Development of Dorsal-Ventral Polarity in the Optic Vesicle and Its Presumptive Role in Eye Morphogenesis As Shown by Embryonic

Home , Eye development, Neural plate, Neural tube, Optic cup (anatomical), Optic cup (embryology), Optic stalk

Developmental Biology 248, 319–330 (2002) doi:10.1006/dbio.2002.0737

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Development of Dorsal–Ventral Polarity in the provided by Elsevier - Publisher Connector Optic Vesicle and Its Presumptive Role in Eye Morphogenesis As Shown by Embryonic Transplantation and in Ovo Explant Culturing

Tomoko Uemonsa,* Kiyo Sakagami,† Kunio Yasuda,† and Masasuke Araki*,1 *Developmental Neurobiology Laboratory, Faculty of Science, Nara Women’s University, Nara 630-8506, Japan; and †Laboratory of Molecular and Developmental Biology, Nara Institute of Science and Technology, Ikoma 630-0101, Japan

Dorsal and ventral specification in the early optic vesicle appears to play a crucial role in the proper development of the eye. In the present study, we performed embryonic transplantation and organ culturing of the chick optic vesicle in order to investigate how the dorsal–ventral (D-V) polarity is established in the optic vesicle and what role this polarity plays in proper eye development. The left optic vesicle was cut and transplanted inversely in the right eye cavity of host chick embryos. This method ensured that the D-V polarity was reversed while the anteroposterior axis remained normal. The results showed that the location of the choroid fissure was altered from the normal (ventral) to ectopic positions as the embryonic stage of transplantation progressed from 6 to 18 somites. At the same time, the shape of the optic vesicle and the expression patterns of Pax2 and Tbx5, marker genes for ventral and dorsal regions of the optic vesicle, respectively, changed concomitantly in a similar way. The crucial period was between the 8- and 14-somite stages, and during this period the polarity seemed to be gradually determined. In ovo explant culturing of the optic vesicle showed that the D-V polarity and choroid fissure formation were already specified by the 10-somite stage. These results indicate that the D-V polarity of the optic vesicle is established gradually between 8- and 14-somite stages under the influence of signals derived from the midline portion of the forebrain. The presumptive signal(s) appeared to be transmitted from proximal to distal regions within the optic vesicle. A severe anomaly was observed in the development of optic vesicles reversely transplanted around the 10-somite stage: the optic cup formation was disturbed and subsequently the neural retina and pigment epithelium did not develop normally. We concluded that establishment of the D-V polarity in the optic vesicle plays an essential role in the patterning and differentiation of the neural retina and pigment epithelium. © 2002 Elsevier Science (USA) Key Words: optic vesicle; dorsal–ventral axis; chick; transplantation; choroid fissure; Pax2; Tbx5.

INTRODUCTION the surface ectoderm. Subsequently, the optic vesicle in- vaginates to form the double-layered structure of the optic Development of the vertebrate eye depends on coordi- cup, while the surface ectoderm, which contacts the optic nated interactions between three distinct tissues: the neu- vesicle, thickens into a placode that will form a lens vesicle roepithelium, overlying surface ectoderm, and mesen- (Grainger, 1992; Saha et al., 1992). chyme (Mikami, 1939; Hyer et al., 1998; Fuhrmann et al., The invagination of the optic vesicle begins at a point 2000). In the early stage of eye development, the optic displaced somewhat toward its ventral surface, rather than vesicle arises as a lateral outgrowth of the neural tube in the beginning at the most lateral point in the optic vesicle, and forebrain region and extends until it makes contact with is directed mediodorsally (Romanoff, 1960; Bellairs and Osmond, 1998). Consequently, the distal and ventral por- 1 To whom correspondence should be addressed. Fax: ϩ81-742- tions of the optic vesicle are considered to organize the 20-3411. E-mail: [email protected]. inner layer of the optic cup fated to become the neural

FIG. 1. (A) Schematic drawings of developing optic vesicles of chick embryos. Transplantation of optic vesicle was performed on embryos between the 6- and 17-somite stages. The lateral walls of the forebrain evaginate bilaterally to form optic vesicles at the 6-somite stage. At the 10-somite stage, the optic vesicle makes contact with the surface ectoderm, which thickens into the lens placode at the 17-somite stage. The optic vesicle then starts invagination to form the optic cup. (B) Diagram of surgical operation. The left optic vesicle from the donor embryo was transplanted inversely in the place of the host right optic vesicle. The D-V polarity was reversed, while the A-P polarity remained normal. Transplantation was usually carried out between embryos at the same stage.

FIG. 3. The location of the optic stalk in transplanted eyes observed on day E2. Transplanted eyes are on the left side, and the upper side corresponds to the dorsal direction. Arrows indicate the presumptive optic stalk. (A) Transplanted at the 6-somite stage. The optic cup connects to the ventral forebrain by the ventrally formed optic stalk (arrow). (B) Transplanted at the 10-somite stage. The optic cup connects to the lateral forebrain. (C) Transplanted at the 14-somite stage. The optic cup connects to the more dorsal region of the forebrain. FIG. 4. Expression of Pax2 and Tbx5 in transplanted optic vesicles. Whole-mount in situ hybridization was performed on day E2.5–E3. (A, B) Expression patterns of Pax2 and Tbx5 in normal embryos. In (A), Pax2 is expressed in the ventral region of the optic cup around the choroid fissure (arrow), while in (B), Tbx5 is expressed dorsally. (C, D) After transplantation at the 6-somite stage, the expression of these genes is similar to that in normally developing eyes. Arrows indicate choroid fissures formed ventrally. (E, F) Transplanted at the 10-somite stage. Pax2 (E) and Tbx5 (F) are expressed in the anterior and posterior sides of the optic cup, respectively. (G, H) Transplanted at the 17-somite stage. Arrows indicate choroid fissures formed dorsally. In (G), Pax2 is expressed around the choroid fissure, and in (H), Tbx5 is expressed in the ventral side of the optic cup.

