2Δ/Δ and RBP-JjΔ/Δ mice. Strikingly, these mutants developed a 581

Sarode et al.

A

B

C

D

E

microphthalmic eye phenotype with 100% penetrance. At 1 month of age, Notch2Δ/Δ and RBP-JjΔ/Δ eyes were significantly smaller in size compared to littermate controls (Figure 1B, C). Moreover, mutant mice exhibited 582

Figure 1. Notch signaling disruption in ocular pigment cells results in ciliary body (CB) aplasia and phthisis bulbi. (A) H & E staining of CB sections from RBP-JΚΔ/Δ and Notch 2Δ/Δ eyes. Lower panel indicates magnified images of selected part in upper panel. Scale bar: 350 lm (upper panel), 50 lm (lower panel). (B) Phenotype of RBP-JΚΔ/Δ and Notch 2Δ/Δ mice (upper panel), and of enucleated eyes (lower panel), demonstrating reduced eye size (Control, n = 18 eyes and mutant, n = 24 eyes). (C) Eye size (left panel) and in vivo intra-ocular pressure (right panel) in control and mutant mice at 4 weeks of age (n = 12 eyes for each group). (D) Left panel shows enucleated eyes of a 5-monthold control and mutant mouse. Eye size measurement is shown in the right panel (n = 12 eyes for each group). (E) H & E staining of sections from 5-month-old RBP-JΚΔ/Δ and control mice. Higher magnification image of RBP-JΚΔ/Δ is shown beside it. Scale bar: 350 lm. All results are mean
SEM. *P < 0.05, ***P < 0.001 (student’s t-test).

reduced IOP when measured in vivo using a rebound tonometer (Figure 1C). These phenotypic abnormalities are consistent with the role of the CB in secreting AH and thus maintaining normal IOP and eye shape. At 5 weeks of age, RBP-JjΔ/Δ mice retained visual function and exhibited normal vascular structure (Figure S5). However, the long-term consequence of CB hypoplasia was progressive hypotony, resulting in shrinkage and disorganization of the eye (Figure 1D, E). Interestingly, a similar pathology, known as phthisis bulbi, is observed in humans. It is the end-stage consequence of prolonged and abnormally low IOP, leading to the shrinkage and disorganization of the eye globe. In humans, there are several causes for ocular hypotony, including increased outflow of AH or CB dysfunction or detachment (Coleman, 1995; Pederson, 2006). Thus, our Notch loss-offunction mouse model could serve as a valuable tool to study progressive ocular hypotony. Notch gain-of-function in ocular pigment cells results in aniridia and ciliary body hyperplasia The data obtained from the Notch loss-of-function experiments demonstrate that canonical Notch signaling is essential for CB morphogenesis but is dispensable for iris development. Similar results have been reported by a recent study, which also demonstrated that conditional ablation of Notch2 in the pigmented epithelium resulted in CB aplasia (Zhou et al., 2013). It was also shown that Notch signaling is induced in the presumptive CB by Jagged-1 expression in the non-pigmented, inner ciliary epithelium, which is an extension of the neural retina. Interaction between pigmented and non-pigmented cells is restricted to the CB, and is not evident in the iris. These findings therefore suggest that the patterning of the anterior pigmented epithelium into CB and iris occurs as a consequence of differential Notch activity imparted by Jag1-expressing non-pigmented epithelial cells. We therefore speculated that enforced Notch signaling throughout the anterior pigmented epithelium would result in CB morphogenesis at the expense of iris formation. To test this hypothesis, conditional Notch gain-of-function studies were performed. Mart-1::Cre ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Notch and ocular pigmented epithelium

transgenic mice were crossed to R26.NICDlox/lox mice (Murtaugh et al., 2003). The R26.NICDlox/lox model is a gene-targeted mouse in which a cDNA coding for a dominant active form of the Notch1 receptor (NICD1) is preceded by a floxed STOP cassette, followed by an IRES-eGFP cDNA, and inserted into the ubiquitously expressed Rosa26 gene locus (Murtaugh et al., 2003). R26.NICD1lox/+Mart1::Cre (hereafter RosaNICΔ/+) developed buphthalmic eyes (Figure 2A) by the age of 3 weeks characterized by a significant increase in eye ball size (Figure 2B) as well as in the IOP (Figure 2C) compared to littermate controls lacking the MART-1::Cre transgene. Interestingly, histological analysis of the anterior pigmented epithelium of RosaNICΔ/+ eyes revealed CB hyperplasia and aniridia (Figure 2D), thus presenting the reciprocal phenotype to that observed upon Notch signaling ablation. This confirmed that Notch signaling promoted formation of the CB at the expense of iris formation. The presence of a hyperplastic CB leads to the closure of the angle of drainage, thereby obstructing outflow of the AH. Currently, we cannot distinguish whether the hyperplastic CB produces AH compared to control animals. But, even if the production rate of AH would be comparable to control animals, the fact that AH can no longer drain due to the large increase in CB mass, obstructing the TM, explains the increase in IOP as well as the buphthalmic eye (Figure S6). Given the hyperplastic nature of the CB in RosaNICΔ/+ mutants, we analyzed proliferation in the anterior pigmented epithelium by Ki67 immunostaining. This revealed a profound increase in actively cycling pigmented epithelial cells (MITF+) in the CB (Figure 2E), indicating that the hyperplastic CB results at least in part from hyperproliferation. This is consistent with the growth promoting and oncogenic functions of excessive Notch signaling reported in other tissues (Koch and Radtke, 2010), and is in accordance with the reduced proliferation observed in Notch2 null CB (Zhou et al., 2013). Proliferation was also analyzed during development, when patterning and specification of the CB and iris is occurring. In control animals, distinct structures resembling the CB and iris were readily apparent at P1, P4, and

