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Develop. Growth Differ. (2005) 47, 523– 536 Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium Blackwell Publishing, Ltd. Yuka Kagiyama,1 Nanaka Gotouda,1 Kiyo Sakagami,2 Kunio Yasuda,2 Makoto Mochii3 and Masasuke Araki1,* 1 Developmental Neurobiology Laboratory, Faculty of Science, Nara Women’s University, Nara 630-8506, Japan Laboratory of Molecular and Developmental Biology, Nara Institute of Science and Technology, Ikoma 630-0101, Japan and 3Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan 2 Dorsal–ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of the eye. To address the questions of how DV specification is determined and how it affects fate determination of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively, when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures. Combination cultures of collagenase-treated optic vesicles with either the dorsal or ventral part of the head indicated that head-derived factors have an important role in the fate determination of the optic vesicle: in the optic vesicles co-cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium, while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and differentiation of the neural retina and pigment epithelium. Key words: chick embryo, cRax, Mitf, optic vesicle, organ culture. Introduction Development of the vertebrate eye begins with optic vesicle formation, which develops by lateral protruding of the diencephalic neuroepithelium. The optic vesicle subsequently invaginates to form a doublelayered optic cup, whose inner and outer layers further develop into the neural retina (NR) and the retinal pigment epithelium (RPE), respectively (Saha et al. 1992; Chow & Lang 2001). At the same time, the invagination of the optic vesicle extends from the distal to the proximal direction at the ventral part to form the future optic fissure in the optic stalk, through which the retinal ganglion axons pass into the optic tectum. Thus, the early optic vesicle appears to consist of at least two discrete dorsal and ventral compartments that show different developmental fates. This is also suggested by the different expression *Author to whom all correspondence should be addressed. Email: [email protected] Received 14 June 2005; revised 26 August; accepted 30 August 2005. patterns of various transcription factors (Torres et al. 1996; Schulte et al. 1999; Koshiba-Takeuchi et al. 2000). The optic vesicle is surrounded by and makes contact with various different tissues, such as the overlying surface ectoderm and periocular mesenchyme, and it has been assumed that the specification of the NR and RPE is influenced by signals from the surface ectoderm and mesenchyme, respectively (Dragomirov 1937; Lopashov 1963; Hyer et al. 1998; Fuhrmann et al. 2000). The extraocular mesenchyme originates from two different sources, the cephalic neural crest cells at the dorsal region and the precaudal mesodermal cells at the ventral region (Johnston et al. 1979). It is not clear whether these differently located mesenchymal tissues play different roles in eye development (Pera & Kessel 1997). Optic vesicle invagination begins at a point displaced somewhat toward its ventral surface, rather than beginning at the most lateral and distal point, and is directed mediodorsally (Romanoff 1960; Bellairs & Osmond 1998). Consequently, the distal and ventral portions of the early optic vesicle are to organize the inner layer of the optic cup, fated to develop into the 524 Y. Kagiyama et al. NR, while the dorsal region will organize the outer layer, fated to develop into the RPE. Fate mapping of the chick optic vesicle with fluorescent dye also reveals different developmental fates of the discrete regions in the optic vesicle according to the dorsal– ventral (DV) and proximal–distal directions (Dütting & Thanos 1995). DV polarity is well understood for its importance in eye morphogenesis, and several transcription factors, such as Pax2 and Pax6, are known to play an essential role in the fate determination of the optic vesicle regions (Schwartz et al. 2000; Baümer et al. 2003). The domain of Pax2 expression is complementary to that of Pax6 (Nornes et al. 1990; Macdonald & Wilson 1996). Several signaling molecules have been implicated in the regulation of ventral optic development. The secreted protein sonic hedgehog (Shh) plays important roles in patterning tissues of the vertebrate embryo, including the eye, and accumulating evidence indicates that Shh activity is also involved in pattern formation of the vertebrate eye (Oliver & Gruss 1997; Jean et al. 1998). Pax2 and Pax6 expression in the optic vesicle are regulated by Shh. Alterations in Shh activity in zebrafish have been shown to perturb Pax6 and Pax2 expression (Macdonald et al. 1995). Bone morphogenetic proteins (BMP) also have profound effects on patterning in the nervous system. BMP4, for instance, is involved in the dorsalization of the retina (Koshiba-Takeuchi et al. 2000). The spatial and temporal expression patterns of Shh and BMP4 suggest a model in which ventral midline-derived Shh and dorsally derived BMP4, either from the dorsal midline of the neural tube or the dorsal optic cup, play roles in establishing the DV polarity of the optic primordium (Schulte et al. 1999; Zhang & Yang 2001; Martí & Bovolenta 2002). To elucidate the mechanism involved in DV polarity formation in the developing optic vesicle, we have previously used an embryonic transplantation technique on the chick embryo, in which the truncated optic vesicle is transplanted inversely without changing the anterior–posterior direction (Araki et al. 2002; Uemonsa et al. 2002). These approaches