retina (NR), while the dorsal-most region is considered to is continued from the ventral side of the optic cup to the organize the outer layer fated to become the retinal pig- region of the optic stalk. As a result, the ventral surface of mented epithelium (RPE). the optic cup and stalk become grooved, forming the future The optic cup is connected to the ventral diencephalon by choroid ﬁssure, in which the optic nerves and blood vessels the optic stalk. The infolding process of optic cup formation come to lie later in development (Bellairs and Osmond,

FIG. 2. Choroid fissure formation in transplanted optic vesicles. Embryos were examined at around day E2.5–E3 for the location of the choroid fissure. (A) Stage of transplantation and location of the choroid fissure. As the stage of grafting became later, the location of the choroid fissure changed from ventral to dorsal. When transplantation was performed between the 8- and 14-somite stages, choroid fissure formation was seen in only a few cases, and was seen particularly rarely in 10-somite embryos. (B–F) Lateral views of the transplanted side, showing the location of choroid fissure. Schematic drawings under the photographs show the configuration and direction of the transplanted optic cups. Transplantation was made at the 6-, 8-, 10-, 14-, and 17-somite stages, as shown in (B–F), respectively. In (B), the choroid fissure formed ventrally (arrow). In (C), the choroid fissure was not formed, and the developing optic cup appears to show a normal configuration with the normal direction. In (D), the choroid fissure was not formed, and most of the optic cups show an oval shape along the A-P direction. In (E), the choroid fissure was not formed, and the shape of the optic cup shows a reversed direction. In (F), the choroid fissure was formed dorsally (arrow).

1998), and retinal axons project in an organized topographic yolk beneath embryos to visualize the embryonic structures. manner through the choroid fissure (Udin and Fawcett, Embryos were staged according to Hamburger and Hamilton (1951) 1988; Holt and Harris, 1993). To accomplish this, the cells (HH). within the developing NR need to acquire distinct posi- Transplantation of the optic vesicle was performed in embryos tional cues specifying the anterior–posterior (A-P) and from the 6- to 17-somite stages (Fig. 1A; 6-somite stage corresponds to HH8, 8-somite to HH9, 10-somite to HH10, 14-somite to HH11, dorsal–ventral (D-V) polarities. Thus, for proper eye devel- and 17-somite to HH12). The left optic vesicle of the donor embryo opment, regional specialization must occur in the optic was truncated at the most proximal part close to the forebrain and vesicle according to the axial patterns. was transplanted to the right cavity of the host embryo, from which In the present study, we performed embryonic transplan- the counterpart optic vesicle had previously been removed. Trans- tation of the chick optic vesicle to investigate how the D-V plantation was always carried out between embryos of the same polarity is formed and what role it plays in eye develop- stages. ment. For this purpose, the left optic vesicle was grafted The surgical operation was performed as follows: donor embryos inversely in the place of the right cavity of host embryos, were placed into round-bottomed dishes filled with Hanks’ solu- resulting in reversed D-V polarity with normal A-P polarity. tion to wash out the yolk and India ink. The embryos were then The position of the choroid fissure, which is normally pinned over a black silicone plate, and the left optic vesicle was cut out by using a fine sharpened needle with the covering ectoderm formed ventrally, was examined with respect to whether it and the mesenchymal tissues (Fig. 1B). The orientation of the graft formed normally or ectopically. At the same time, we made was clearly distinguishable by the shape. Grafts were kept in in ovo explant cultures of the isolated optic vesicle to ice-chilled Hanks’ solution until transplantation. Subsequently, determine the time when the D-V regions are specified. In the right optic vesicle of the host embryo was excised in ovo after addition to morphologic observation, we examined the the vitelline membrane was opened. The graft was placed in the expression patterns of Pax2 and Tbx5 as marker genes for corresponding cavity of the host embryo (Fig. 1B). A close match in ventral and dorsal regions, respectively. Pax2, a member of the size between the graft and the host cavity was very important the paired box gene family, is first expressed at stage 10 in for the successful integration of the graft. Particular care was taken the ventral half of the optic vesicle (Nornes et al., 1990; to maintain the correct anteroposterior orientation of the graft. Krauss et al., 1991; Puschel et al., 1992). The chick Tbx5 After transplantation, the windows of the host eggs were sealed with sealing tape, and the eggs were incubated at 37.6°C. One hour gene, a member of the T-box transcription factor family, is later, the sealing tape was removed to confirm that the graft was expressed in the dorsal side of the developing eye and is firmly attached to the host brain vesicle. The egg was sealed again homologous to the Drosophila optomotor blind (omb) gene, and incubated until embryonic day 2–3 (day E2–E3, corresponding which regulates optic lobe development. Tbx5 expression to stages 15–18), at which stage the choroid fissure is already in the chick eye was first detected at stage 11 throughout formed in unoperated eyes. In some cases, embryos were incubated the retina, with the strongest signal in the dorsal retina. until day E4–E 5 (stage 21–23). The Tbx5 gene is considered to be a strong candidate for a In some graft experiments, the optic vesicles of embryos at determinant of the D-V axis of the eye (Gibson-Brown et al., 10-somite stage were truncated at more distal locations and in- 1998; Chapman et al., 1996; Jeremy et al., 1998; Koshiba- versely transplanted in a similar way, and the results were com- Takeuchi et al., 2000). pared with those obtained for embryos truncated at a more proximal location. The present results showed that the D-V polarity of the For in ovo explant cultures, optic vesicles were cut from em- optic vesicle is gradually established during early optic bryos at the 8-, 10-, 14-, and 17-somite stages and placed into the vesicle development and that the inversion of the D-V amnion of host embryos. They were allowed to develop further in polarity at particular stages causes the failure of optic cup ovo, and optic cup formation as well as choroid fissure formation formation, followed by a loss of organized structures of the were observed. To allow determination of the orientation of NR and RPE. This suggests that the establishment of the explants, charcoal-activated powder was sprinkled on the most D-V polarity is a prerequisite for the proper development dorsal part of the graft. and organization of the NR and RPE. Our results also suggest that presumptive signal(s) derived from the midline portion of the embryo and transmitted within the optic In Situ Hybridization vesicle play a significant role in the determination of the D-V polarity in the optic vesicle. Plasmids containing Tbx5 and Pax2 cDNAs in pBluescript SK(Ϫ) were linearized with EcoRV and EcoRI and transcribed in vitro with T3 RNA polymerase to generate digoxigenin-labeled anti- MATERIALS AND METHODS sense and sense RNA probes, respectively (Promega). The Tbx5 cDNA was obtained from Dr. T. Ogura (Nara Institute of Science Surgical Manipulation of Embryos and Technology) and the Pax2 cDNA from Dr. H. Nakamura (Tohoku University). Whole-mount in situ hybridization was per- Fertilized eggs were incubated in a humidified atmosphere at formed as described previously (Henrique et al., 1995). After 37.6°C. All operations were carried out according to the procedures hybridization, embryos were incubated with 1/2000-diluted alka- previously described (Alvarado-Mallart and Sotelo, 1984; Bronner- line phosphatase-conjugated anti-DIG antibody (Roche) overnight, Fraser, 1996; Araki et al., 2002). A small window was made in the and NBT (nitroblue tetrazolium)/BCIP (5-bromo-4-chloro-3-indoyl shell, and India ink diluted in Hanks’ solution was injected into the phosphate) staining was employed for the detection of hybridiza-