P14 (Figure 3A). In addition, a small but consistent proportion of pigmented epithelial cells in both structures were proliferating at these stages (Figure 3A, B). However, in RosaNICΔ/+ mutants, only enlarged, hyperprolifA

B

C

D

E

Figure 2. Notch gain-of-function in ocular pigment cells results in buphthalmic eye and glaucoma-like disease. (A) In situ (upper panel) and enucleated (lower panel) buphthalmic eyes are shown. (Control, n = 30 eyes and RosaNICΔ/+, n = 40 eyes). (B) Size measurement of enucleated eyes in control and RosaNICΔ/+ mice (n = 12 eyes for each group). (C) In vivo measurement of intra-ocular pressure in RosaNICΔ/+ and control mice (n = 12 eyes for each group). (D) H & E staining of whole eye sections (upper panel) and ciliary body (lower panel) in control and RosaNICΔ/+ mice. Scale bar: 350 lm (upper panel) and 50 lm (lower panel). (E) MITF (red) and Ki67 (green) staining of ciliary body in control and RosaNICΔ/+ mice. Inset is magnified view of selection. Nuclei were counterstained with DAPI (blue). Scale bar: 20 lm. All results are mean
SEM. ***P < 0.001 (student’s t test).

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

583

Sarode et al.

A

B

erative CB structures were present, and the iris was completely absent (Figure 3A, B). Interestingly, the RPE in RosaNICΔ/+ mutants did not exhibit increased proliferation during development or in the adult eye (Figure S7), despite the activity of the Mart::Cre transgene within these cells. This is in contrast to previous findings in which expression of NICD from the Tyrp1 promoter resulted in hyperproliferation of the RPE (Schouwey et al., 2011). These differences may reflect variations in the level of expression of NICD, as the Rosa promoter used to drive NICD expression in our models is relatively weak. It is therefore possible that the pigmented epithelial cells in the CB/iris are more sensitive to the effects of Notch signaling than those in the RPE. To establish whether the effects of Notch overexpression were RBP-Jj dependent, and were thus a consequence of canonical Notch signaling, we generated RBP-Jjlox/lox:RosaNICDlox/+:Mart1::Cre compound mutants (herein referred to as RBP-JjΔ/Δ:RosaNICΔ/+) and analyzed the ocular phenotype at 3 weeks of age. Strikingly, RBP-JjΔ/Δ:RosaNICΔ/+ mice exhibited CB aplasia and reduced eye size and IOP (Figure S8), recapitulating the phenotype observed in RBP-JjΔ/Δ single mutants. These data confirm that the effects elicited by NICD over-expression are RBP-Jj dependent and thus demonstrate that the reciprocal phenotypes observed in Notch loss- and gain-of-function mutants reflect aberrations in canonical Notch signaling. The above findings suggest that Notch signaling in pigmented epithelial cells induces CB morphogenesis via the induction of proliferation, resulting in the folded 584

Figure 3. Notch gain-of-function results in hyperproliferation of ciliary body. (A) H & E and Ki67 (Red) staining of control and RosaNICΔ/+ eyes at P1, P4, and P14. Dotted lines demarcate CB. Nuclei were counterstained with DAPI (blue). Scale bar: 20 lm. (B) Quantification of Ki67 positive cells in (A). Proportion of Ki67-positive cells in pigmented epithelium of control and RosaNICΔ/+ CB. PE, pigmented epithelium. CB, ciliary body. (n = 8 eyes for P1 and P4; n = 7 for P14; n = 10 for 1M). All results are mean
SEM. *P < 0.05, ***P < 0.001 (student’s t-test).