revealed that DV polarity is established gradually between the 10- and 14-somite stages under the influence of signals derived from the midline portion of the forebrain, and that the presumptive signals appear to be transmitted from proximal to distal regions within the optic vesicle. Disturbance of DV polarity formation causes the failure of optic cup formation, as well as patterning of the NR and RPE. Accordingly, two new questions arose: which tissue is responsible for the presumptive dorsal or ventral signal(s), and how are they transmitted to the optic vesicle? To answer these questions, we performed organotypic culture of the optic vesicle, either with or without the surrounding tissues. The optic vesicle was cut into its dorsal and ventral halves, which were cultured separately to determine their developmental fates. The optic vesicle was also cultured in combination with the dorsal or ventral half of the head portion, to examine whether the head-derived factors affect the development of the optic vesicles. The present results indicate that optic vesicle development is regulated by the surrounding tissue, probably the neural crest-derived dorsal mesenchyme. This tissue appears to induce RPE fate in the optic vesicle, which otherwise develops into the NR as a default pathway. The results also indicate that the dorsal part of the head suppresses Pax2 expression in the optic vesicle, and that the dorsal and ventral halves of the optic vesicle are committed to develop mainly into the RPE and NR, respectively. Materials and methods Preparation of chick embryos Fertilized eggs were incubated in a humidified atmosphere at 37.8°C. All operations were carried out according to the procedures previously described (Araki et al. 2002; Uemonsa et al. 2002). A small window was made in the shell, and India ink, diluted in Hanks’ solution, was injected into the yolk beneath the embryos to visualize the embryonic structures (Fig. 1). Embryos were staged according to Hamburger and Hamilton (1951). Preparation of optic vesicles Embryos at given stages were placed into roundbottomed dishes filled with Hanks’ solution to wash Fig. 1. Chick embryos at the 10-somite stage. (A) Dorsal view showing the lateral protrusion of the optic vesicle. Bar, 300 µm. (B) Transverse section of the proencephalon. The optic vesicle makes contact with different types of tissues at the dorsal and ventral regions. Arrow indicates the dorsal mesenchymal tissue. Bar, 100 µm. Extraocular signal and eye development out the yolk and India ink. The embryos were then pinned over a black silicone plate, and optic vesicles with the covering ectoderm and mesenchyme were truncated using a finely sharpened hand-made knife. The surrounding tissues, like the surface ectoderm and mesenchyme, were removed by using a fine tungsten needle after a treatment with 0.03% collagenase (Sigma, St Louis, MO, USA) for 60 min at 25°C. To obtain the dorsal and ventral halves of the whole head portion, the head was cut transversely at the level at the rhombomere, and then a careful lateral incision was made exactly along the horizontal plane that divides the dorsal from the ventral half. Particular attention was paid to cut the optic vesicle precisely into the dorsal and ventral halves along the horizontal level. The dorsal or ventral halves were cultured on a filter membrane cup (Millicell-CM, 0.4 µm, Millipore, Bedford, MA, USA) by placing the cut surface facing the membrane. Fig. 2. Two examples of embryonic transplantation of the optic vesicle into the distal wing bud. Transplantation was performed as shown in the schematic illustration. Optic vesicle at the 10-somite stage was inserted into an incision made at the distal part of a wing bud of a host embryo. In (A), a well developed eye is seen embedded in the mesodermal tissue of the wing bud. In this case, quail optic vesicle was transplanted to chick wing bud. At a higher magnification numerous chicken (host) cells are intermingled among quail cells (C, arrowheads) in the periocular connective tissue. In the other case (D), a less developed eye is found at the wing bud in chick–chick transplantation. The outer retinal pigment epithelium layer appears multilayered epithelial form with pigment granules as shown in (E). Bar in (A) is 250 µm and applies to (D). Bar in (B) is 40 µm, and Bar in (C) is 20 µm and applies to (E). 525 In ovo transplantation of the optic vesicle Optic vesicles were cut from embryos at 8-, 10-, 14and 16-somite stages and placed into the wing buds of host embryos at 3 days of incubation without further treatment with collagenase. A small cut was made at the distal part of the wing bud and the optic vesicle was inserted into the limb mesenchymal tissue (Fig. 2). The grafts were allowed to develop further in ovo for 3–4 days until fixed for histological preparation. In some cases, optic vesicles from quail embryos were transplanted to the wing buds of chick embryos to discriminate donor cells from the host. Organotypic culture of the optic vesicle Optic vesicles were cut from embryos at the 8-, 10-, 14-, 16- and 18-somite stages and were used for organotypic culture. Two types of organ culture methods were used. Isolated optic vesicles were first 526 Y. Kagiyama et al. laid on a filter membrane cup (Millicell-CM, pore size 0.4 µm, Millipore) and cultured with or without embedding in a collagen gel (Cellgen I-AC, Koken, Tokyo, Japan). They were cultured for 3 days, and fixed for histological observation in the case of those cultured in collagen gel. Optic vesicles cultured without collagen gel were used mainly for immunocytochemistry and in situ hybridization. The standard medium was Dulbecco’s MEM (Nissui, Tokyo, Japan) supplemented