Development 127, 3581-3591 (2000) 3581 Printed in Great Britain © The Company of Biologists Limited 2000 DEV4340

Signaling and transcriptional regulation in early mammalian eye development: a link between FGF and MITF

Minh-Thanh T. Nguyen and Heinz Arnheiter* Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA *Author for correspondence (e-mail: [email protected])

Accepted 27 May; published on WWW 20 July 2000

SUMMARY

During vertebrate eye development, the optic vesicle is Conversely, the removal of the surface ectoderm results in partitioned into a domain at its distal tip that will give rise the maintenance of MITF in the distal optic epithelium, to the neuroretina, and another at its proximal base that lack of expression of the neuroretinal-specific CHX10 will give rise to the pigmented epithelium. Both domains transcription factor, and conversion of this epithelium are initially bipotential, each capable of giving rise to either into a pigmented monolayer. This phenomenon can be neuroretina or pigmented epithelium. The partitioning prevented by the application of fibroblast growth factor depends on extrinsic signals, notably fibroblast growth alone. In Mitf mutant embryos, parts of the future factors, which emanate from the overlying surface pigment epithelium become thickened, lose expression of ectoderm and induce the adjacent neuroepithelium to a number of pigment epithelium transcription factors, assume the neuroretinal fate. Using explant cultures of gain expression of neuroretinal transcription factors, and mouse optic vesicles, we demonstrate that bipotentiality eventually transdifferentiate into a laminated second of the optic neuroepithelium is associated with the retina. The results support the view that the bipotential initial coexpression of the basic-helix-loop-helix-zipper optic neuroepithelium is characterized by overlapping gene transcription factor MITF, which is later needed solely expression patterns and that selective gene repression, in the pigmented epithelium, and a set of distinct brought about by local extrinsic signals, leads to the transcription factors that become restricted to the separation into discrete expression domains and, hence, to neuroretina. Implantation of fibroblast growth factor- domain specification. coated beads close to the base of the optic vesicle leads to a rapid downregulation of MITF and the development of Key words: Retinal pigment epithelium, Neuroretina, an epithelium that, by morphology, gene expression, and Neuroepithelium, Optic vesicle culture, Transcription factor, lack of pigmentation, resembles the future neuroretina. Transcriptional repression, Mouse, Cell-fate specification

INTRODUCTION Here we focus on the factors that specify the neuroretina (NR) and the retinal pigment epithelium (RPE). These two The development of the vertebrate eye, much as that of many tissues, though distinct in form and function, have a common other organs, is the result of complex, reciprocal interactions precursor, the cells of the anterior neuroepithelium that become between tissues of distinct embryological origin. The lens, for specified in an eye field. Normally, the epithelium forms an NR instance, is derived from the surface ectoderm that overlies the in the distal (inner) layer of the eye cup and an RPE in the optic vesicle, but without the juxtaposition of a normal optic proximal (outer) layer. However, there is a species-specific vesicle lens development is perturbed (Spemann, 1901; Lewis, period during which the fates of these two tissues remain 1904; Li et al., 1994; Porter et al., 1997; Furuta and Hogan, reversible. For instance, when in amphibians the eye is 1998; Kamachi et al., 1998). Conversely, without a proper lens experimentally inverted so that the outer layer comes to lie placode the optic vesicle remains rudimentary, and an optic cup adjacent to the lens primordium, this outer layer may develop with a well-defined neuroretinal layer and a pigmented layer into a complete retina rather than an RPE (Mikami, 1939). does not form (Holtfreter, 1939; Hyer et al., 1998). Even These and many other observations (Pittack et al., 1997; Hyer though these tissue interactions have long been noted and et al., 1998) suggest that the neuroepithelium of the optic characterized extensively, it remains a challenge to sort out the vesicle is initially bipotential and that its separation into NR molecular mechanisms responsible for them. In particular, and RPE domains is mediated by extrinsic factors that emanate much needs to be learned about the nature of the relevant from the surface ectoderm. signaling molecules and how the cells interpret them to develop Among the factors potentially responsible for this effect, into the eye’s many distinct structures. fibroblast growth factors (FGFs) are prime candidates. Both 3582 M.-T. T. Nguyen and H. Arnheiter