structures typical of the CB. In addition, we propose that Notch-induced proliferation throughout the pigmented epithelium promotes ectopic folding and thus prevents formation of the iris. Alternatively, Notch may promote CB fate in bipotent progenitors, and in this way regulate binary cell fate decisions as demonstrated in other tissues. At present, we have been unable to identify markers that are differentially expressed between the CB and iris (Figure S9) and thus cannot formally exclude the latter hypothesis. However, it is clear that a key function of Notch signaling during development of the anterior pigmented epithelium is to promote proliferation. The mechanism by which Notch signaling induces cell cycle progression is unclear. Reduced phospho-Smad activity has been reported in the Notch2-deficient CB, suggesting that Notch may induce proliferation by promoting BMP signaling (Zhou et al., 2013). Consistent with this, inhibition of ocular BMP signaling results in perturbed CB development, recapitulating aspects of the phenotype in Notch2 mutants (Chang et al., 2001; Zhao et al., 2002). However, in our investigations, Notch signaling does not appear to influence phospho-Smad expression, which is evident throughout the CB and iris in both WT and Notch mutant mice (Figure S9). This therefore suggests that Notch signaling promotes proliferation via mechanisms in addition to regulation of BMP signaling. RosaNICΔ/+ mice develop closed angle glaucoma-like disease The blockage of the aqueous outflow structures in RosaNICΔ/+ mice resembles the human eye disease ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Notch and ocular pigmented epithelium

1980), but in general, it is only infrequently observed in human glaucoma (Satofuka et al., 2008). Furthermore, we also observed high levels of apoptosis in the photoreceptors of RosaNICΔ/+ retinas (Figure S10), consistent with previous reports which demonstrate that loss of these cells is a consequence of retinal detachment (Arroyo et al., 2005; Cook et al., 1995). Together these results are consistent with RosaNICΔ/+ mice developing several features of a glaucoma-like disease.

known as closed angle glaucoma, which represents approximately one-third of all glaucoma cases and is often associated with increased IOP (Quigley, 2011). In the RosaNICΔ/+ mice, the outflow tract is blocked by the hyperplastic CB, whereas in human disease, blockage results from adherence of the iris to the TM. Nevertheless, the consequences of developing increased IOP are the same. Although chronically increased intra-ocular pressure is one of the major risk factors for developing glaucoma, it is not sufficient to develop the disease. An additional histological hallmark of glaucoma is the loss of a substantial number of retinal ganglion cells in the inner retina and loss of their axons in the optic nerve (Quigley et al., 1981; Ruberte et al., 2004). Interestingly, RosaNICΔ/+ mice fulfill this criterion and show reduced numbers of retinal ganglion cells (Figure 4A). Moreover, all of the RosaNICΔ/+ mice analyzed showed exudative retinal detachment (Figure 4A), which has been reported for congenital glaucoma cases (Cooling et al.,

RosaNICΔ/+ mice exhibit impaired visual function and vascularization The reduction in retinal ganglion cells and the retinal detachment observed in RosaNICΔ/+ mice prompted us to perform functional in vivo analysis of the retina and its associated vasculature. Scoptic (rods) and photopic (cones) electroretinographies (ERGs) were recorded from 5-week-old control and RosaNICΔ/+ mice. While the rod

A

B

C

E

D

F

G

Figure 4. ROSANICΔ/+ mice develop glaucoma-like disease. (A) H & E staining of eye sections from 6-month-old control and RosaNICΔ/+ mice. Higher magnification of selected areas is shown in lower panels. More than 30 lm separation of retinal ganglionic cells in RosaNICΔ/+ mice is shown by arrows (lower panels). Scale bar: 350 lm (upper panel) and 50 lm (lower panel). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; R & C, rode & cone; RPE, retinal pigment epithelium. All results are mean
SEM. ***P < 0.001 (student’s t-test). (B) Representative in vivo scotopic (rod) and photopic (cone) ERG recording of control and RosaNICD/+ mice. Near complete absence of rod and cone activity in RosaNICD/+ eyes indicates affected visual function (n = 8 eyes for each group). (C) Representative in vivo evaluation of control and RosaNICD/+ retina with SD-OCT. Circled part indicates optic nerve. Retinal folding and damage is clearly distinct in RosaNICD/+ eyes (n = 8 eyes for each group). (D) Representative in vivo fluorescein angiography images of control and RosaNICD/+ eyes. Note that retinal vasculature is collapsed in RosaNICD/+ eye in comparison with control (n = 8 eyes for each group). (E) Enucleated eyes from non-treated control, vehicle-treated RosaNICΔ/+, and Azarga-treated RosaNICΔ/+ mice (5-week treatment starting at 3 weeks of age). (F) Eye size measurement of non-treated control, vehicletreated RosaNICΔ/+, and Azarga-treated RosaNICΔ/+ mice. (G) In vivo intra-ocular pressure measurements of non-treated control, vehicle-treated RosaNICΔ/+, and Azarga-treated RosaNICΔ/+ mice. All results are mean
SEM. *P < 0.05. ***P < 0.001 (student’s t-test; n = 8 eyes for nontreated control and n = 14 eyes each for vehicle-treated RosaNICΔ/+ and Azarga-treated RosaNICΔ/+ mice). LOF: loss of function, GOF: gain of function.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