with sodium pyruvate (55 mg/mL), glucose (6 mg/mL) and 8% fetal bovine serum (FBS; Micro, Walkersville, MD, USA). For the combination culture of the optic vesicle with the head halves, the right optic vesicle was first cut off, and then the head portion of embryos was divided into two halves as described above. The optic vesicle was laid down at the original position of the removed optic vesicle in close contact with the head half. Immunocytochemistry Cultures were fixed with ice-chilled 2% paraformaldehyde in 80 mM phosphate buffer (pH 7.4) for 6 h. They were then stained for Mitf, cRax, HNK-1 or QCPN, either by using the indirect method or the ABC method according to the procedure previously described (Araki et al. 2002). Briefly, cultures were first immersed in phosphate-buffered saline (50 mM, pH 7.4) containing 0.2% Triton X-100 (PBS-TX) for 60 min. For the indirect method, the immunoreactive sites were detected using a fluorescein-conjugated secondary antibody (Alexa-488, Molecular Probes, Eugene, OR, USA), and for the ABC method, they were visualized by using hydrogen peroxide and diaminobenzidine. The specificity of the antibodies had been confirmed previously (Mochii et al. 1998; Sakagami et al. 2003). In situ hybridization Plasmids containing Tbx5 and Pax2 cDNA in pBluescript SK(-) were linearized with EcoRV and EcoRI, and transcribed in vitro with T3 RNA polymerase to generate digoxigenin-labeled antisense and sense RNA probes, respectively (Promega, Madison, WI, USA). The Tbx5 cDNA was obtained from Dr. T. Ogura (Nara Institute of Science and Technology), and the Pax2 cDNA from Dr. H. Nakamura (Tohoku University). Whole-mount in situ hybridization was performed as described previously (Henrique et al. 1995). After hybridization, embryos were incubated with 1/2000diluted alkaline phosphatase-conjugated antiDIG antibody (Roche Diagnostics, Mannheim, Germany) overnight, and nitroblue tetrazolium (NBT)/5-bromo- 4-chloro-3-indoyl phosphate (BCIP) staining was employed for the detection of hybridization signals. Embryos were photographed with a camera attached to a stereomicroscope (Olympus, Tokyo, Japan). Histological preparation For histological observation, embryos were fixed with Bouin fixative for 3–10 h depending on their size, dehydrated in a graded ethanol series, and finally embedded in paraffin. Serial sections of 6 µm thickness were cut in a transverse plane and stained with hematoxylin and eosin. Results In ovo transplantation of the optic vesicle: Optic cup formation and retinal pigment epithelium differentiation Our previous experiments suggested that diffusible factors from the forebrain region of the embryo act on the optic vesicles and contribute to the delineation of the territories fated to form RPE and NR. To further define the source of this activity, we performed a series of in ovo transplantation and in vitro co-culture experiments. To examine their capability of autonomic development in a foreign milieu, isolated optic vesicles were transplanted into the wing buds of host embryos without further treatment with collagenase. Histological examination of the optic vesicle at the 10-somite stage (HH stage 10) revealed that it faces dense mesenchymal tissues at the dorsal region (Fig. 1). Isolated optic vesicles therefore contained both the epidermis and the dorsal mesenchymal tissue. After 3 days of in ovo culture, approximately onethird of the grafts (19 out of 60 grafts) remained at the grafting position (the distal part of the wing buds). Some of them had grown considerably, even to a size similar to that of normal eyes at the corresponding stage (Fig. 2A). We further examined the histological structures of grafts with respect to the differentiation of the NR, RPE and the distribution of the mesenchymal cells (Fig. 2B,C). In all cases, a well developed lens and the inner and outer epithelial layers of the optic cup were found. The inner layer was usually much thicker than the outer layer, which often showed a single or multilayered epithelial form with pigment granules (Fig. 2C,E). In all cases, these pigmented epithelial layers were surrounded with mesenchymal cells, suggesting that mesenchymal cells in the optic vesicle have an inductive role in RPE development. The mesenchymal cells surrounding RPE were often of the host origin, Extraocular signal and eye development as shown by the histological appearance of their nuclei in the chimeric transplantation (Fig. 2C). Organotypic culture of the optic vesicle To examine whether in vitro culture conditions enabled isolated optic vesicles to develop autonomously, they were maintained in vitro under two different culture conditions: gel-embedded or simply laid on filter membrane. In cultures of gel-embedded optic vesicles, similar results were obtained as seen in the in ovo transplants. After 3 days of culture, the lens, as well as the inner and outer layer, were observed in approximately half of the explants (29 out of 61 grafts; Fig. 3). The inner and outer layer had no distinct difference in thickness. Mesenchymal cells were generally poorly developed in comparison with those of the in ovo transplants. RPE layers were found in only a few cases (8 out of 29) and were surrounded by mesenchymal cells (Fig. 3A,B). These observations were mostly the same regardless of the stages (10-, 12-, 14-, 18- and 20somite stages) of the grafts. In another culture condition, optic vesicles isolated from 10-somite-stage embryos were laid on the membrane filter cup and cultured for 3–4 days without being embedded in a gel. This culture method allowed us to perform immunocytochemical staining of the whole-mount tissues without preparing sections, and in