© 2002 Elsevier Science (USA). All rights reserved. D-V Polarity in Optic Cup Formation 323 tion signals. Embryos were photographed with a camera attached to and 17-somite stages were connected to the dorsal forebrain a stereomicroscope (Olympus). by a dorsally formed optic stalk (Fig. 3C). To see whether the procedure of truncation affects the Histological Preparation development of the optic cup and choroid fissure, truncated optic vesicles were transplanted to host embryos at the For the histological observation, embryos were fixed with Bouin 10-somite stage without inversion. The eyes developed fixative for 3–10 h (depending on their size), dehydrated in a graded normally and no abnormal features were noticed, indicating series of ethanol, and finally embedded in paraffin. Serial sections that the truncation procedure had no apparent effect on eye of 6-␮m thickness were cut in a transverse plane and stained with hematoxylin and eosin. development in agreement with the results in a previous study (Araki et al., 2002). Expression patterns of Pax2 and Tbx5. We next exam- RESULTS ined the expression patterns of two genes, Pax2 and Tbx5, markers for the ventral and dorsal regions of the optic cup, D-V Polarity-Reversed Transplantation respectively. Pax2 is expressed in ventral structures, such as the optic stalk and early retinal cells around the choroid Choroid fissure formation in the D-V polarity-reversed fissure (Fig. 4A), while Tbx5 is restricted to the dorsal transplantation. The location of a choroid fissure in the region of the optic (Fig. 4B). We performed whole-mount in graft was examined at day E2.5 in embryos that underwent situ hybridization of these genes on transplanted embryos reverse transplantation of the optic vesicle between the 6- at day E2.5. and 17-somite stages. The choroid fissure was formed When grafting was done at the 6-somite stage (n ϭ 4), ventrally when the transplantation was done at earlier Pax2 was expressed in the ventral region of the optic cup stages (6- and 8-somite stages), while it was formed dorsally and Tbx5 was expressed in the dorsal region (Fig. 4D), as when transplantation was done at later stages (14- and seen in the normally developing eye. When grafting was 17-somite stage; Fig. 2A). done at the 10-somite stage (n ϭ 12), Pax2 was expressed in When transplantation was performed at the 6-somite the anterior region of the optic cup, and Tbx5 was expressed stage (n ϭ 12), the grafted optic vesicle developed to an optic in the posterior region (Fig. 4E), and in embryos grafted at cup with a ventral choroid fissure in all cases (Figs. 2A and the 17-somite stage (n ϭ 4), Pax2 was expressed around the 2B). When grafting was performed at the 8-somite stage (n ϭ choroid fissure formed dorsally and Tbx5 was expressed in 11), a normally appearing choroid fissure or, in some cases, the ventral region (Fig. 4H). It was confirmed again that in a groove-like choroid fissure was formed ventrally in a most cases of transplantation at 10-somite stage no choroid small fraction (36%) of embryos (Figs. 2A and 2C), while in fissure was formed and the optic cup showed an oval shape most cases, no choroid fissure was formed. At both stages extending anteroposteriorly. Pax2 was expressed in the (6- and 8-somite stages), the optic cup was connected to the Tbx5 ventral forebrain by the optic stalk, which was formed anterior region, while expression was seen in the ventrally (Fig. 3A). posterior region, indicating that segregated localization of It is interesting to note that no choroid fissure was the two gene transcripts was achieved within the optic cup. formed in most cases (95% of embryos) when grafted at the Thus, the expression of the two marker genes changed in 10-somite stage (n ϭ 18) (Figs. 2A and 2D). In one excep- the direction from the normal D-V to the inverted V-D tional case out of 18 specimens, a choroid fissure was location via the A-P location. formed anteriorly (data not shown). All of the transplanted Effect of the truncation site along the proximodistal eyes were connected to the lateral forebrain (data not length. To clarify whether there is any temporal delay in shown). Invagination, a necessary step for optic cup forma- the determination of the D-V polarity related to the proxi- tion, was not obvious in half the cases, and in the rest of the modistal location of the optic vesicle, optic vesicles were cases, the grafts developed to optic cups, most of which truncated in 10-somite-stage embryos at increasingly distal showed an oval shape extending anteroposteriorly rather sites and transplanted reversely into the corresponding than in the D-V direction. It appeared as if the D-V axis of cavity of the host embryos (Fig. 5). When optic vesicles were the optic vesicle had rotated 90° anteriorly. truncated in the middle of the proximodistal length (Fig. In embryos grafted at the 14-somite stage (n ϭ 12), similar 5A, indicated by line b), a choroid fissure was formed results were obtained to those for embryos grafted at the ventrally in some cases (38% of embryos, n ϭ 8), while in 10-somite stage: a choroid fissure was not formed in most the rest of the cases, no choroid fissure was formed. When cases (83% of embryos) (Figs. 2A and 2E), and in the rest of the vesicles were truncated at a more distal location (Fig. the cases (17% of embryos), either a normal choroid fissure 5A, line c), a choroid fissure was formed ventrally in half of or a groove-like choroid fissure was formed at the dorsal– the cases (58% of embryos, n ϭ 12), and in the rest of the anterior location of the optic cup. When embryos were cases, no choroid fissure was formed. These results indicate grafted at the 17-somite stage (n ϭ 15), a choroid fissure was that optic vesicles cut at a more distal location form a formed dorsally in most cases (93% of embryos) (Figs. 2A ventral choroid fissure more frequently and suggest that and 2F). The optic cups of embryos transplanted at the 14- development of the choroid fissure is affected by some