585

Sarode et al.

and cone activity of control animals was normal, RosaNICΔ/+ mice exhibited a clearly diminished ERG response for both rods and cones, indicating that their function is severely impaired (Figure 4B). The detachment of the retina in RosaNICΔ/+ mice was further validated through in vivo imaging using spectral domain optical coherence tomography (SD-OCT; Figure 4C). The optic nerve contains the axons running to the brain as well as the incoming blood vessels that open into the retina to vascularize and nourish retinal layers. As a retinal detachment would also impact on the vasculature, RosaNICΔ/+and control animals were analyzed by in vivo fluorescein angiography. Control mice showed a normal structure of venules and arterioles along with the optic nerve head surrounded by retinal vasculature. In contrast, the retinal vasculature of RosaNICΔ/+ mice was collapsed, and the structure of venules and arterioles was severely disturbed (Figure 4D). Together these results demonstrate that RosaNICΔ/+ mice exhibit a severe impairment of visual function. Pharmacological treatment of ocular hypertension in RosaNICΔ/+ mice Currently, glaucoma cannot be cured and the standard of care is treating patients with eye drops containing either a single drug or a combination of several drugs such as prostaglandin analogs, b-blockers, adrenergic agonists, or carbonic anhydrase inhibitors to lower the intra-ocular pressure (Quigley, 2011). Other forms of treatment include laser-driven surgery to allow drainage of the AH. As RosaNICΔ/+ mice develop ocular hypertension with several glaucoma-like features, we investigated their responsiveness to glaucoma hypotensive agents currently used in the clinics. We used Azarga (Alcon) eye drops, which contain two active components, timolol and brinzolamide. Timolol is a b-blocker and brinzoamide is a carbonic anhydrase inhibitor, both of which function to reduce AH production and/or facilitate its drainage through the outflow tract. Three-week-old RosaNICΔ/+ mice were treated daily on both eyes for 5 weeks with vehicle or Azgara eye drops. Control animals were treated with vehicle only. Subsequent analysis showed that Azarga-treated RosaNICΔ/+ mice exhibited a significantly reduced eye globe diameter and IOP compared to vehicle-treated Notch mutant mice (Figure 4E–G). These results suggest that our RosaNICΔ/+ mouse model might be a valuable tool for screening new hypotensive drugs. Other mouse models that develop increased IOP or glaucoma-like phenotypes have been reported. However, these mouse models often exhibit longer latencies and lower penetrance for disease development (Libby et al., 2005; Sarfarazi and Stoilov, 2000). In conclusion, our results demonstrate that de-regulation of Notch signaling specifically in the pigmented epithelium of the anterior eye segment induces both ocular hypo- and hypertension. The mouse models described here therefore represent useful tools to study 586

aspects of human pathology such as phthisis bulbi and closed angle glaucoma. In addition, they demonstrate that the function of Notch signaling in the pigmentary system of the eye and the skin differs. While in the skin, Notch signaling is required for melanocyte development (AubinHouzelstein et al., 2008; Kumano et al., 2008; Moriyama et al., 2006; Schouwey et al., 2007), in the eye, it regulates the patterning of the anterior pigmented epithelium by promoting CB development via the induction of proliferation.

Methods Mice MART1::Cre (Aydin and Beermann, 2011) transgenic mice carrying floxed alleles of R26.NICD, Notch1, Notch2, or RBP-Jk were used. All mice used for breeding and analyses were pigmented (Tyr+), nonagouti (a), and black (Tyrp1+). Moreover, these mice were equally tested for the absence of the retinal degeneration mutation Pde6brd1 (Gimenez and Montoliu, 2001) and Crb1rd8(Mattapallil et al., 2012) mutation as well. After breeding, the pups were weaned at P21, and ear biopsies were taken for genotype analysis. To amplify the floxed (500 bp) and the wild-type (450 bp) alleles of Notch1, we used the primers 50 -CTGAGGCCTAGAGCCTTGAA-30 and 50 -TGTGGGACC CA GAAGTTAGG-30 . Primers 50 -GTGAGATGTGACACTTCTGAGC-30 and 50 -GAGAAGCAGAGATGAGCAGATG-30 amplified the floxed (296 bp) and wild-type (236 bp) alleles of Notch2, while the floxed (880 bp) and wild-type (720 bp) alleles of RBP-Jj were detected using the primers 50 -CTTGATAATTCTGTAAAGAGA-30 and 50 -ACATTGCATTTT CACATAAAAAAGC-30 . To identify R26.NICD mice, the wild-type (600 bp) and R26 (200 bp) fragments were identified by PCR using 50 -AAAGT CGCTCTGAGTTGTTAT-30 , 50 -GCGAAGAGTTTGTCCTCAACC-30 , and 50 -GGAGCGGGAGAAATGGATATG-30 . The MART-1::Cre transgene (339 bp fragment) was detected using primers 50 -CTAGAGCCTGTTTTGCACGTTC-30 and 50 - GTTCGC AAGAACCTGATGGACA-30 . The mouse colonies were maintained in the animal facility of the EPFL. All mouse work has been performed under authorization of cantonal authorities and was conducted according to Swiss guidelines.