the following culture study we used this method to detect Mitf and cRax immunocytochemical staining. Detection of melanin granules under a light microscope was also a good indication of RPE differentiation. We treated optic vesicles with collagenase to remove the ectodermal and mesenchymal tissues, and compared the results with those obtained in untreated optic vesicles. Fig. 3. Gel-embedded culture of the optic vesicle removed from embryos at the 10-somite stage. The outer layer is facing mesenchymal tissue and contains pigment granules as shown by arrowheads in (B). The amount of mesenchymal cells is much less than found in the embryonic transplanted optic vesicle. Bar (A), 100 µm; bar (B), 40 µm. 527 Untreated optic vesicles, which still contained surrounding tissues, the surface ectoderm and mesenchyme, normally became pigmented, though not very intensely, and expressed both Mitf and cRax (Fig. 4A–C). In contrast, collagenase-treated optic vesicles seldom showed melanin deposition (3 out of 22) (Fig. 4D). Mitf was not usually detected, while cRax was always detected (15 out of 15), but less intensely than that found in the untreated optic vesicles (Fig. 4E,F). These observations indicate that without the surrounding tissues the optic vesicle neuroepithelium differentiates into NR but not into RPE. Organotypic culture of dorsal and ventral halves of the embryonic head portion To examine whether the dorsal and ventral parts of the optic vesicle at these stages were already committed or determined to develop along separate fates, we cultured dorsal and ventral halves of embryonic heads removed from 10- to 16-somite stage embryos. After 3 days in culture, the explants grew considerably, and the epidermis and neuroepithelial cells intensively extended outward at the periphery of the explants (Fig. 5A–C). The optic vesicles also extended outward and occasionally they fused with the brain vesicles, such as the telencephalon and diencephalon (Fig. 5C). It was found that, in the dorsal brain culture, melanin deposition was always seen in both sides of the optic vesicle (Fig. 5D), while it was observed only rarely, if at all, in the optic vesicle areas of the ventral head culture (Fig. 5G). Cultures were fixed after 3 days in vitro to examine RPE and NR differentiation. In the dorsal head culture, intense Mitf staining was always found in both the right and left optic vesicles, but only a very faint staining for cRax was observed. In contrast, in the ventral head culture, intense cRax staining was always observed, while Mitf immunostaining was very faint. In cases where it was detected, Mitf staining was always found in the posterior part of the optic vesicle. These results indicate that the dorsal and ventral parts of the optic vesicle are committed to differentiate mainly into the RPE and NR, respectively, as early as at the 10-somite stage. Taken together the above results, it was suggested that the dorsal portion of the optic vesicle is committed to develop into RPE, and the dorsal mesenchymal tissue plays a role in fate determination. Combination culture of the optic vesicle with the head portion To investigate factors involved in the fate commitment or fate determination of NR and RPE, we cultured 528 Y. Kagiyama et al. Fig. 4. Organotypic culture of the optic vesicle. Isolated optic vesicle was laid onto a membrane filter cup for culture. (A,B,C) Untreated optic vesicle. (D,E,F) Collagenase-treated optic vesicle in which surrounding tissues were removed. In A, melanin deposition can be seen (arrow). (B,E) cRax (C,F) Mitf immunostaining. No Mitf positive nuclei are found in cultures of collagenase-treated optic vesicle. Bar in (A) is 250 µm and applies to (D). Bar in (B) is 50 µm and applies to (C,E,F). the isolated optic vesicle in combination with either the dorsal or ventral half of the head portion. For this purpose, collagenase-treated optic vesicles (without the surrounding tissues) were placed in close contact with the dorsal or ventral half of the head at the corresponding position of the head explant from which the right optic vesicle had been removed in advance (Fig. 6). As a control, untreated optic vesicles were cultured with the head half in a similar way, and the results showed that both NR and RPE developed as seen in the single culture of the untreated optic vesicle. Neither the dorsal (Fig. 6D–F; Fig 6A–C for comparison) nor the ventral (data not shown) half of the head significantly affected the results. When the treated optic vesicle (without the surrounding tissues) was co-cultured with the head half, we obtained different results depending on whether it was co-cultured with the dorsal or ventral half. The optic vesicle developed into both NR and RPE when it was co-cultured with the dorsal half of the head (Fig. 6G–I). This was confirmed by intense immunostaining for cRax and Mitf. In contrast, the treated optic vesicle developed only into NR when co-cultured with the ventral half of the head (Fig. 6J–L): no Mitf immunostaining was detected, while cRax staining increased in its intensity. These results suggest that the dorsal area of the head portion plays an inductive role in RPE differentiation of the optic vesicle. The ventral area of the corresponding portion did not have such an activity, but it enhanced NR differentiation. The effect of the dorsal head on RPE induction in the optic vesicles was examined at different stages of the co-cultured head portions, and dorsal head portions at 8-, 10- and 14-somite stages showed the same effect, while those at the 16-somite stage did not show this effect. The statistical counting of the number of optic vesicles with melanin deposition also supported the results (Fig. 7). In order to exclude the possibility that some part of the neuroepithelium of the dorsal brain vesicle cocultured with the optic vesicle may differentiate into RPE, we co-cultured quail optic vesicle with chicken dorsal head. The results showed that the Mitf-expressing domain was all stained with the QCPN antibody (Fig. 8A–D), verifying that it was from quail optic cells and not from chicken dorsal brain cells. Thus, it was concluded that the differentiated RPE was derived Extraocular signal and eye development 529 Fig. 5. Organotypic culture of dorsal or ventral half of the optic vesicle. (A) The embryonic brain was cut into the dorsal and ventral halves which were then laid onto the membrane filter cup and cultured for 3 days. (B) Dorsal half on the filter cup immediately after transferred on the cup. (C) The same dorsal half cultured for 3 days. Melanin deposition can be seen at the both sides (arrows). (B,C) are shown at the same magnification. (D,E,F) Dorsal half culture. Melanin deposition can be seen (arrows in C and D) and Mitf expression is also found (F), while cRax can be seldom found (E). (G,H,I) Ventral half culture. cRax immunostaining is usually found in the explant (H), but Mitf staining is rarely observed. In case it was positively reacted, it was found only in a small area (I). Bar in (D) is 250 µm and applies to (G). Bar in (E) is 50 µm and applies to (F,H,I). from the optic vesicle. Similarly, in quail optic vesicle co-cultured with chicken ventral head half, the cRaxexpressing domain was stained with QCPN antibody (Fig. 8E–H). Pax2 and Tbx5 expression in the optic vesicle co-cultured with the head portion Pax2 is expressed in the optic stalk and ventral-most region of the optic cup and is involved in the development of ventral structures of the eye (Schwartz et al. 2000), while Tbx5 is found in the dorsal region of the optic cup (Koshiba-Takeuchi et al. 2000; Fig. 9). To investigate if their expression was also influenced by signals derived from the dorsal or ventral periocular tissues, we examined Pax2 and Tbx5 expression in optic vesicles that were co-cultured with dorsal or ventral head portions, respectively (Fig. 9). In the culture of untreated optic vesicles, expression of both Pax2 and Tbx5 was clearly detected by the in situ hybridization technique (Fig. 9A,E). Pax2 expression was still detected in the collagenase-treated optic vesicles (Fig. 9B), but Tbx5 was not detected (Fig. 9F). In the co-culture experiments, we did not observe Pax2 expression when collagenase-treated optic vesicles were co-cultured with the dorsal head explant, but clear Pax2 expression was found when co-cultured with the ventral head explant (Fig. 9C,D). The effect of Pax2 suppression by the dorsal head was examined at different stages. Dorsal heads from the 18-somite stage did not suppress Pax2 expression (data not shown). In contrast, Tbx5 expression could not be seen in collagenase-treated optic vesicles even when co-cultured either with the dorsal or ventral head (Fig. 9G,H). These results suggest that the dorsal signals deriving from the dorsal part of the head induce dorsalization of the optic vesicle as well as RPE differentiation, while they suppress ventralization. The surrounding tissue is required for Tbx5 gene expression, but the dorsal head explant was not sufficient to induce Tbx5 expression. 530 Y. Kagiyama et al. Fig. 6. Co-culture of the optic vesicle with either the dorsal or ventral half of the head. Note that one of optic vesicle had been removed from the head portion before it was co-cultured with optic vesicle graft. Four types of combination cultures were performed as seen in the figure. (A,B,C) Cultures of untreated optic vesicles without additional tissue. (D,E,F) Untreated optic vesicles were cocultured with the dorsal head explant. Arrow in (D) indicates melanin deposition of grafted optic vesicle. (G,H,I) Collagenasetreated optic vesicles were cocultured with dorsal head explant. Arrow in (G) indicates the melanin deposition of the grafted optic vesicle and Mitf immunostaining was also detected in the optic vesicle as shown in (I). (J,K,L) Collagenase-treated optic vesicles were co-cultured with ventral head explant. Nither pigmentation nor Mitf staining was seen in the graft (J and L). Bar in (A) is 250 µm and applies to (D,G,J). Bar in (B) is 50 µm and applies to (C,E,F,H,I,K,L). Topographical relationship between migrating neural crest cells and retinal pigment epithelium differentiation A previous study by Fuhrmann et al. (2000) suggested that cranial neural crest cells inhibit NR differentiation. To clarify whether neural crest cells migrating to the eye have any interaction with RPE (Bronner-Fraser 1996), we examined the localization of migrating neural crest cells in cultures of dorsal or ventral halves of the head by using HNK-1 immunocytochmisty. In normally developing chick embryos, HNK-1 positive cells extend antero-laterally from the dorsal midline area of the future diencephalon towards the optic vesicle. At the 10-somite stage, positive cells are distributed in the upper dorsal part of the optic vesicle, and then migrate ventrally through the optic vesicle in either the anterior or posterior direction (Fig. 10A– C). No positive cells were found in the distal part of the optic vesicle. In the culture of the dorsal half of the head, numerous HNK-1 positive cells migrated over the explant and further outward. Deposits of melanin granules Fig. 7. Melanin deposition in optic vesicles which were cultured alone, with the dorsal half head, or with the ventral half head. Untreated optic vesicles always show melanin deposition whether or not they were cultured with the head half. Melanin