FGF1 and FGF2 are expressed in the surface ectoderm (de regulating the expression of Mitf. The results suggest a model Longh and McAvoy, 1993; Pittack et al., 1997) and their in which the initial coexpression of transcription factors marks receptors, FGFR1 and FGFR2, are expressed in the developing undifferentiated, bipotential neuroepithelial precursor cells, optic vesicle (Wanaka et al., 1991; Tcheng et al., 1994). RPE, and the region-specific repression of distinct transcription when exposed to FGF2, develops into NR (Park and factors leads to lineage commitment. Hollenberg, 1989; Pittack et al., 1991, 1997; Guillemot and Cepko, 1992; Zhao et al., 1995), and FGF1 is involved in early optic vesicle patterning in the chick (Hyer et al., 1998). MATERIALS AND METHODS Furthermore, FGFs have well-established roles in patterning of other tissues in both invertebrates and vertebrates. Whether Mice and harvest of embryos FGFs play similar roles in the patterning of the mammalian C57BL/6J embryos were used as Mitf wild-type controls. Mitf mutant optic vesicle, however, remains unknown. Neither FGF1 or embryos were of the genotypes Mitfvga−9/vga9, Mitfmi-ew/mi-ew (eyeless- FGF2 knockout mice nor FGF1/FGF2 double knockout mice white) and MitfMi-wh/Mi-wh (white), whose molecular lesions and have overt eye defects (Dono et al., 1998; Ortega et al., 1998; genetic backgrounds have been described (Hodgkinson et al., 1993; Miller et al., 2000). Thus, in the mouse, FGFs may either have Hemesath et al., 1994; Steingrímsson et al., 1994; Nakayama et al., redundant functions or may collectively be unimportant for 1998). For timed pregnancies, the noon when a vaginal plug was found was defined as embryonic day 0.5 (E0.5). In embryos younger cell-fate decisions in the optic vesicle. than E10.5, somite numbers were counted for accurate staging. Much as determination of the nature of the relevant signaling molecules is crucial to our understanding of eye development, Optic vesicle explant culture and manipulations so is identification of the cell-intrinsic factors, including Embryos were harvested at E9-9.5 in Hepes-buffered DMEM particular transcription factors, through which these signaling supplemented with 10% fetal bovine serum (FBS). The head bands molecules may act. A multitude of transcription factors containing the optic vesicles were dissected and cultured in 10% FBS- specifically involved in the various developmental steps of eye supplemented UltraCulture medium (BioWhittaker) on floating 0.45 formation have recently been identified from spontaneous and µm Nucleopore membranes (Corning #110607) coated with 10 ng/ml poly-D-Lysine (Sigma). Heparin-coated acrylic beads (125-200 µm induced mutations, predominantly in mice. For instance, the µ homeodomain protein RX is needed for optic vesicle formation diameter, Sigma H5263) were soaked in 5 l of 1 mg/ml of human recombinant FGF1, FGF2, murine recombinant EGF (all from Gibco) (Mathers et al., 1997), the lim-homeodomain protein LHX2 or bovine serum albumin (BSA, fraction V, Sigma) for 1 hour (Porter et al., 1997) and the paired-domain protein PAX6 (Hill (Niswander et al., 1993) and implanted into specific locations around et al., 1991) are needed for optic cup formation, and the the optic vesicle, as indicated in the text. Removal of the surface homeodomain protein CHX10 controls the growth of the NR ectoderm was done as described (Furuta et al., 1998). For and the differentiation of retinal interneurons (Burmeister et immunolabeling, the cultured explants were fixed in 4% al., 1996). Based on analyses in dissociated cultures, several paraformaldehyde overnight, cryoprotected in sucrose, embedded in transcription factors, including MITF on which the present OCT compound and sectioned at −14°C. study focuses, have been implicated in cell-fate decisions in Tissue preparations for histological analysis and in situ cultures of dissociated optic cups (Mochii et al., 1998a; Holme hybridization et al., 2000), but none so far has been shown to regulate the Embryo preparations, immunolabeling and in situ hybridizations were separation into NR and RPE in the intact optic vesicle. done as described (Hodgkinson et al., 1993; Chan et al., 1995; We have recently demonstrated that the basic-helix- Nakayama et al., 1998). The following mouse probes, all described loop-helix-zipper (bHLH-Zip) transcription factor MITF previously, were used: Mitf (Hodgkinson et al., 1993), Dct (Opdecamp (Hodgkinson et al., 1993; Hughes et al., 1993), in addition et al., 1997), Chx10 (Liu et al., 1994), Crx (Furukawa et al., 1997), to regulating the development of neural crest-derived Six3 (Oliver et al., 1995), Pax6 (Walther and Gruss, 1991). The melanocytes, also controls the development of the RPE. Such templates for Otx2 and FGF15 were EST cDNA clones (accession a role is not surprising given Mitf’s prominent expression in numbers AA199520 and AA051675, respectively) obtained from the RPE (Hodgkinson et al., 1993; Nakayama et al., 1998). Genome Systems Inc. and confirmed by sequencing. In fact, when rendered non-functional in embryos with Antibodies and immunohistochemistry microphthalmia mutations, the RPE hyperproliferates, remains The following antibodies were used: for horizontal cells, NF160 unpigmented and displays areas developing into a second (Sigma); for amacrine cells, syntaxin, VC1.1 (Sigma); for bipolar retina (Packer, 1967; Scholtz and Chan, 1987; Mochii et al., cells, anti-Chx10 (R. McInnes); for rod outer segments K42-41c; for 1998b; Nakayama et al., 1998; Bumsted and Barnstable, 2000). rod outer and inner segments, R2-15 and R2-12 (Röhlich et al., 1989) Intriguingly, in the mouse, Mitf mRNA expression starts in the (P. Hargrave). Rabbit antiserum to chicken PAX6 (cross-reacting with entire optic vesicle and thus precedes the separation into mouse PAX6) was a gift from S. Saule. Monospecific Anti-FGF1 and presumptive NR and RPE; only later is its expression restricted anti-FGF2 rabbit sera were from R&D, and rabbit anti-calretinin to the presumptive RPE (Nguyen et al., 1997; Bora et al., 1998; serum was from Chemicon. Rabbit anti-DCT was a gift of V. Hearing. Nakayama et al., 1998). It is likely, therefore, that the All primary antibodies were used at 1:100 dilution in 10% normal progressive restriction of Mitf expression to the future RPE is goat serum. Secondary antibodies were appropriate FITC-conjugated instrumental for the separation of the mammalian optic vesicle affinity-purified goat immunoglobulins (Sigma). Sections were viewed using a Reichert-Jung Polyvar microscope equipped for into NR and RPE. epifluorescence and differential interference contrast and In the present paper we use wild-type and Mitf mutant mouse photographic images were processed digitally. embryos and explant cultures of optic vesicles to demonstrate that FGFs from the surface ectoderm partition the DiI labeling neuroepithelium into NR and RPE, and that they do so by For cell labeling in optic vesicle cultures, DiI crystals (D3911, Role of FGF and MITF in eye development 3583