Immunohistochemistry Eyes were fixed in 4% paraformaldehyde for 1 h on ice before embedding in paraffin and sectioning (4 lm). After rehydration of the sections through xylol and ethanol, antigen retrieval was performed in 10 mM Tris-EDTA (pH 9) for 20 min at 95°C. After washing in phosphate-buffered saline (PBS), the sections were incubated for 30 min in a blocking solution (BSA 2%, Tween 0.05%, goat serum 1%, in PBS) at RT, followed by overnight incubation with primary antibody at 4°C. Tissue sections were then treated with the secondary antibody conjugated to a fluorescent dye (45 min, 37°C), and counterstained on slides with DAPI (40 -6Diamidino-2-phenylindole, 1:4000) for 5 min at RT. Slides were mounted in DABCO. Paraffin sections and antibody staining were performed according to the standard procedures. Primary antibodies and dilutions were as follows: rabbit anti-GFP (Life Technologies, Lucern, Switzerland, #A11122; 1:100), rabbit anti-Mitf (Sigma, Buchs, Switzerland, #HPA003259; 1:1000), mouse anti-Ki67 (1:500), mouse anti-RPE65 (Santa Cruz, TX, USA, #sc-53489; 1:1000), rabbit anti-Notch 2 (Abcam, Lucern, Switzerland, #ab118824; 1:500 & #ab 8926; 1:400), rabbit anti-pSmad1/5/8 (Cell

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Notch and ocular pigmented epithelium Signaling, Allschwil, Switzerland, #ab 9511; 1:100), rabbit antiClaudin1 (Fisher Scientific, Basel, Switzerland #ab RB9209-P0), mouse anti-Pax6 (Developmental Studies Hybridoma Bank, IA, USA, 1:500), and Rabbit anti-Dct (provided by V. Hearing, Bethesda, USA, PEP8; 1:1000). Alexa Fluor conjugated secondary antibodies (Molecular Probes, Lucern Switzerland) and dilutions were as follows: goat anti-rat IgG Alexa 488 (1:1000), donkey antirabbit IgG Alexa 568 (1:1000), goat anti-mouse IgG Alexa 488 (1:1000). For IHC, biotinylated donkey anti-rabbit (1:500) was used and revelation was performed with fast red. Mayer’s hematoxyline was used for nuclear staining.

Electroretinography Electroretinographic recordings were performed as described recently (Zencak et al., 2013). For scotopic conditions, mice were dark-adapted overnight. Subsequent procedures were undertaken under dim red light. Mice were anaesthetized with a mixture of ketamine (100 mg/kg) and xylazine (15 mg/kg), and pupils were dilated by topical administration of 0.5% tropicamid (Novartis Pharma, Bern, Switzerland). Mice were then positioned on a heated platform to prevent a drop in body temperature. A reference and a ground needle electrode were inserted below the skin of the scalp and the back of the mouse. Active ring electrodes were put in contact with the surface of the cornea. The cornea was covered with a carboxymethylcellulose sodium solution (Celluvisc, Allergan, Irvine, CA, United States) to prevent drying of the cornea and to ensure maximal contact with the electrodes. The mouse was placed in the center of a Ganzfeld stimulator from a Espion E3 apparatus (Diagnosys llc, Lowell, MA, USA). The corneal ERG was then recorded in scotopic conditions in response to single flashes of a mix of green (520 nm; half-bandwidth 35 nm) and UV (365 nm, halfbandwidth 9 nm) light of the following power: 0.00002952, 0.0002952, 0.002952, 0.008856, 0.02952, 0.08856, 0.08856, 0.2952, 0.8856, 2.952, and 8.856 lW, generated by a stroboscopic light located in the upper part of the Ganzfeld stimulator. At the end of the examination, the eyes were covered with Viscotears (Novartis Pharma, Bern, Switzerland), and the mice were kept under surveillance until they regained consciousness.

