The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2013) 55, 668–675 doi: 10.1111/dgd.12074

Original Article The expression of LIM-homeobox genes, Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation

Junji Inoue,1 Yuuki Ueda,2 Tetsuya Bando,1 Taro Mito,2 Sumihare Noji2 and Hideyo Ohuchi1* 1Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama, 700-8558, and 2Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, 2-1 Minami-Josanjima-cho, Tokushima, 770-8506, Japan

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye-related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for genera- tion of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differ- entiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM-homeobox genes, induced the formation of a second neural retina from the pre- sumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle-like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the develop- ment of the retinal pigment epithelium, before the formation of the OV. Key words: chick, eye development, LIM-homeobox, neural retina, optic vesicle.

factor (TGF)-b molecules, including activin and bone Introduction morphogenetic proteins (BMPs), from periocular mes- Understanding the detailed mechanisms of retinal enchyme (Hyer et al. 1998; Fuhrmann et al. 2000; development is essential for the treatment of retinal Muller€ et al. 2007). These signals are thought to pro- diseases, including the formation of the retinal sheet mote RPE specification by inducing the expression of used for retinal transplantation (Eiraku et al. 2011). The microphthalmia-associated transcription factor (Mitf), eye originates from a bilateral evagination of the fore- which is involved in the acquisition and maintenance brain, called the optic vesicle (OV). Subsequently, the of RPE identity (Martınez-Morales et al. 2004; Horsford distal part of the OV begins to invaginate to form the et al. 2005). Simultaneously, Otx2, which is induced optic cup with two layers. These inner and outer layers by Mitf, is also required for RPE specification (Crossley develop into neural tissues, including the neural retina et al. 2001; Martinez-Morales et al. 2001; Martınez- (NR) and the retinal pigmented epithelium (RPE) (Chow Morales et al. 2003). In addition, the surface ectoderm, & Lang 2001). which abuts the OV, produces several fibroblast Differentiation of the RPE depends on exposure to growth factor (FGF) signals that confer prospective NR extracellular signals, such as transforming growth characteristics at the distal region of the OV (Pittack et al. 1997; Hyer et al. 1998; Nguyen & Arnheiter 2000; Chow & Lang 2001; Kurose et al. 2004; Kurose *Author to whom all correspondence should be addressed. et al. 2005). FGF signals then upregulate the expres- Email: [email protected] sion of Chx10, which represses Mitf and induces the Received 26 June 2013; revised 22 July 2013; NR to undergo the normal specification of retinal cell accepted 22 July 2013. ª types (McFarlane et al. 1998; Nguyen & Arnheiter 2013 The Authors € Development, Growth & Differentiation ª 2013 Japanese 2000; Vogel-Hopker et al. 2000; Rowan et al. 2004; Society of Developmental Biologists Horsford et al. 2005; Martinez–Morales et al. 2005). Lhx1 and Lhx5 in retinal development 669

The interaction between Mitf and Chx10 confers the differences between the NR and RPE (Muller€ et al. 2007). In the present study we focus on the LIM class homeodomain transcription factors, Lhx1 and Lhx5 (Fujii et al. 1994; Tsuchida et al. 1994; Sun et al. 2008) . We have previously shown that both Lhx1 and Lhx5 are expressed in the boundary region of the fore- (A) (B) brain and optic vesicle, while overexpression studies have suggested that Lhx1 and Lhx5 facilitate NR development (Kawaue et al. 2012). However, the phe- notypes of RNA interference (RNAi) against Lhx1 are inconsistent and the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNAi of Lhx5 has not been previ- ously reported. Therefore, the role for Lhx5 in early (C) chicken retinal development has not yet been revealed Fig. 1. Electroporation targeting the presumptive forebrain region by RNAi. In this study, we demonstrate that a new in the chicken embryo. (A) Old electroporation method. Electro- method of electroporation which can introduce more poration of the stage 10 optic vesicle. (B) Electroporation of a RNAi vector DNA into the embryo, thereby providing a stage 8 forebrain region. A tungsten needle electrode (cathode) consistent Lhx1/Lhx5 silencing effect than has been and a platinum wire electrode (anode) were used to achieve previously shown. We found that silencing Lhx5 region-specific gene knockdown. Placing the anode more poste- riorly to the embryo enhances the effect of Lhx1- or Lhx5-RNA expression, as early as Hamburger and Hamilton’s interference. Injected DNA solution is shown in green. (C) Antero- (HH) stage 8, inhibits NR formation, resulting in the lateral view of the embryo with both electrodes. The cathode conversion of the NR region into a pigment vesicle-like pierces the anterior neural fold while the anode is vertical to the tissue. vitelline membrane.