Molecular Probes) were placed in specific locations as indicated in portions of E9.5 embryos and established explant cultures that the text. retained the morphology of the budding optic vesicles. Over a period of 3 days, each culture formed two optic cups, which became pigmented in their proximal parts (Fig. 3A,C,E). RESULTS Immunolabeling of cryosections of these cultures showed that the eye cups were composed of an MITF-negative lens vesicle Expression of MITF and FGF during early eye (lv, Fig. 3B) that later detached from the surface ectoderm to development form a lens (l, Fig. 3D,F), an MITF-positive layer reminiscent We first determined the expression patterns of MITF RNA and of RPE, and an MITF-negative layer reminiscent of NR (Fig. protein during early eye development. Fig. 1A shows that 3B,D,F). Pigmentation of the RPE started at 2 days and became initially, at the 22-somite stage, wild-type MITF RNA was prominent at 3 days. At 2 days, the NR layer became weakly expressed throughout the optic vesicle (OV). Shortly thereafter positive for calretinin, a neuronal marker (not shown). Staining (Fig. 1B, 26-somite stage), its levels were reduced in the distal for PAX6 (NR and RPE) and CHX10 (NR) (see Figs 4 and 5) optic vesicle (dOV) adjacent to the surface ectoderm (SE) confirmed previous in vivo observations (Li et al., 1994; Liu et but maintained in the proximal optic vesicle (pOV). This al., 1994). differential expression pattern was further accentuated at the cup stage (Fig. 1C) where the future NR did not express MITF while the future RPE did. Protein expression patterns faithfully tracked RNA expression (Fig. 1D-F). In optic vesicles of Mitf null embryos (Mitfvga−9/vga−9), no immunofluorescent label was detected, indicating that the antiserum was specific (Fig. 1G). In Mitfmi-ew/mi-ew embryos, which synthesize an MITF protein deficient in DNA binding, the shift from expression in the entire optic vesicle to the presumptive RPE was as in wild type, indicating that the mechanisms regulating MITF expression remained intact (Fig. 1H-I). Even though a cup was formed, however, it was misshapen, and the mutant protein, rather than being concentrated in the cell nuclei, filled both nuclei and cytoplasm as observed previously in the RPE at E14.5 (Nakayama et al., 1998). In addition, some areas in the dorsal part appeared thickened and displayed reduced levels of MITF (Fig. 1I, arrow, see below). We then labeled similar sections for FGF1 and FGF2 protein (Fig. 2A,B). The antibodies used in these studies recognize these proteins selectively and gave low level staining in the entire vesicle area. However, the surface ectoderm overlying the optic vesicle, though not the more dorsal and ventral surface ectoderm, was particularly intensely stained. Similar expression patterns have been seen for FGF2 in chick (Pittack et al., 1997) and FGF1 in rat (de Longh and McAvoy, 1993). In addition, in situ hybridizations (not shown) confirmed previous observations (McWhirter et al., 1997) that another member of the FGF family, FGF15, is expressed in the distal optic vesicle beginning at E9.5. Thus, several Fig. 1. Expression patterns of MITF in the mouse embryonic eye. (A-C) RNA FGFs are expressed in the surface ectoderm or the expression in wild-type embryos. (D-F) Protein expression in wild-type embryos distal optic vesicle and thus at the right time and and (G-I) protein expression in mutant embryos. (A,D,G,H) Early optic vesicle stage (22 pairs of somites). (B,E) Late optic vesicle stage (24-26 pairs of somites). place to potentially play a role in MITF regulation vga−9/vga−9 and optic vesicle patterning. (C,F,I) Optic cup stage (E10.5). (G) Mitf (Mitf null mutation). (H,I) Mitfmi-ew/mi-ew (coding region mutation). Note that MITF is initially expressed FGFs downregulate MITF in the throughout the optic vesicle but then is downregulated in the distal optic vesicle. Arrow in I marks beginning downregulation of mutant MITF in dorsal RPE. SE, presumptive RPE surface ectoderm; OV, optic vesicle; dOV, distal optic vesicle (presumptive To test the potential role of FGFs in neuroretina); pOV, proximal optic vesicle (presumptive RPE); lv, lens vesicle; NR, downregulating MITF, we dissected the head neuroretina; RPE, retinal pigment epithelium. Bar, 50 µm. 3584 M.-T. T. Nguyen and H. Arnheiter