Spectral domain – optical coherence tomography Spectral domain optical coherence tomography was performed as described recently (Pennesi et al., 2012). For SD-OCT imaging, mice were anesthetized and pupils were dilated by topical administration of 0.5% tropicamid (Novartis Pharma, Bern, Switzerland). The cornea was covered with a carboxymethylcellulose sodium solution (Celluvisc, Allergan, Irvine, CA, USA) to prevent drying of the cornea. Spectral domain optical coherence tomography images were obtained using the Bioptigen spectral domain ophthalmic imaging system (SDOIS Envisu R2200-HR SD-OCT device Bioptigen, Durham, NC, USA) with the reference arm placed at approximately 1185 mm. Horizontal and vertical linear scans through the optic nerve, and annular scans around the optic nerve were obtained. After the examination, anesthesia was reverted by injection of Antisedan (1 mg/kg). Mice were kept under surveillance on a heating pad until they regained consciousness, and the eyes were covered with Viscotears (Novartis Pharma, Bern, Switzerland).

Fluorescein angiography Fluorescein angiography was performed as previously described (Marcelli et al., 2012). Mice were anaesthetized with ‘ON’ phase anesthetics at 10 ll/g of body weight (12%, 50 mg/ml ketamine and 10%, 1 mg/ml medetomidine), and pupils were dilated by topical administration of 0.5% tropicamid (Novartis Pharma, Bern, Switzerland). Eyes were kept moist by adding a drop of 0.3% hypromellose

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

gel (GenTeal Gel, Novartis, Bern, Switzerland) for subsequent in vivo analysis. Anesthetized mice were injected intraperitoneally with 12.5% sodium fluorescein solution (HUB Pharmaceuticals) at the dose of 10 ll per 5–6 g of body weight. Retinal angiography was performed using a retinal imaging microscope for small animals containing a built in exciter and barrier filter for fluorescein (Micron III, Phoenix Research Laboratories, CA, USA). Retinal vessels start filling about 20 s after fluorescein administration, and imaging should be completed 2 min later. Once the imaging is done, anesthesia was reversed with intraperitoneal injection of the antidote for the ‘OFF’ phase at a concentration of 10 ll/g of body weight (5 mg/ml Atipamezole, Antisedan, Pfizer, Zurich, Switzerland). Mice were kept under surveillance on heating pad until they regained consciousness.

Intra-ocular pressure measurement Intra-ocular pressure was measured using TonoLab rebound tonometer for rodents (iCARE, Helsinki, Finland) according to the manufacturer’s recommended procedures and as described earlier (Wang et al., 2005). Mice were gently restrained by first being placed in a transparent plastic cone then secured in a custom-made restrainer. Once mice are calm, IOP measurements were then performed using the rebound tonometer. Intra-ocular pressure measurement using the rebound tonometer appeared to be well tolerated by mice. The animals exhibited no signs of irritation or discomfort during or after the procedure.

Treatment of buphthalmic eyes Mart-1Cre::R26.NICD lox/+ (RosaNICΔ/+) mice manifest a big eye phenotype and were treated with Azarga (Alcon, Bosch, Switzerland) eye suspension to reduce intra-ocular pressure. Treatment was started at the age of 3 weeks and continued for the next 5 weeks. One drop of Azarga was added in each eye, every day in the morning. Intra-ocular pressure was measured at the end of the treatment, and mice were then sacrificed.

Morphological examination of mouse eyes To measure eye growth of Mart-1Cre::R26.NICD lox/+ and MART1:: Cre, RBP lox/lox mice with littermate control mice, the eye diameter was measured by determining the distance from the optic nerve head to the center of the cornea using an electronic digital caliper (5– 10 animals per group). Digital camera (18X, SZ-20, Olympus) was used to image enucleated eyes for further macroscopic evaluation.

Acknowledgements We thank Sylvain Crippa for his technical assistance for the ERG le Ferrand for technical recording, Jessica Sordet-Dessimoz and Gise assistance, and Sylvain Roy and Adan Villamarine for helpful discussion. This work was in part supported by the Swiss Cancer League, the Swiss National Science Foundation, and School of Life Science of EPFL.

Conflict of Interest The authors declare that they have no conflict of interest.

References Arroyo, J.G., Yang, L., Bula, D., and Chen, D.F. (2005). Photoreceptor apoptosis in human retinal detachment. Am. J. Ophthalmol. 139, 605–610.