deposition in the collagenase-treated optic vesicles is found only when they were co-cultured with the dorsal half. were usually observed at the optic vesicle of the dorsal half culture. Distribution of HNK-1 positive cells obviously overlapped partially on the pigmented area, and positive cells were often found to surround the pigmented areas (Fig. 10D,E). Extraocular signal and eye development 531 Fig. 8. Double immunofluorescence of quail-chick chimeric culture; optic vesicles from quail embryos at 10-somite stage were cocultured with the chicken dorsal half (A,B,C,D) or with the chicken ventral half (E,F,G,H). Green fluorescence in (A) and (C) shows Mitf immunostaining and that in (E) and (G) shows cRax staining. Red fluorescence is by QCPN antibody. Bar in (B) is 250 µm and applies to (A,E,F). Bar in (D) is 50 µm and applies to (C,G,H). Discussion Morphogenesis of the vertebrate eye is regulated by progressive interactions between the optic vesicle neuroepithelium and the surrounding tissues, and our previous study indicated that the dorsal and ventral specification within the optic vesicle is crucial, not only for optic cup formation, but also for the patterning of the neural retina and RPE (Uemonsa et al. 2002). DV specification was also suggested to be determined by factors originating in the midline region of the forebrain. The present study was intended to address the question of how these factors regulate the development of the eye anlagen. Previous studies have shown that BMP and Shh are among the most likely candidates, and ventrally derived Shh signals and dorsally restricted BMP4 signals appear to act antagonistically to regulate the growth and specification of the optic primordium (Huh et al. 1999; Zhang & Yang 2001; Adler & Belecky-Adams 2002). The precise mechanism involved in the DV specification of the optic vesicle, however, is still not clear. In the present study, we addressed this primary question using our new organ culture method for chick embryos. Mesenchymal tissue regulates retinal pigment epithelium and neural retina development by directing the retinal pigment epithelium domain We performed a series of different cultures of the optic vesicle: in ovo culture (embryonic transplantation), collagen gel-embedded culture and filter membranesupported culture. We evaluated RPE differentiation by direct histological observations, in addition to Mitf expression. Histological observations revealed that in ovo transplanted optic vesicles developed similarly to the normal eye, forming an ectopic eye on the distal tip of the wing bud. The eye had a lens, neural retina and RPE and was surrounded by mesenchymal tissues. This indicates that the optic vesicle at the 10-somite stage can develop autonomously and needs no further influence from the forebrain, but requires certain factors deriving from mesenchymal cells. We have done careful histological observations of the grafted and cultured optic vesicles and found that differentiating RPE always faced mesenchymal tissues, suggesting that they play an important role in RPE differentiation. In the chimeric transplantation experiment, in which quail 532 Y. Kagiyama et al. Fig. 9. In situ hybridization of Pax2 and Tbx5 expression in cultured optic vesicles. Embryos at HH stage 18 show dorsally and ventrally located staining profiles of Tbx5 and Pax2 expression, respectively. (A,E) Untreated optic vesicles from 10-somite embryo showed intense staining of both Pax2 and Tbx5. (B,F) In collagenase-treated optic vesicles Pax2 expression is found, though less intense than found in untreated optic vesicle, while no Tbx5 expression could be found. (C) No Pax2 expression can be seen in collagenase-treated optic vesicle when co-cultured with dorsal half, while it is upregulated when cultured with ventral half as shown in (D). (G,H) Tbx5 expression can not be found in collagenase-treated optic vesicles whether they are co-cultured with dorsal half or ventral half. Note that collagenase-treated optic vesicle did not show Tbx5 expression but displayed pigmentation (asterisk in G). Asterisks indicate grafts of optic vesicles. Arrowheads in (D) and (G) indicate optic vesicle portions of co-cultured ventral and dorsal heads, respectively. Bar (A), 250 µm. Fig. 10. Neural crest cell migration in developing chick embryos and in cultures of the dorsal half as shown by HNK-1 immunostaining. (A,B,C) 8-, 10- 13-somite stages of embryos. Positive cells became to gradually cover the developing optic vesicles. At 13-somite stage, most regions of the optic cup are covered with positive cells except future lens-forming area and ventral area. (D,E) Dorsal half head removed from 10-somite stage embryo and cultured. Arrowheads in (D) indicate areas of melanin deposition. Arrowheads in (E) indicate the area of melanin deposition shown at a higher magnification. HNK-1 positive neural crest cells appear to surround the area of melanin deposition. Bar in (A) is 250 µm and applies to (B,C,D). Bar (E), 100 µm. optic vesicle was transplanted into the chick wing bud, numerous chick mesenchymal cells surrounded the RPE (Fig. 2C). This indicates that the mesenchymal cells need not necessarily be derived from the periocular mesenchyme. Members of the BMP family are expressed in the apical ectodermal ridge of developing limbs throughout their life, as well as in the mesenchyme (Pizette & Niswander 1999, 2001). Since BMP4 appears to function in RPE development, the limb mesenchyme may play a role as a source of BMP signal. It is, however, still unclear whether, without any periocular mesenchymal tissue, Extraocular signal and eye development the optic vesicle can develop normally, or whether a small amount of