vesicle and no optic cup were formed. Immunolabeling showed that the PAX6 positive surface ectoderm had been removed entirely without damage to the distal layer (Fig. 5F,K,P). The distal layer soon became covered with unlabeled mesenchymal cells from the surrounding tissue (see for instance Fig. 5G, arrow). More importantly, the distal layer, which over 2-3 days should have developed into the MITF-negative presumptive NR and should have stayed unpigmented, remained a single cell layer, retained expression of MITF (Fig. 5G-I), and starting at day 2 became pigmented (Fig. 5H,I). This layer remained positive for Fig. 2. FGF protein expression in the mouse embryonic optic vesicle area (wild PAX6 (Fig. 5K-N) but soon lost the initial staining for type, E9.5). (A) FGF1, (B) FGF-2. Note predominant expression in the surface CHX10 (compare Fig. 5P with Q-S). Thus, rather than ectoderm overlying the optic vesicle. Bar, 50 µm. developing into NR, the distal layer switched its fate to that of RPE. With this system in hand, we then implanted growth factor- coated acrylic beads immediately after establishing the cultures. The beads were placed close to the area where the outer MITF-positive layer would normally develop and were left in place until the cultures were harvested 1-3 days later. In all of 21 vesicles given beads coated with BSA and 21 of 22 vesicles given beads coated with EGF, development of a pigmented layer was not perturbed (a typical example is shown in Fig. 4A, arrow). With FGF1-coated beads, however, 16 of 18 bead-exposed vesicles showed no pigmentation, and with FGF2-coated beads, 25 of 29 remained unpigmented (a typical example is shown in Fig. 4B, arrow). To test whether the lack of pigmentation was correlated with a disturbance in gene expression, the cultures were harvested at various time points, cryosectioned, and the sections stained for MITF, PAX6 and CHX10 protein. As shown in Fig. 4C for a culture implanted with a control bead and harvested at 3 days, MITF staining was, as expected, restricted to the RPE layer, PAX6 staining (Fig. 4E) was seen in the surface ectoderm, lens vesicle, NR and RPE, and CHX10 staining was found only in the NR (Fig. 4G). In contrast, when the bead was coated with FGF1, there was no MITF staining and the RPE layer became thickened, resembling the NR layer in appearance and thickness (Fig. 4D). Both NR and RPE layer were positive for PAX6 (Fig. 4F) and CHX10 (Fig. 4H). These results suggest that in the presumptive RPE, FGFs downregulate MITF, upregulate CHX10, and lead to increased cell numbers, thus allowing the RPE to initiate differentiation along the neuroretinal pathway. Removal of the surface ectoderm leads to inversion of the optic vesicle If exposure to FGFs downregulates MITF expression, upregulates CHX10 expression, and eliminates pigmentation in the presumptive RPE, then the removal of the surface ectoderm, i.e. the removal of a physiological source of FGF along with any other signals emanating from this part of the Fig. 3. Development of pigmented eye structures in embryonic ectoderm, should have the opposite effects. We thus removed explant cultures. The head bands of wild-type embryos were the surface ectoderm by a combination of enzymatic and harvested at E9.5 and cultured for 1 day (A,B), 2 days (C,D) or 3 physical means from E9 (20-22 somite stage) wild-type days (E,F). (A,C,E) View in dissection scope; (B,D,F) crytostat sections immunostained for MITF protein (green). Arrows in A mark embryos. This time point marks a stage at which there is no lens placodes, and arrowheads in A,C and E the progressively direct contact between optic vesicle and surface ectoderm. The pigmented optic cup. Note similarities to the in vivo situation (Fig. 1) procedure left the optic vesicle intact (compare Fig. 5A with in morphology, MITF expression, and RPE pigmentation. SE, B) and allowed for pigmentation to develop over a 3-day period surface ectoderm; lv, lens vesicle; l, lens; NR neuroretina; RPE, (Fig. 5C-E, left side) but had the consequence that no lens retinal pigment epithelium. Bar, 50 µm (B,D,F). Role of FGF and MITF in eye development 3585