587

Sarode et al. Aubin-Houzelstein, G., Djian-Zaouche, J., Bernex, F., Gadin, S., Delmas, V., Larue, L., and Panthier, J.J. (2008). Melanoblasts’ proper location and timed differentiation depend on Notch/RBP-J signaling in postnatal hair follicles. J. Invest. Dermatol. 128, 2686– 2695. Aydin, I.T., and Beermann, F. (2011). A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium. Genesis 49, 403–409. Beebe, D.C. (1986). Development of the ciliary body: a brief review. Trans. Ophthalmol. Soc. U. K. 105(Pt 2), 123–130. Besseyrias, V., Fiorini, E., Strobl, L.J., Zimber-Strobl, U., Dumortier, A., Koch, U., Arcangeli, M.L., Ezine, S., Macdonald, H.R., and Radtke, F. (2007). Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation. J. Exp. Med. 204, 331–343. Camacho-Hubner, A., and Beermann, F. (2001). Increased transgene expression by the mouse tyrosinase enhancer is restricted to neural crest-derived pigment cells. Genesis 29, 180–187. Chang, B., Smith, R.S., Peters, M. et al. (2001). Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure. BMC Genet. 2, 18. Coca-Prados, M., and Escribano, J. (2007). New perspectives in aqueous humor secretion and in glaucoma: the ciliary body as a multifunctional neuroendocrine gland. Prog. Retin. Eye. Res. 26, 239–262. Coleman, D.J. (1995). Evaluation of ciliary body detachment in hypotony. Retina 15, 312–318. Cook, B., Lewis, G.P., Fisher, S.K., and Adler, R. (1995). Apoptotic photoreceptor degeneration in experimental retinal detachment. Invest. Ophthalmol. Vis. Sci. 36, 990–996. Cooling, R.J., Rice, N.S., and Mcleod, D. (1980). Retinal detachment in congenital glaucoma. Br. J. Ophthalmol. 64, 417–421. Do, C.W., and Civan, M.M. (2009). Species variation in biology and physiology of the ciliary epithelium: similarities and differences. Exp. Eye Res. 88, 631–640. Fine, H.F., Biscette, O., Chang, S., and Schiff, W.M. (2007). Ocular hypotony: a review. Compr. Ophthalmol. Update 8, 29–37. Gimenez, E., and Montoliu, L. (2001). A simple polymerase chain reaction assay for genotypingthe retinal degeneration mutation (Pdeb (rd1)) in FVB/N-derived transgenic mice. Lab. Anim. 35, 153–156. Han, H., Tanigaki, K., Yamamoto, N., Kuroda, K., Yoshimoto, M., Nakahata, T., Ikuta, K., and Honjo, T. (2002). Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int. Immunol. 14, 637–645. Hanssen, E., Franc, S., and Garrone, R. (2001). Synthesis and structural organization of zonular fibers during development and aging. Matrix Biol. 20, 77–85. Koch, U., and Radtke, F. (2010). Notch signaling in solid tumors. Curr. Top. Dev. Biol. 92, 411–455. Koch, U., Lehal, R., and Radtke, F. (2013). Stem cells living with a Notch. Development 140, 689–704. Kumano, K., Masuda, S., Sata, M. et al. (2008). Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis. Pigment Cell Melanoma Res. 21, 70–78. Lee, H.Y., Wroblewski, E., Philips, G.T., Stair, C.N., Conley, K., Reedy, M., Mastick, G.S., and Brown, N.L. (2005). Multiple requirements for Hes 1 during early eye formation. Dev. Biol. 284, 464–478. Lee, H., Khan, R., and O’keefe, M. (2008). Aniridia: current pathology and management. Acta Ophthalmol. 86, 708–715. Libby, R.T., Anderson, M.G., Pang, I.H. et al. (2005). Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration. Vis. Neurosci. 22, 637–648.