ocular mesenchymal cells are needed only in the beginning of development. Fuhrmann et al. (2000) reported that extraocular mesenchyme, but not lateral plate mesoderm, is required for the expression of the RPE-specific genes. Our present data indicates that the proper morphogenesis of the eye anlage may depend on the extraocular mesenchyme for the initial step of eye morphogenesis, and that once it is initialized the non-specific mesodermal tissue can take over the further eye development. The significant role of the extraocular mesenchyme was also supported by an organ-culture method. RPE differentiated in the outer layer, making contact with the mesenchymal tissue, while the inner layer differentiated into NR facing the lens. This was also evidenced by immunostaining for Mitf and cRax, suitable markers for RPE and NR differentiation, respectively (Mochii et al. 1998; Ohuchi et al. 1999; Sakagami et al. 2003). When the mesenchymal tissue was removed by enzymatic treatment, no Mitf immunostaining could be detected but cRax staining was still found, suggesting the possibility that NR is a default pathway of optic vesicle development. Extraocular mesenchyme has two different sources, the dorsally located neural crest cells and ventrally located precaudal mesodermal cells. Although it is difficult to clearly distinguish these two cells in the histological sections, the former type of cells are considered to play the essential role in the patterning of the optic vesicle. This was drawn from the culture experiments: the collagenase-treated optic vesicle seldom showed Mitf expression nor melanin deposition (Fig. 4), but they developed into both NR and RPE when co-cultured with the dorsal head explant (Fig. 6). Developmental fates of the dorsal and ventral optic vesicle The dorsal and ventral parts of the optic vesicle appear to develop somewhat differently from each other (Romanoff 1960; Bellairs & Osmond 1998). The distal and ventral parts are to organize the inner layer of the optic cup and develop as NR, while the dorsalmost part is considered to organize the outer layer, fated to become RPE. To determine the developmental fates of the dorsal and ventral parts of the optic vesicle, we separated the head portion into its dorsal and ventral halves by making an incisor cut at the horizontal midline of the head, and cultured them in an organotypic condition. The results clearly showed that intense Mitf staining was seen in the cultured 533 dorsal part, whereas cRax staining was found only in the ventral part, suggesting that these two regions are specified to develop along separate fates in the early stage of development by inductive signals from surrounding tissues. Our recent study also supports this notion by an experiment lesioning either the dorsal or ventral half of the chick optic vesicle (manuscript in preparation). In contrast to the chick, Mitf expression in the mouse starts throughout the optic vesicle and later becomes restricted to the presumptive RPE (Bora et al. 1998). In a developing mouse optic vesicle, Mitf and Chx10, a neuroretina-specific marker, were initially coexpressed and later the surface ectoderm-derived factor downregulated Mitf, suggesting a somewhat different inductive mechanism from that of chick eye development (Nguyen & Arnheiter 2000; Vogel-Hopker et al. 2000). Recent studies have also implicated fibroblast growth factors (FGF) as candidate factors released from the lens ectoderm that pattern the distal optic vesicle (Pittack et al. 1997; Hyer et al. 1998). A study by Dütting and Thanos (1995) revealed the fate mapping of the chick optic vesicle at the HH 11 stage (approximately corresponding to 13-somite stage) by injecting DiI, and showed that the distal sector of the dorsal part is destined to become the dorsal-most region of the retina. This appears to be inconsistent with the present result that positive staining for cRax was rarely found in the organ-cultured dorsal half of the head portion (Fig. 5). Although results obtained by a fate-mapping study have a different meaning from those by explant culture experiments, several possibilities can be considered for that inconsistency: isolation and culturing of the dorsal head explant may alter the geographic relations among the neuroepithelium, mesenchymal tissue and surface ectoderm, and this will affect the developmental fate of the dorsal optic vesicle. Alternatively, the optic vesicle may be patterned by diffusible factors emanating from the dorsal and ventral poles of the vesicle (or the surrounding tissue at these poles) and that separation of the vesicle into dorsal and ventral halves depletes the dorsal explant from the ventrally derived signal and vice versa. The expression pattern of transcription factors such as Pax2, cVax, and Tbx5 in the optic cup indicates that the optic cup can be divided into three sectors in relation to the DV direction (Schulte et al. 1999; Peter & Cepko 2002). The medial part is devoid of the expression of these genes, and may not be rigidly committed as early as at the stage of the optic vesicle. Their fates may be influenced by the reciprocal signals from the dorsal and ventral parts of the optic vesicle, as mentioned above. 