Interestingly, during this time period, the normally cell-free also acquired CHX10 expression (Fig. 5Q-S). Serial sectioning space in the vesicle’s interior became progressively filled with of vesicles harvested over a 3-day time period confirmed that MITF-negative, unpigmented cells (Fig. 5G-I). These cells these cells resulted from progressive proliferations and were positive for PAX6 (Fig. 5L-N), suggesting they were infoldings of the proximal layer, i.e. the presumptive RPE. This contributed from the PAX6-positive proximal layer, and they interpretation was consistent with numerous DiI tracing experiments (examples of which are depicted in Fig. 6A,B), which suggested that the inner cells (x in Fig. 6A,B) are neither derived from the distal layer nor the surrounding mesenchyme nor from the more proximal neuroepithelium of the optic stalk and brain. Thus, by morphology and gene expression, these cells resembled an NR that was derived from the presumptive RPE and was localized on the wrong (proximal) side of the pigmented layer. If the interpretation is correct that FGF signals from the surface ectoderm are thus responsible for optic vesicle patterning, then replacing the removed surface ectoderm with FGF alone should at least partially restore the development of the distal optic vesicle into presumptive NR. This was indeed the case. While BSA-coated control beads placed in front of an optic vesicle lacking a surface ectoderm did not inhibit the distal pigment formation (Fig. 5C, arrow), placing an FGF1- or FGF2-coated bead in this location resulted in a thickened distal layer that lacked MITF expression, retained PAX6 and CHX10 expression, and remained unpigmented (Fig. 5D,J,O,T; arrow indicates FGF2-coated bead). Not unexpectedly, however, simply replacing the removed surface ectoderm with a source of FGF did not restore the normal formation of a cup and did not allow the proximal layer to become pigmented. Interestingly, EGF- coated beads reduced distal pigmentation almost to the same extent as FGF (Fig. 5E, arrowheads) and led to changes in gene expression (not shown) similar to those observed with FGF. This result is different from those obtained with EGF- coated beads placed in the back of vesicles with intact surface ectoderm where pigmentation was not disturbed (see above). The difference in EGF response between the proximal layer that would go on to form the pigmented RPE in the presence of the surface ectoderm, and the distal layer that would become pigmented after removal of the Fig. 4. FGF downregulates MITF in RPE and interferes with pigmentation. Wild- surface ectoderm, suggests that the two layers, type cultures were established as for Fig. 3, but single BSA- or FGF-coated beads though each bipotential, are not entirely were implanted at the beginning of the culture period proximal to the area of the equivalent. This non-equivalence is reflected in optic vesicle. The cultures were harvested for cryosectioning at 3 days. the fact, for instance, that expression of CHX10 (A,C,E,G) Implantation of BSA-coated control bead (arrows). or FGF15 is restricted to the distal layer even (B,D,F,H) Implantation of FGF2-coated bead (arrows). (A,B) View in dissection before MITF downregulation effects the scope. Note the presence of pigmentation on both implanted and unimplanted sides observed cell-fate switches. in A and on the unimplanted side but not on the side implanted with the FGF2- Nevertheless, the two layers were similar coated bead in B. (C,D) Staining for MITF. Note absence of MITF and relative with respect to the non-uniform intracellular thickening of both neuroretina (NR) and retinal pigment epithelium (RPE) in D compared to C. (E,F) Immunostaining for PAX6. Note PAX6 expression in surface distribution of the pigment granules. As is well ectoderm (SE), lens (l), NR and RPE in vesicles implanted with control or FGF2- known for the in vivo situation at the cup stage, coated beads. (G,H) Immunostaining for CHX10. Note staining in NR but not RPE RPE pigment granules are concentrated at the in control and staining in both NR and RPE in FGF2 bead-implanted side. Bar, epithelium’s apical side, adjacent to the NR (see 50 µm (C-H). for instance, Fig. 4C). In the pigmented cells that 3586 M.-T. T. Nguyen and H. Arnheiter were generated from the distal optic vesicle after surface cells was maintained irrespective of whether they were derived ectoderm removal, the granules were still concentrated at the from the proximal optic vesicle, as is normally the case, or apical side (see for instance, Fig. 5I). This observation from its distal part, as was experimentally achieved. suggested that the basal-to-apical polarity of these pigmented Taken together, these results suggest that removing the

Fig. 5. After removal of the surface ectoderm, the distal optic vesicle maintains MITF expression, loses CHX10 expression, and becomes a pigmented monolayer. Wild-type cultures were established as for Fig. 3. The surface ectoderm was removed on both sides of the explants and in some cultures, growth factor-coated beads were implanted on one side (arrows in C-E,J,O,T) at the beginning of the culture period. (A-E) View in dissection scope. (A) Fresh explant, OV not manipulated. (B) Appearance immediately after removal of the surface ectoderm. (C) At 3 days, note pigment on both sides, including where a BSA-coated control bead had been implanted after surface ectoderm removal. (D) Note the presence of pigment on unimplanted side but its absence where an FGF2-coated bead had been implanted. (E) Note the presence of pigment on the unimplanted side but near absence of pigment where an EGF-coated bead had been implanted. (F-T) Immunolabeled cryosections. (F-I) Development of optic vesicle after surface ectoderm removal, labeled for MITF. Note the maintenance of MITF expression and development of pigmentation in distal optic vesicle, which becomes covered with unlabeled mesenchymal cells (arrow in G). (K-N) Similar sections, labeled for PAX6. Note PAX6-positive cells filling the originally cell-free space proximal to the distal layer. (P-S) Similar sections, labeled for CHX10. At 0 days, CHX10 is expressed in the distal layer but later is downregulated in this layer and upregulated in the PAX6- positive proximal cells. (J,O,T) Effect of FGF2-coated bead after surface ectoderm removal. FGF2 interferes with MITF expression (J), but PAX6 (O) and CHX10 (T) staining are seen in the entire optic vesicle. dOV, distal optic vesicle; pOV, proximal optic vesicle; me, mesenchyme. Bar, 50 µm (F-T). Role of FGF and MITF in eye development 3587