FIG. 5. Effect of the truncation site along the proximodistal length of the optic vesicle. (A) Diagram of operation: optic vesicles were truncated at three different sites. (a) the most proximal, (b) the middle, and (c) the most distal sites. They were transplanted in reverse in host embryos in which the corresponding regions had been removed. (B) Summary of the results. When transplanted at the most proximal site, no choroid ﬁssure was formed in any of the cases.

factor(s) in the optic vesicle that are present in a proximo- 14-somite stage (n ϭ 18) and cultured developed into optic distal gradient. cups with a ventral choroid fissure or a groove-like choroid fissure in most cases (Fig. 6D). In some cases, no choroid fissure was formed in the optic cup. Optic vesicles trun- In Ovo Explant Culture cated at the 17-somite stage (n ϭ 10) developed into optic We next performed in ovo explant culturing of optic cups with a ventral choroid fissure in most cases. These vesicles to determine the time when the D-V regions are optic vesicles truncated at the 14- and 17-somite stages specified in the optic vesicle. Optic vesicles truncated at the developed into oval-shaped optic cups extending in the D-V 6-, 8-, 10-, 14-, and 17-somite stages were placed within the direction, while those truncated at the 10-somite stage amnion of host embryos and cultured in ovo for 2–3 days formed a round-shaped optic cup, if any. These results and examined for the location of a choroid fissure and Pax2 indicate that optic vesicles at the 10-somite stage develop expression site (Fig. 6). The original dorsal position had into optic cups autonomously without any further influ- been marked with charcoal-activated powder before graft- ence from the neighboring brain regions. The results also ing. suggest that the distinct capacity for choroid fissure forma- Choroid fissure formation in the in ovo-cultured optic tion is gradually acquired in the ventral region between the vesicles. Optic vesicles truncated at the 6-somite stage 10- and 14-somite stages under the influence of the neigh- (n ϭ 10) underwent a slight but incomplete invagination, boring tissues. and no choroid fissure was formed in any of the cases (Fig. Expression pattern of Pax2 and Tbx5 as marker genes 6B). Optic vesicles truncated at the 10-somite stage (n ϭ 25) for D-V regions. In explants taken from embryos at the developed into an optic cup with a round shape. In half of 6-somite stage (n ϭ 3), Pax2 was expressed in the ventral the specimens, optic vesicles developed into optic cups region of developing optic cups in all cases (Fig. 6B). The with a ventrally formed choroid fissure or groove-like same result was obtained in all cases (n ϭ 4) when explants choroid fissure (Fig. 6C), and in the rest of the cases, optic were made at the 8- and 10-somite stages. On the other cups had no choroid fissure. Optic vesicles truncated at the hand, no Tbx5 expression was seen in any cases of explants

FIG. 6. Specification of the choroid fissure formation and the expression of Pax2 and Tbx5. Optic vesicles from various embryonic stages were cultured in ovo to examine the stage when D-V polarity is specified intrinsically. (A) Summary of the results. In explant cultures of optic vesicles from the 6- and 8-somite stages, no choroid fissure is formed. At the 10-somite stage, choroid fissures are formed ventrally in half of the cases. (B, C, E, F) Expression of Pax2/Tbx5 in explant cultures. (B) In explants cultured at the 6-somite stage, Pax2 is expressed in the ventral half. Invagination of the optic vesicle is incomplete and no choroid fissure is formed. (C) In explants cultured at the 10-somite stage, Tbx5 is expressed in the dorsal half. (D–F) Explants cultured at the 17-somite stage. In (D), a choroid fissure is formed ventrally (arrow). In (E), Pax2 is expressed around the ventral choroid fissure, and in (F), Tbx5 is expressed in the dorsal half. Charcoal powder (arrowheads in C and E) indicates the original dorsal region of explants. L, lens vesicle; cf, choroid fissure.