534 Y. Kagiyama et al. Dorsal head-derived factor regulates dorso-ventral specialization We hypothesized factors that may be derived from the dorsal part of the head and play a role in RPE determination. This was examined in the present study by co-culturing the optic vesicle with the dorsal head (Fig. 6). The optic vesicle was treated to remove the surrounding tissues and was juxtaposed in vitro with the dorsal head. It was shown that this treatment induced Mitf expression in the optic vesicle that otherwise could not be found. The ventral head did not have this effect on Mitf but it apparently upregulated cRax expression to some extent. These results clearly indicate the existence of a dorsal factor(s) in the dorsal head that upregulates Mitf expression. Interestingly, this dorsal factor also suppressed Pax2 expression in the optic vesicle. Pax2 plays an important role in the development of the ventral structure of the eye, such as the optic stalk and ventral fissure (Torres et al. 1996; Schwartz et al. 2000). Pax2 appears to repress the expression of Otx2, a gene that has a crucial role for the determination of the presumptive RPE territory (Martinez-Morales et al. 2001; Bovolenta et al. 1997). Pax2 and Pax6 seem to cooperate to establish the boundary between the optic stalk and optic cup by reciprocally controlling their expression levels (Schwartz et al. 2000). In the present study, Pax2 expression was not affected by the removal of the surrounding tissues, but was repressed by co-culturing with the dorsal head (Fig. 9). Thus, the dorsal factors appear to have two effects on the optic vesicle: induction of Mitf expression and suppression of Pax2, leading the optic vesicle to be dorsalized. In contrast, the ventral head appears not to have a crucial role in the development of the NR and RPE, although it enhanced cRax expression in addition to Pax2 expression to a certain extent. With respect to dorsalizing factors, it has been reported that BMP play crucial roles in the regional morphogenesis of the dorsal forebrain in mouse embryos by regulating specific gene expression, cell proliferation and local cell death, and BMP4 is expressed in the dorsal region of the optic cup in the mouse embryo (Furuta et al. 1997). It has also been reported that when mouse BMP4 is misexpressed in the ventral half of the optic cup in chick embryos, round eyes are formed with the expansion of Tbx5 expression in the ventral half, and that expression of Vax and Pax2 is repressed (KoshibaTakeuchi et al. 2000). When BMP4-soaked beads were implanted into the head region at HH stage 11/ 12 of the chick embryo, the resulting optic tissue was entirely pigmented with a complete absence of neural retinal tissue (Hyer et al. 2003). The present culture study indicates that the surrounding tissue is essential for Tbx5 expression in the optic vesicle and the dorsal head explant can not compensate for the tissue. The previous in ovo explant culture study showed that in explants taken from embyros earlier than the 10-somite stage Tbx5 expression could not be seen, while Pax2 was expressed. Explants taken from embryos at the 14-somite stage normally expressed Tbx5 (Uemonsa et al. 2002). These results suggest that Tbx5 expression is specified between the 10- and 14-somite stages under the influences coming from both the surrounding (mesenchymal) tissues and brain portions. It is also suggested that Tbx5 does not directly suppress Pax2 expression, since loss of Tbx5 expression did not cause expansion of Pax2 positive areas as shown presently and in the previous study. The previous work by Fuhrmann et al. (2000) indicated that the extraocular mesenchymal cells inhibited the expression of the NR-specific transcription factor, Chx10, and that these cells may originate from the cranial neural crest. From our present observations that HNK-1 positive cells were always gathering at the area with pigmented granules (Fig. 10), we can also suppose that the neural crest-derived mesenchymal cells, densely distributed at the dorsal part of the optic vesicle, must direct the optic vesicle to develop into RPE. Neural crest cell migration to the optic vesicle still continues up to later stages (18somite stage), and these migrating cells may reinforce the fate of the dorsal optic vesicle. In early Xenopus embryos, BMP-2,4 are expressed in the head neural crest cells (Fainsod et al. 1994; Clement et al. 1995). BMP-regulated Smad proteins, intracellular signaling molecules specific for BMP, are also localized in the neural crest-derived ocular mesenchymal cells (Kurata et al. 2001). These studies indicate a possibility that ocular neural crest cells may be the source of BMP signals. Tissue interaction between the neuroepithelium and the surface ectoderm may also bring about a separation into a discrete expression domain, and FGF signaling plays an important role in this process (Hyer et al. 1998; Nguyen & Arnheiter 2000; VogelHopker et al. 2000; Zhao et al. 2001). Conditional deletion of Pax6 expression in the surface ectoderm by using a Pax6 surface ectoderm-specific Cre mouse indicated that the developing lens is required for the proper organization of the NR and RPE (Ashery-Padan et al. 2000). Hyer et al. (2003) reported that surgical removal of the pre-lens ectoderm of the 11-somitestage embryo resulted in a persistent optic vesicle that initiated NR differentiation but failed to undergo Extraocular signal and eye development invagination. Such an ectoderm-less optic vesicle still showed patterned development of RPE and NR, indicating that the mesenchymal tissue has the primary function in RPE differentiation, as shown in the present study. It is plausible to consider that the surface ectoderm that covers the optic vesicle also plays some role in the DV patterning of the eye primordium, but this must be examined in future experiments. Acknowledgements We are sincerely grateful to Dr Dorothea Schulte for valuable discussion and critical reading of the manuscript. This work was supported in part by a Grant-in-Aid and Special Coordination Funds for Brain Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to M. Araki. 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