the previously described semidominant Mitf alleles (see below). To characterize the events in cell-fate changes in Mitf mutant RPE in more detail, we analyzed in vivo RNA or protein expression of several marker genes at various stages of embryonic and postnatal development. Besides the markers used above in explant cultures (Mitf, Pax6 and Chx10), we included Otx2, which encodes a homeodomain transcription factor expressed in RPE (Bovolenta et al., 1997), Six3, the mammalian homolog of the Drosophila eye developmental gene sine oculis, which at E9.5 is expressed in the surface ectoderm and the entire optic vesicle but at E14.5 is restricted to the NR and lens (Oliver et al., 1995) and Crx, which is required for the differentiation of rod and cone photoreceptor cells and normally begins to be expressed at E12.5 in the neuroblast layer of the NR but is negative in RPE (Furukawa et al., 1997). For postnatal stages, we used markers for specific Fig. 6. Cells filling the inner vesicle space after surface ectoderm retinal interneurons such as amacrine, horizontal and bipolar removal are not derived from the distal neuroepithelium or from the cells, and antibodies labeling rods and, selectively, rod outer proximal mesenchyme or optic stalk. DiI crystals were placed over segments. (A) the distal optic neuroepithelium (for 1-2 minutes) or (B) behind The results of the analysis in embryos are shown in Fig. 7. the optic vesicle (left in place) and the cultures were kept for 2 days In the leftmost column, sections through wild-type eyes are before cryosectioning. Note that the PAX6/CHX10-positive cells identified in Fig. 5 (marked x) are not labeled, suggesting they are shown for comparative NR labeling, not RPE labeling, since neither derived from the distal layer nor from the proximal RPE labeling is obscured by pigmentation. However, the mesenchyme or adjacent areas such as the optic stalk. Note that, at mutant eyes shown in the middle and rightmost column are the stage shown, the distal optic vesicle has not yet accumulated unpigmented, and hence RPE labeling in these eyes is specific pigment. for the genes in question. The results, shown for either Mitfvga−9 or Mitfmi-ew as indicated in Fig. 7, were confirmed for MitfMi-wh and can be summarized as follows. While the surface ectoderm, and with it a source of FGFs, leads to the principle separation into a retinal and RPE layer was formation of an inverted optic vesicle whose distal part maintained in mutant, the RPE progressively thickened, resembles RPE and whose proximal part resembles NR. The particularly in the dorsal part. The first sign of abnormal gene formation of a distal pigment layer can be prevented with just expression was the downregulation of MITF in the dorsal part, FGF alone. which had started already at E10.5 (see Fig. 1I, arrow), became prominent at E12.5 (Fig. 7B), and was complete at E14.5 (Fig. Mitf mutant RPE proliferates, expresses neuroretinal 7C). Concomitantly, the thickened dorsal and proximal parts markers and transdifferentiates into a second retina of the mutant RPE also lost expression of Otx2 (Fig. 7E,F), While the above experiments showed that FGF signals from similar to observations recently made in E13 embryos the surface ectoderm regulate MITF expression and affect cell homozygous for Mitfmi (Bumsted and Barnstable, 2000). fate decisions, they did not formally establish a causal link Expression of DCT, an enzyme involved in pigment synthesis, between the two events. The view that MITF is the crucial cell- was likewise lost (not shown). Of note, however, the areas of intrinsic factor through which extrinsic signals work would be the RPE that escaped the dorsal thickening retained expression supported, though, if deliberate MITF expression, on its own, of all of these RPE markers, suggesting that functional MITF turned NR into RPE, or if elimination of MITF turned RPE is not required for their expression. Expression of Pax6, which into NR. Since ectopic MITF expression may not easily be is normally progressively lost in wild-type RPE, stayed high achieved at physiological levels and may not readily result in throughout the mutant RPE, and the neuroretinal markers Six3, a NR-to-RPE transition against the physiological action of Chx10 and Crx, not found in wild-type RPE, were all expressed FGF, we restricted our analysis to the fate of the Mitf mutant throughout the mutant RPE. These results suggested that the RPE. mutant RPE, particularly its thickened dorsal parts, initiates the Using cyclinD1 staining, we first confirmed that excessive differentiation along the NR fate. RPE proliferation is an early sign of RPE pathology, not only To determine the further fate of the mutant RPE, we in embryos homozygous for the semidominant Mitf alleles examined subsequent embryonic stages and labeled postnatal Mitfmi and MitfMi-wh (Packer, 1967) but also for the null allele day (PN) 0-14 eyes for retinal interneurons including amacrine, Mitfvga−9. CyclinD1 marks cells in S phase and is horizontal and bipolar cells. The expanded dorsal region of the downregulated in mitosis. It is prominently expressed in many RPE remained easily identifiable and at PN 0 showed labeling cells in the NR for whose development it is required (Fantl et for the three retinal cell types, as did the retina in wild type al., 1995; Sicinski et al., 1995), but is seen in relatively few (Fig. 8A-C) and in mutant (Fig. 8D-F). In fact, the thickened cells in the wild-type RPE. In mutant RPE, however, the RPE (labeled ‘second retina’ in Fig. 8D-F) appeared layered frequency of cyclinD1 positive cells was similar to that in NR but the layers were topologically inverted with respect to those (not shown). Furthermore, an RPE-to-retina transition was also of the primary retina. This topological inversion was consistent seen in Mitfvga−9/vga−9 mice and thus was not a peculiarity of with earlier observations in Mitfmi/mi mutant mice (Scholtz and 3588 M.-T. T. Nguyen and H. Arnheiter

Chan, 1987) and a recent study (Bumsted and Barnstable, Taken together, the above results suggest that irrespective of 2000), where in addition rods lacking outer segments were whether Mitf is downregulated experimentally by FGF, or identified at PN 17. Beyond PN 14, however, the second retinae rendered non-functional by mutation, presumptive RPE cells showed complex foldings, making their clear identification change their gene expression profile and may switch their fate difficult, and they soon degenerated. In fact, in the absence of towards that of neuroretina. In vivo, the loss of wild-type Mitf a normal RPE, the primary retina became disorganized as well may eventually lead to the development of a second retina, and lacked expression of rod outer segments (not shown). albeit one that is restricted to the dorsal area of the RPE, suggesting that the loss of functional Mitf, though crucial, is not sufficient for this in vivo transdifferentation.