taken from embryos at the 6- (n ϭ 2) and 8- (n ϭ 3) somite (NR) and pigment epithelium (RPE) (Fig. 7). At this stage, a stages. In explants taken from embryos at the 10-somite choroid fissure can be recognized as a white line at the stage (n ϭ 2), Tbx5 expression was observed in the dorsal ventral location (Fig. 7A), and the inner layer of the optic region of developing optic cups, but only at a low level (Fig. cup has clearly differentiated as the NR, and the outer layer 6C). In explants taken at the 14-somite stage (n ϭ 3), Tbx5 as the RPE (Fig. 7E). was normally expressed. These results suggest that Pax2 When optic vesicles were reversely transplanted at the expression is already specified in the optic vesicle prior to 6-somite stage (n ϭ 10), the optic cup developed normally in the 6-somite stage, while Tbx5 expression is specified later, most cases (90% of embryos; data not shown), and both the at around the 10-somite stage. Tbx5 expression in the dorsal NR and RPE differentiated normally. When transplanted at region also appears to be correlated with the development of the 8- (n ϭ 11) and 10- (n ϭ 16) somite stages, approximately morphological polarity of the eyecup. half of the grafts (55% from 8-somite embryos and 44% from 10-somite embryos) developed into optic cups without a choroid fissure, and both the NR and RPE were normally Histological Observation of Reversely developed. In the rest of the cases (45% from 8-somite Transplanted Optic Vesicle embryos; 56% from 10-somite embryos), invagination of Reversely transplanted eyes were fixed at day E4.5 (stage optic vesicles was incomplete and the bilayered optic cup 23) and examined for the development of the neural retina failed to develop (Fig. 7D) and lacked the normal organiza-

© 2002 Elsevier Science (USA). All rights reserved. 326 Uemonsa et al. tion of the NR and RPE. Instead, the wall of the optic cup fied around the 10-somite stage. It seems that the expres- consisted of a multistratified cell layer which often pos- sion of a ventral marker gene is specified first, and during sessed pigmented granules (Figs. 7F and 7G). subsequent development the ventral region is specified to When transplanted at the 14- (n ϭ 14) and 17- (n ϭ 6) form the choroid fissure by the 10-somite stage. The ques- somite stages, optic cups developed normally with a dorsal tions then arise as to why the optic vesicle development is choroid fissure (86% from 14-somite embryos; 100% from severely affected and the choroid fissure formation is dis- 17-somite embryos). The morphology of these eyes, includ- rupted when the optic vesicle is reversely transplanted ing the NR and RPE, was normal except for the location of between the 8- and 14-somite stages. Our presumption is the choroid fissure. that some factor(s) derived from the midline region of the forebrain may have a substantial role in the development of the D-V polarity in the optic vesicle. By reverse transplan- DISCUSSION tation, the ventral part of an optic vesicle comes into contact with the dorsal part of the host forebrain and vice The structures of the vertebrate eye are organized accord- versa. As a result, regional signals from the host embryo ing to three distinct axes, and these axes are determined may be transmitted to the opposite region of the graft, during early embryonic development under the influence of which has in part already been specified, counteracting the the neighboring regions. The A-P and D-V axes appear to D-V polarity. Subsequently, the presumed regionalizing play important roles not only in development of the neural factor in the transplanted optic vesicle may be neutralized retina (NR) and pigment epithelium (RPE), but in the to a certain extent, causing the disorder of D-V polarity and establishment of the retinotectal projection. Because axis failure of choroid fissure formation. It is significant to note formation is a crucial process for the early development of that the shape of the optic cup was oval and oriented in an the eye, we addressed this issue by using an embryonic A-P direction when transplanted at the 10-somite stage, and in ovo transplantation technique and organ culture com- that the expression pattern of Pax2 and Tbx5 also changed bined with the expression of region-specific marker genes. as if the D-V axis had rotated 90° anteriorly. Since the A-P With these approaches, we investigated how the D-V polar- axis appears to be determined at or prior to the 10-somite ity is specified and determined during the development of stage (Matsuno et al., 1992; Dutting et al., 1995), our the optic vesicle. We also considered what role this polarity findings suggest that formation of the D-V polarity may be plays in the development of the NR and RPE. coordinated with the A-P axis formation, although the mechanism is totally unknown at the moment. Development of the D-V Polarity in the Optic In a different series of transplantation, a more distal Vesicle portion of the optic vesicle was truncated and transplanted reversely at the 10-somite stage. The results showed that When the optic vesicle was reversely transplanted at the 6-somite stage, a choroid fissure was formed ventrally (in the more distally it was truncated, the less disrupted was the normal position), and the expression of both Pax2 and the optic vesicle in the development of the optic cup and Tbx5 was seen in a region-specific manner similar to that in the formation of the choroid fissure. As discussed above, we the normally developing eye. In embryos with optic vesicles suppose that regionalizing signals may come from the host reversely transplanted at between the 8- and 14-somite forebrain toward the optic vesicle. If the signals are diffus- stages, a choroid fissure was developed in only a few cases ible and transmitted gradually through the optic vesicle in and was located ectopically, depending on the stage. It is of the proximodistal direction, the distal portion of the optic particular interest that, in embryos with optic vesicles vesicle might be less specified than the proximal portion. reversely transplanted at the 10-somite stage, no choroid A similar embryonic transplantation study in chick em- fissure was formed, except in one case, where it was formed bryos was previously reported, in which an optic vesicle anteriorly. When vesicles were transplanted at the 17- from various stages between HH stages 9 and 17 was somite stage, a choroid fissure was always formed dorsally. transplanted reversely into a host embryo at stage 11 The expression pattern of Pax2 and Tbx5 was also changed (Silver, 1977). In that study, when the grafts were from concurrently with the change of the choroid fissure loca- younger embryos, a choroid fissure was formed ventrally, tion. These results indicate that the D-V polarity in the whereas it was formed dorsally when grafts were from older optic vesicle is formed gradually during early embryonic embryos. Goldberg (1976) also performed a similar work development, and the crucial period seems to be between and obtained results similar to the present observations. the 8- and 14-somite stages; at the 6-somite stage, the D-V However, our study is the first to reveal that the D-V polarity of the optic vesicle appears not to be yet deter- polarity is gradually specified and determined around at the mined, while it appeared to be completely determined at 10-somite stage under the influence of the presumed mid- the 17-somite stage. line signals and that the polarity plays an essential role in Separate experiments using in ovo explant cultures indi- the subsequent eye morphogenesis. The positional specific- cated that Pax2 expression is already specified by the ity along the A-P axis also appears to be determined in the 6-somite stage and that choroid fissure formation is speci- optic vesicle at or prior to HH stages 10–11 (Matsuno et al.,