588

Marcelli, F., Escher, P., and Schorderet, D.F. (2012). Exploration of the Visual System: Part 2. In Vivo Analysis Methods: Virtual-Reality Optomotor System, Fundus Examination, and Fluorescent Angiography. Curr. Protoc. Mouse Biol. 3, 207–218. Martinez-Morales, J.R., Rodrigo, I., and Bovolenta, P. (2004). Eye development: a view from the retina pigmented epithelium. BioEssays 26, 766–777. Mattapallil, M.J., Wawrousek, E.F., Chan, C.C., Zhao, H., Roychoudhury, J., Ferguson, T.A., and Caspi, R.R. (2012). The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Invest. Ophthalmol. Vis. Sci. 53, 2921– 2927. Moriyama, M., Osawa, M., Mak, S.S. et al. (2006). Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells. J. Cell Biol. 173, 333–339. Murisier, F., and Beermann, F. (2006). Genetics of pigment cells: lessons from the tyrosinase gene family. Histol. Histopathol. 21, 567–578. Murisier, F., Guichard, S., and Beermann, F. (2007). Distinct distal regulatory elements control tyrosinase expression in melanocytes and the retinal pigment epithelium. Dev. Biol. 303, 838–847. Murtaugh, L.C., Stanger, B.Z., Kwan, K.M., and Melton, D.A. (2003). Notch signaling controls multiple steps of pancreatic differentiation. Proc. Natl Acad. Sci. U. S. A. 100, 14920–14925. Papayannopoulos, V., Tomlinson, A., Panin, V.M., Rauskolb, C., and Irvine, K.D. (1998). Dorsal-ventral signaling in the Drosophila eye. Science 281, 2031–2034. Tasman, W. and Jaeger, E.A., eds. (2006). Duane’s Opthalmology on CD- ROM, edition. (Philadelphia PA: Lippincott Williams & Wilkins) Clinicial Opthalmology Volume 3 (Glaucoma), Chapter 58. Pennesi, M.E., Michaels, K.V., Magee, S.S. et al. (2012). Long-term characterization of retinal degeneration in rd1 and rd10 mice using spectral domain optical coherence tomography. Invest. Ophthalmol. Vis. Sci. 53, 4644–4656. Quigley, H.A. (2011). GlaucomaLancet 377, 1367–1377. Quigley, H.A., Addicks, E.M., Green, W.R., and Maumenee, A.E. (1981). Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch. Ophthalmol. 99, 635–649. Rowan, S., Conley, K.W., Le, T.T., Donner, A.L., Maas, R.L., and Brown, N.L. (2008). Notch signaling regulates growth and differentiation in the mammalian lens. Dev. Biol. 321, 111–122. Ruberte, J., Ayuso, E., Navarro, M. et al. (2004). Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease. J. Clin. Invest. 113, 1149–1157. Sarfarazi, M., and Stoilov, I. (2000). Molecular genetics of primary congenital glaucoma. Eye (Lond) 14(Pt 3B), 422–428. Satofuka, S., Imamura, Y., Ishida, S., Ozawa, Y., Tsubota, K., and Inoue, M. (2008). Rhegmatogenous retinal detachment associated with primary congenital glaucoma. Int. Ophthalmol. 28, 369–371. Schouwey, K., and Beermann, F. (2008). The Notch pathway: hair graying and pigment cell homeostasis. Histol. Histopathol. 23, 609–619. Schouwey, K., Delmas, V., Larue, L., Zimber-Strobl, U., Strobl, L.J., Radtke, F., and Beermann, F. (2007). Notch1 and Notch2 receptors influence progressive hair graying in a dose-dependent manner. Dev. Dyn. 236, 282–289. Schouwey, K., Aydin, I.T., Radtke, F., and Beermann, F. (2011). RBP-Jj-dependent Notch signaling enhances retinal pigment epithelial cell proliferation in transgenic mice. Oncogene 30, 313–322. Singh, A., Tare, M., Puli, O.R., and Kango-Singh, M. (2012). A glimpse into dorso-ventral patterning of the Drosophila eye. Dev. Dyn. 241, 69–84.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Notch and ocular pigmented epithelium Wang, W.H., Millar, J.C., Pang, I.H., Wax, M.B., and Clark, A.F. (2005). Noninvasive measurement of rodent intraocular pressure with a rebound tonometer. Invest. Ophthalmol. Vis. Sci. 46, 4617–4621. Yu, E.N., Paredes, I., and Foster, C.S. (2007). Surgery for hypotony in patients with juvenile idiopathic arthritis-associated uveitis. Ocul. Immunol. Inflamm. 15, 11–17. Zencak, D., Schouwey, K., Chen, D., Ekstrom, P., Tanger, E., Bremner, R., Van Lohuizen, M., and Arsenijevic, Y. (2013). Retinal degeneration depends on Bmi1 function and reactivation of cell cycle proteins. Proc. Natl Acad Sci. U. S. A. 110,, E593–601. Zhao, S., Chen, Q., Hung, F.C., and Overbeek, P.A. (2002). BMP signaling is required for development of the ciliary body. Development 129, 4435–4442. Zheng, M.H., Shi, M., Pei, Z., Gao, F., Han, H., and Ding, Y.Q. (2009). The transcription factor RBP-J is essential for retinal cell differentiation and lamination. Mol. Brain 2, 38. Zhou, Y., Tanzie, C., Yan, Z. et al. (2013). Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye. Proc. Natl Acad. Sci. U. S. A. 110, 8966–8971.

Supporting information Additional Supporting Information may be found in the online version of this article: Figure S1. The MART-1::Cre transgene induces recombination in ciliary body, iris, and RPE, and does not cause

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

ocular abnormalities. Figure S2. Normal RPE structure in 5-week-old RBPJjΔ/Δ mice. Figure S3. Normal ciliary body in Notch 1Δ/Δ mice. Figure S4. Notch 2 expression in human and mouse ciliary body. Notch 2 expression (pink) is shown by immunohistochemistry in mouse and human ciliary body. Figure S5. Normal retinal vasculature and function in 5week-old RBP-JjΔ/Δ mice. Figure S6. Mechanistic explanation for small and big eye phenotype. Figure S7. Normal proliferation in the RPE of RosaNICD/+ mice. Figure S8. CB hyperplasia in RosaNICD/+ mutants is RBP-JK dependent. Figure S9. Shared marker expression in the CB and iris. Figure S10. Apoptosis of photoreceptors in the detached retinas of RosaNICd/+ mice. Movie S1. Intact retinal structure and morphology in control mice through in vivo imaging using spectral domain optical coherence tomography (SD-OCT). Movie S2. Detached and damaged retina in RosaNICΔ/ + mice through in vivo imaging using SD-OCT.

589