DISCUSSION

It is well established that the division of the optic neuroepithelium into two domains, the presumptive NR and the RPE, is critical for the development of the vertebrate eye. Here we describe experiments whose design was prompted by the notion that the early optic neuroepithelium is bipotential, capable of giving rise to either NR or RPE, and that this bipotentiality may be reflected in the fact that distinct transcription factors, which are later needed separately in NR and RPE, are initially coexpressed. The results of these experiments support the view that in the mouse, the division into NR and RPE is effectively controlled by extrinsic factors, notably FGFs, which emanate from the surface ectoderm and regulate an intrinsic factor, the transcription factor MITF, which ultimately specifies the RPE. A first set of experiments addressed the

Fig. 7. Molecular markers of in vivo RPE transdifferentiation in Mitf mutant embryos. Pictures in the left column represent wild-type embryos and those in the right two columns embryos homozygous for the Mitf alleles indicated. The rightmost column shows higher magnifications of areas of transition between RPE and thickened parts that transdifferentiate into neuroretina. All pictures represent embryonic eyes from E14.5 except (A,B,G,H), which are from eyes at E12.5. Labeling was for the markers indicated on the left. MITF and PAX6 were labeled by the immunoperoxidase reaction, the remainder of markers by in situ hybridization. Note that in wild-type RPE, pigmentation obscures specific labeling, and so the insets in A and G show a small area labeled by immunofluorescence. Based on earlier studies of the genes labeled by in situ hybridization, only Otx2 is expressed in the RPE at the developmental stage shown (E14.5) while Six3, Chx10 and Crx are not expressed in the RPE. Mitf mutant RPE is not pigmented and so does not obscure the label. Arrows in B and C show downregulation of MITF expression (see text for details). Bar, 100 µm (left and middle column), 40 µm (right column). Role of FGF and MITF in eye development 3589