1992; Dutting and Meyer, 1995). Hence, that stage seems to vesicle developed to an abnormal pigmented vesicle com- be very critical for the early development of the eye. posed of a multistratified epithelium. These pigmented vesicles seem to be identical to the microphthalmia that was observed by Goldberg (1976) in a similar transplanta- Presumptive Regionalizing Signals Which tion work. Our histological observations suggested that the Determine the D-V Polarity NR and RPE cells might have intermingled within the Although the precise mechanism of the determination of epithelium. Our preliminary experiments showed that Rx, the D-V polarity in the optic vesicle is unknown, molecules a marker gene for NR differentiation, was expressed in that emanating from the midline of the forebrain are thought to pigmented epithelium (unpublished observations). The es- affect the D-V patterning of the eye. It has been reported tablishment of the D-V polarity in the optic vesicle appears that signals from the prechordal mesoderm control the to be closely related to the determination and separation of patterning of the ventral forebrain (Pera and Kessel, 1997; the NR and RPE domains within the optic vesicle. Dale et al., 1997, 1999) and that the notochord induces floor Tissue interaction between the optic vesicle and sur- plate cells caudally. Shh is a signaling molecule that is rounding tissues is crucial for the patterning of the eye. The expressed in cells along the ventral midline of the CNS optic vesicle is in contact with both the head ectoderm and (Fietz et al., 1994; Johnson and Tabin, 1995), including cells mesenchymal tissue, and classical experiments suggest that at the base of the optic stalks (Krauss et al., 1993). The interactions among these tissues are essential for early eye prechordal mesoderm expresses Shh and BMP7, which development. For instance, the overlying surface ectoderm appear to induce the differentiation of the ventral midline is considered to polarize the distal optic vesicle so that it cells in the diencephalon (Dale et al., 1997). In cyclops becomes the NR (Dragomirov, 1937; Lopashov, 1963). Re- mutant embryos of zebrafish, in which midline signaling is cent studies have shown that fibroblast growth factors severely perturbed, an absence of ventral midline CNS (FGFs) are candidate factors which induce NR differentia- tissue occurs, which subsequently results in the fusion of tion and suppress Mitf and pigmentation in the distal optic the bilateral eyes (Krauss et al., 1993; Macdonald et al., vesicle (Hyer et al., 1998; Pittack et al., 1997; Nguyen and 1995). In these embryos, Shh transcripts are absent in the Arnheiter, 2000). Extraocular mesenchyme appears to be neuroepithelium at the early developmental stages. In necessary for the induction and maintenance of expression mouse embryos, Shh injection expands the Vax1 and Pax2 of RPE-specific genes Mitf and Wnt13 (Fuhrmann et al., territory at the expense of the Pax6 and Rx region (Hallonet 2000). When optic vesicles are subjected to reverse trans- et al., 1999). These facts suggest that the midline signaling plantation, the local interaction with surrounding tissues is involved in the determination of the D-V patterning and must be altered, because the mesenchymal cells are largely the development of eye structures. distributed in the dorsal portion rather than the ventral With respect to the dorsalizing factors, it has been re- part. The dorsal mesenchymal cells are the neural crest ported that bone morphogenetic proteins (BMPs) play cru- origin, and numerous additional crest cells migrate to the cial roles in regional morphogenesis of the dorsal forebrain optic vesicle during subsequent development (Araki et al., in mouse embryos by regulating specific gene expression, 2002). Therefore, it is plausible that alteration of the mes- cell proliferation, and local cell death (Furuta et al., 1997). enchymal cell distribution after reverse transplantation at Expression of BMP4 or BMP7 in the ectoderm activates the 10-somite stage may cause the disruption of the D-V dorsal genes in the neural tube (Liem et al., 1995). BMP4 is polarity and severely affect the determination and develop- expressed in the dorsal region of the optic cup in the mouse ment of the NR and RPE. embryo (Furuta et al., 1997). Ectopic expression of BMP5 The expression patterns of marker genes for NR and RPE and BMP4 in the chicken forebrain leads to cyclopia differentiation have been determined in the anlagen of the (Golden et al., 1999). It has also been reported that when eye in early development. The expression of Six3, Rx, and mouse BMP4 is misexpressed in the ventral half of the optic Optx2 is first observed in the entire optic vesicle, but soon cup in chick embryos, round eyes are formed with expan- becomes restricted to the distal ventral region, which is sion of Tbx5 expression in the ventral half, and that thought to differentiate into the NR (Bovolenta et al., 1998; expression of Vax and Pax2 is repressed (Koshiba-Takeuchi Furukawa et al., 1997; Ohuchi et al., 1999; Toy et al., 1998). et al., 2000). These genes involved in the D-V patterning of Otx2 expression is diffusely distributed throughout the the rostral neural tube are also expected to have important optic vesicles at first but becomes restricted to the dorsal roles in the D-V patterning of the optic vesicle. region when the optic vesicle makes contact with the surface ectoderm. As optic cup formation proceeds, Otx2 expression becomes restricted to the outer layer, which Role of the D-V Polarity and Tissue Interaction in gives rise to the RPE (Bovolenta et al., 1997). These expres- Eye Organogenesis sion patterns of NR/RPE-specific genes within the optic As mentioned above, when the optic vesicle was re- vesicle clearly indicate that the developmental fate to versely transplanted at around the 10-somite stage, devel- become either NR or RPE is regionally determined within opment of the vesicle was disrupted in half of all cases: the the optic vesicle. The present observations indicate that optic cup formation did not proceed, and instead, the optic disruption or loss of the D-V regionalization in the optic

FIG. 7. Optic cup formation in the transplanted eyes and their histological observations. (A) Whole-mount view of a normal embryo at day E4. A choroid fissure can be recognized as a white line in a ventral location (arrow). (B) Eye reversely transplanted at the 10-somite stage. Optic cup formation is disrupted and the eye is considerably smaller than the normal eye. (C) Reversely transplanted eye at the 17-somite stage. The choroid fissure is seen dorsally (arrow), and the optic cup develops normally. cf, choroid fissure; mb, midbrain. (D) Transverse view of an operated embryo like that shown in (B) fixed on day E4. The transplanted eye is on the right side. Invagination is incomplete and the eye fails to form a bilayered optic cup. The parts indicated by rectangles are shown in (E–G) at a higher magnification. (E) In the unoperated eye, the NR and RPE are well developed. (F, G) The transplanted eye. The wall of the optic vesicle consists of a multistratified epithelium which often possesses pigment granules (arrowheads). (H) Summary of the optic cup formation in the reversely transplanted eyes. Eyes were examined to determine whether the optic cup is developed normally (as shown in A or C) or anomalously (as seen in B). In the latter case, the optic cup formed a pigmented multistratified epithelium and is referred to as a pigmented vesicle. Optic cup formation was disrupted in half of the cases transplanted reversely at 8- and 10-somite stages. When transplanted at the 6- and 14-somite stages, such anomalies were seldom observed. L, lens; NR, neural retina; RPE, retinal pigmented epithelium.

© 2002 Elsevier Science (USA). All rights reserved. D-V Polarity in Optic Cup Formation 329 vesicle causes severe failure of fate determination of the NR Dutting, D., and Meyer, S. U. (1995). Transplantation of the chick and RPE. Since Pax2 and Tbx5 appear to be localized in a eye anlage reveal an early determination of nasotemporal polar- segregated manner along the A-P length, even in such ity. Int. J. Dev. Biol. 39, 921–931. anomalous optic vesicles, localized expression of the two Fietz, M. J., Concordet, J.-P., Barbosa, R., Johnson, R., Krauss, S., genes is not sufficient for the determination of the NR and McMahon, A., Tabin, P. C., and Ingham, P. W. (1994). The RPE domains. Further investigations are needed to clarify hedgehog gene family in Drosophila and vertebrate development. the molecular mechanisms involved in such inductive Dev. Suppl.,43–51. sequences during eye development. Fuhrmann, S., Levine, E. M., and Reh, T. A. (2000). Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development 127, 4599–609. Furukawa, T., Kozak, C. A., and Cepko, C. L. (1997). rax, a novel ACKNOWLEDGMENTS paired-type homeobox gene, shows expression in the anterior neural fold and developing retina. Proc. Natl. Acad. Sci. USA 94, We thank Dr. H. Nakamura (Tohoku University) and Dr. T. 3088–3093. Ogura, J. K. Takeuchi, and K. K. Takeuchi (Nara Institute of Furuta, Y., Piston, D. W., and Hogan, B. L. M. (1997). Bone Science and Technology) for providing plasmids. We also thank N. morphogenetic proteins (BMPs) as regulators of dorsal forebrain Shimada for technical assistance. This work was supported in part development. Development 124, 2203–2212. by a Grant-in-Aid and Special Coordination Funds for Brain Re- Gibson-Brown, J. J., Agulnik, S., Silver, L. M., and Papaioannou, search from the Ministry of Education, Culture, Sports, Science and V. E. (1998). Expression of T-box genes Tbx2–Tbx5 during chick Technology of Japan (to M.A.). organogenesis. Mech. Dev. 74, 165–169. Goldberg, S. (1976). Polarization of the avian retina. Ocular transplantation studies. J. Comp. Neurol. 168, 379–392. REFERENCES Golden, J. A., Bracilovic, A., Mcfadden, K. A., and Beesley, J. S. (1999). Ectopic bone morphogenetic proteins 5 and 4 In the chicken forebrain lead to cycropia and holoprocencephaly. Proc. Alvarado-Mallart, R. M., and Sotelo, C. (1984). Homotopic and Natl. Acad. Sci. USA 96, 2439–2444. heterotopic transplantation of quail tectal primordia in chick Grainger, R. M. (1992). Embryonic lens induction: Shedding light embryos: Organization of the retinotectal projections in the on vertebrate tissue determination. Trends Genet. 8, 349–355. chimeric embryos. Dev. Biol. 103, 378–398. Araki, M., Takano, T., Uemonsa, T., Nakane, Y., Tsudzuki, M., and Hallonet, M., Hollemann, T., Pieler, T., and Gruss, P. (1999). Vax1, Kaneko, T. (2002). Epithelia-mesenchyme interaction plays an a novel homeobox-containing gene, directs development of the essential role in transdifferentiation of retinal pigment epithe- basal forebrain and visual system. Genes Dev. 13, 3106–3114. lium of silver mutant quail: Localization of FGF and related Hamburger, V., and Hamilton, H. L. (1951). A series of normal molecules and aberrant migration pattern of neural crest cells stages in the development of the chick embryo. J. Morphol. 88, during eye rudiment formation. Dev. Biol. 244, 358–371. 49–92. Bellairs, R., and Osmond, M. (1998). “The Atlas of Chick Develop- Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., and ment.” Academic Press, London. Ish-Horowicz, D. (1995). Expression of a Delta homologue in Bovolenta, P., Mallamaci, A., Briata, P., Corte, G., and Boncinelli, prospective neurons in the chick. Nature 375, 787–790. E. (1997). Implication of OTX2 in pigment epithelium determi- Holt, C. E., and Harris, W. A. (1993). Position, guidance, and nation and neural retina differentiation. J. Neurosci. 17, 4243– mapping in the developing visual system. J. Neurosci. 24, 1400– 4252. 1422. Bovolenta, P., Mallamaci, A., Puelles, L., and Boncinelli, E. (1998). Hyer, J., Mima, T., and Mikawa, T. (1998). FGF1 patterns the optic Expression pattern of cSix3, a member of the Six/sine oculis vesicle by directing the placement of the neural retina domain. family of transcription factors. Mech. Dev. 79, 201–203. Development 125, 869–877. Bronner-Fraser, M. (1996). Manipulation of neural crest cells or Jeremy, J., Gibson-Brown, J. J., Sergei, I., Agulnik, S., Silver, L. M., their migratory pathways. Methods Cell Biol. 51, 61–80. Virginia, E., and Papaioannou, V. E. (1998). Expression of T-box Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, genes Tbx2–Tbx5 during chick organogenesis. Mech. Dev. 74, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, 165–169. L. M., and Papaioannou, V. E. (1996). Expression of the T-box Johnson, D. E., and Tabin, C. (1995). The long and short of family genes, Tbx1-Tbx5, during early mouse development. Dev. hedgehog signalling. Cell 81, 313–316. Dyn. 206, 379–390. Koshiba-Takeuchi, K., Takeuchi, J. K., Matsumoto, K., Momose, Dale, J. K., Vesque, C., Lints, T. J., Sampath, K., and Placzek, M. (1997). Cooperation of BMP7 and SHH in the induction of T., Uno, K., Hoepker, V., Ogura, K., Takahashi, N., Nakamura, forebrain ventral midline cells by prechordal mesoderm. Cell 90, H., Yasuda, K., and Ogura, T. (2000). Tbx5 and the retinotectum 257–269. projection. Science 287, 134–137. Dale, J. K., Sattar, N., Heemskerk, J., Placzek, J. D. W., Clarke, M., Krauss, S., Johansen, T., Korzh, V., and Fjose, A. (1991). Expression and Dodd, J. (1999). Differential patterning of ventral midline of the zebrafish paired box gene pax [zf-b] during early neurogen- cells by axial mesoderm is regulated by BMP7 and chordin. esis. Development 113, 1193–1206. Development 126, 397–408. Krauss, S., Concordet, J. P., and Ingham, P. W. (1993). A function- Dragomirov, N. I. (1937). The Influence of the neighboring ecto- ally conserved homolog of the Drosophila segment polarity gene derm on the organization of the eye rudiment. Dokl. Akad. hh is expressed in tissues with polarizing activity in zebrafish Nauk. 15, 61–64. embryos. Cell 75, 1431–1444.