NR may help to mutually reinforce the specification of these two layers (Orts- Llorca and Genis-Galvez, 1960; Sheedlo and Turner, 1996) and thus, it is conceivable that after removal of the surface ectoderm, the newly formed distal RPE may cooperate with proximal signals to induce the proximal cells to express NR markers. Intriguingly, in the chick, in contrast to our observations in the mouse, removal of the surface ectoderm led to the generation of an epithelium in which neuronal cells were interspersed with pigmented cells, which were not arranged as cuboidal epithelium (Hyer et al., 1998). This difference may reflect the fact that chick embryos do not show the initial pan- vesicular expression of Mitf (Mochii et al., 1998a), and since the removal of the surface ectoderm may not induce Mitf, just not suppress it, presumptive chicken NR cells may not differentiate into pigmented cells without the action of additional RPE inducing signals. A second set of experiments suggested that the relevant extracellular signals from the surface ectoderm are members Fig. 8. Neuroretinal markers in wild-type (WT, A-C) and Mitf mutant eye (Mitfvga−9/vga−9, of the FGF family of ligands. Both FGF1 D-F), labeled at postnatal day 0 for the markers indicated. (D-F) Note that in the dorsal and FGF2 were expressed at higher part of the Mitf mutant eyes, the layers of the specific retinal cells are duplicated. The concentrations in the surface ectoderm duplicated layers are derived from the thickened part of the transdifferentiated RPE than in the neuroepithelium itself or the (second retina) and are contiguous with parts of the RPE that retain a more RPE-like, surrounding mesenchyme. In addition, though unpigmented, appearance (not shown). Arrows indicate interneurons in the inner while the neuroepithelium was thickened in nuclear layer, arrowheads (A and D) indicate displaced amacrine cells in the ganglion cell layer. rpe, retinal pigment epithelium; inl, inner nuclear layer; gcl, ganglion cell layer. response to FGFs in the distal as well as the Bar, 40 µm (A-C); 50 µm (D-F). importance of the surface ectoderm in principle. Normally, the optic vesicle forms an NR domain at the distal tip where it comes in contact with the surface ectoderm, and an RPE domain at its proximal base where it connects to the optic stalk. When the surface ectoderm was physically ablated in explant cultures, the two domains still formed, only they were now inverted, with a pigmented monolayer resembling RPE located at the vesicle’s tip and a multicellular layer resembling NR located at its base. This inversion occurred rapidly and cannot be explained by an involution of the vesicle followed by de novo generation of the two layers from pre-existing, committed precursors. Rather, it appears that the optic neuroepithelium of mammals, much as that of lower vertebrates, is capable of giving rise to either NR or RPE and, consequently, needs to be Fig. 9. Schematic representation of how selective gene repression instructed by local signals to develop into the types of cells leads to domain specification in the optic vesicle. In both (A) and appropriate for their anatomical location. If the surface (B), the optic vesicle initially expresses the RPE transcription factor ectoderm were the only source of local signals for NR MITF along with NR transcription factors. As the vesicle comes into specification, however, its removal should leave the optic contact with the surface ectoderm (SE), signals from the ectoderm vesicle incapable of forming NR. Since NR still formed, it downregulate MITF in the future NR. Where MITF tf expression is not downregulated, an RPE develops. In model A, FGF1 and FGF2 follows that NR inducing signals must originate at other sites directly downregulate MITF. The more general model in B suggests as well and gain in prominence in the absence of the surface that other surface ectodermal factors (marked X) induce in the ectoderm. Such signals might emanate from the surrounding adjacent neuroepithelium one or several factors (marked mesenchyme, the neuroepithelium of the brain, or the novel, Y, potentially including FGF15), which in turn lead to distal RPE. In fact, it is thought that the apposition of RPE and downregulation of MITF. 3590 M.-T. T. Nguyen and H. Arnheiter proximal domain, suggesting that the appropriate receptors and transcription factors that serve as transcriptional repressors of signal transduction machineries were available at either Mitf, and conversely, that MITF may directly or indirectly lead location, only the domain closest to the surface ectoderm to transcriptional repression of NR-inducing factors. normally gives rise to the NR. Furthermore, the development The fact that undifferentiated, bi- or multipotential precursor of a pigmented monolayer, either starting out from the cells initially show overlapping gene expression patterns, presumptive RPE or, after ablation of the surface ectoderm, which for subsequent lineage commitment are sorted out by from the presumptive NR, could be prevented by the local selective gene repression, is reminiscent of other systems in application of FGF alone. Consistent with this observation, both invertebrates and vertebrates. During vertebrate activation of FGF receptors in the presumptive RPE by ectopic neurulation, for instance, transcription factor genes such as expression of FGF9 in transgenic mice has recently been Pax3 and Pax7 initially have broad expression patterns that shown to result in a second retina (Zhao and Overbeek, 1999), encompass both the future dorsal and future ventral domains; suggesting that FGFs may be sufficient to trigger the only later do they become restricted to the dorsal neural tube presumptive RPE to form a retina. As discussed below, through repression by sonic hedgehog signaling in the ventral however, other signals from the surface ectoderm may neural tube and maintenance by BMPs in the dorsal neural tube conceivably have similar effects as FGFs. (for a review, see Lee and Jessell, 1999). Singling-out of A third set of experiments led to the conclusion that FGFs (or distinct cell fates by repression of transcription factors also ligands with similar activities) effect these developmental cell seems to operate, for instance, in the vertebrate hematopoietic fate choices by regulating the expression of Mitf. This conclusion system (Nutt et al., 1999). was based on the following lines of reasoning. Where FGF, Equally consistent with our results, and representing a more whether derived from the surface ectoderm or from general explanation, is the model depicted in Fig. 9B. Here, the experimentally implanted beads, were able to prevent the relevant factors from the surface ectoderm need not be FGFs; formation of an RPE, they also induced the disappearance of they only need to be capable of activating signaling pathways Mitf, and where Mitf stayed on, an RPE developed. Beyond this with similar consequences in the adjacent neuroepithelium. This correlation, we also found that when Mitf was missing, as in view is supported by the demonstration that EGF was apparently Mitfvga−9/vga−9 mice, or was crippled in its DNA binding and as potent as FGF in inhibiting pigment formation in the distal transcriptional activity, as in MitfMi-wh/Mi-wh or Mitfmi-ew/mi-ew optic vesicle after surface ectoderm removal (although EGF had mice, the RPE, at least its dorsal part, lost the expression of RPE- no such effect on the normal future RPE). According to this specific genes, including that of Mitf itself, and gained the scenario, then, the experimental application of FGFs would expression of NR-specific genes, including that of Six3, Chx10 simply mimic the signaling effects of other factors. Evidently, and Crx. In fact, this dorsal part thickened and then became a such factors could induce distinct FGFs or functionally related laminated second retina not unlike that induced by the above ligands in the adjacent neuroepithelium. An excellent candidate mentioned ectopic FGF9 expression (Zhao and Overbeek, for an FGF interposed between the surface ectoderm and MITF 1999). The results suggest that the formation of a second retina is FGF15, which is expressed specifically in the NR domain from mutant RPE depends on additional local signals, such as (McWhirter et al., 1997). Although the model in Fig. 9B may BMP7 or other factors expressed on the dorsal side of the cup not seem conceptually different from that in Fig. 9A, it would (Wawersik et al., 1999; Koshiba-Takeuchi et al., 2000). accommodate the fact that mice with null mutations in FGF1 or Nevertheless, the fact that mutations in a single gene, Mitf, are FGF2, alone or even in combination, apparently do not show capable of inducing the transdifferentiation of the presumptive overt alterations in eye development. RPE into an NR suggests that Mitf is pivotal in RPE In conclusion, in the mouse, the division of the optic specification. It remains an open question, though, whether neuroepithelium into a future NR domain and an RPE domain, ectopic expression of wild-type Mitf, experimentally uncoupled and hence the formation of a proper eye, critically depends on from its negative control by FGFs, would be sufficient to induce the negative regulation of Mitf. The results suggest that when the presumptive NR to form an RPE against the action of FGFs. left unchecked, MITF might usurp the future NR and at best The results can be interpreted as schematically depicted in disturb its proper growth, but at worst turn it into a pigmented Fig. 9. The model builds on previous models in the chick, layer unsuitable to function as a retina. which highlighted the importance of the surface ectodermal FGFs in the induction of the NR (Pittack et al., 1997; Hyer et We thank Drs Constance Cepko, Peter Gruss, Paul Hargrave, al., 1998), and extends these models to the mouse and to the Vincent Hearing, Roderick McInnes and Simon Saule for probes and regulation of distinct transcription factors involved in cell-fate antibodies, Story Landis and Ronald McKay for discussions, and determination. The simplest explanation (Fig. 9A) is that Monique Dubois-Dalcq, Lynn Hudson and Ward Odenwald for critically reading the manuscript. the distal neuroepithelium, which initially coexpresses transcription factors that later become NR- and RPE-specific, becomes activated by surface ectodermal FGFs, which leads to REFERENCES transcriptional repression of a presumably eye-specific Mitf promoter and the concomitant disappearance of MITF protein. 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