Liem, K. F., Jr., Tremml, G., Roelink, H., and Jessel, T. (1995). Pera, E. M., and Kessel, M. (1997). Patterning of the chick forebrain Dorsal differentiation of neural plate cells induced by BMP- anlage by prechordal plate. Development 124, 4253–4262. mediated signals from epidermal ectoderm. Cell 82, 969–979. Pittack, C. B., Grunwald, G. B., and Reh, T. A. (1997). Fibroblast Lopashov, G. V. (1963). “Developmental Mechanisms of Vertebrate growth factors are necessary for neural retina but not pigmented Eye Rudiments.” Macmillan, New York. epithelium differentiation in chick embryo. Development 124, Macdonald, R. K., Barth, A., Xu, Q., Holder, N., Mikkola, I., and 805–816. Wilson, S. W. (1995). Midline signaling is required for Pax gene Puschel, A. W., Westerfield, M., and Dressler, G. R. (1992). Com- regulation and patterning of the eyes. Development 121, 3267– parative analysis of Pax-2 protein distributions during neurula- 3278. tion in mice and zebrafish. Mech. Dev. 38, 197–208. Matsuno, T., Itasaki, N., Ichijo, H., and Nakamura, H. (1992). Romanoff, A. L. (1960). “The Avian Embryo.” The Macmillan Retinotectal projection after partial ablation of chick optic Company, New York. vesicles. Neurosci. Res. 15, 96–101. Saha, M. S., Servetnick, M., and Grainger, R. M. (1992). Vertebrate Mikami, Y. (1939). Reciprocal transformations of the parts in the eye development. Curr. Opin. Genet. Dev. 2, 582–588. developing eye-vesicle, with special reference to the inductive Silver, P. H. S. (1977). Experiments concerning the initiation of the influence of lens-ectoderm on the retinal differentiation. Zool. choroid fissure in Gallis domesticus. J. Anat. 123, 219–225. Mag. (Japan) 51, 253–256. Toy, J., Yang, J.-M., Leppert, G. S., and Sundin, O. H. (1998). The Nguyen, M.-T. T., and Arnheiter, H. (2000). Signaling and tran- optx2 homeobox gene is expressed in early precursors of the eye scriptional regulation in early mammalian eye development: A and activates retina-specific genes. Proc. Natl. Acad. Sci. USA link between FGF and MITF. Development 127, 3581–3591. 95, 10643–10648. Nornes, H. O., Dressler, G. R., Knapik, E. W., Deutsch, U., and Udin, S. B., and Fawcett, J. W. (1988). Formation of topographic Gruss, P. (1990). Spatially and temporally restricted expression of maps. Annu. Rev. Neurosci. 11, 289–327. Pax2 during murine neurogenesis. Development 109, 797–809. Ohuchi, H., Tomonari, S., Itoh, H., Mikawa, T., and Noji, S. (1999). Received for publication March 12, 2002 Identification of chick rax/rx genes with overlapping patterns of Revised May 13, 2002 expression during early eye and brain development. Mech. Dev. Accepted May 24, 2002 85, 193–195. Published online July 22, 2002