Materials and methods 8 V, driving pulse, pulse width 30-msec duration, pulse interval 50-msec, four pulses) were applied by a In ovo electroporation of RNAi vectors pulse generator CUY21EX (BEX, Tokyo, Japan). Win- Chicken eggs (Goto Co., Gifu, Japan) were used for dows of these eggs were covered with parafilm and Lhx1/Lhx5-RNAi experimentation. Embryos were placed at 38°C until desired stages. grown in a humidified incubator at 38°C for 30 h. Thereafter, these were staged according to Hamburger Immunostaining, hematoxylin-eosin (HE) staining, and and Hamilton (Hamburger & Hamilton 1992) and har- terminal deoxynucleotidyl transferase-mediated dUTP vested after a specified period of time post-fertilization. nick end labeling (TUNEL) staining Subsequently, the eggs were used for in ovo electro- poration. The pRFPRNAiA vector (Das et al. 2006; Embryos were fixed in 4% paraformaldehyde/phos- Mende et al. 2008), which includes Lhx1 or Lhx5 short phate buffer saline (PBS) on ice for 1 h. They were hairpin RNA target sequence (cLhx1-1/pRFPRNAi or washed in PBS. Embryos were then equilibrated in cLhx5-1/pRFPRNAi; Kawaue et al. 2012), was electro- 15% sucrose in PBS until they sank, and stored over- porated into the right prospective forebrain region at night in 30% sucrose in PBS, at 4°C. Embryos were HH stage 8. A sharpened tungsten needle (CUY614T; viewed under a stereomicroscope to determine the Unique Medical Imada, Miyagi, Japan) was used as a morphology of the retina. After observation, fixed tis- cathode and inserted into the center of the prospective sues were frozen in optimal cutting temperature com- forebrain region (Fig. 1). A platinum wire electrode pound (Sakura, Tokyo, Japan), and sectioned at (1 mm, CUY611P3-1; Unique Medical Imada), which 14 lm thickness. Sections were used for immuno- acted as an anode, was placed diagonally to the cath- staining and HE staining. For immunostaining, sections ode electrode. Placing the anode more posteriorly to were treated with PBS containing 0.2% Triton X-100 the embryo resulted in efficient gene transfer. After the (PBST) for 3 min at room temperature three times. DNA solution (50 nL) with fast green (0.1%) was Subsequently, they were incubated overnight at 4°Cin injected into the forebrain of the embryo, electric the appropriate primary antibody diluted in PBS con- pulses (50 V, poration pulse, 5-msec duration, 1 pulse; taining 5% normal goat serum. The antibodies used

2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists 670 J. Inoue et al. were as follows: anti-Sox2 (Merck Millipore, Billerica, We found that placing the anode diagonally to the MA, USA: AB5603, rabbit immunoglobulin G [IgG], cathode, that is, more posteriorly to the embryo, sup- diluted at 1:1000), anti-HuC/D (Life Technologies, pressed the expression of Lhx1 or Lhx5 efficiently than Carlsbad, CA, USA: 16A11, mouse IgG, 1:500), anti- previous methods. After 48 h of electroporation, we Otx2 (Abcam, Cambridge, UK: ab21990, rabbit IgG, observed the embryos and found that over 90% of 1:1000). Sections were rinsed in PBST and incubated them exhibited severe small eye phenotypes (class III, with Alexa Fluor 488-conjugated secondary antibody > 61% reduction according to Kawaue et al. 2012). (Life Technologies, 1:250) at room temperature. After washing, the sections were mounted in Vectashield Retinal development after Lhx1-RNAi containing 4′,6-diamidino-2-phenylindole (DAPI: Vector Laboratories, Peterborough, UK). For histology, sec- At 96 h incubation after electroporation, we examined tions were washed three times in PBST, and HE stain- morphology of the developing eye. In most cases, the ing was performed according to the standard operated eye was smaller and darker than that on the procedure. TUNEL staining was performed using a contralateral side (Fig. 2). Histological analysis revealed Click-iT TUNEL Assay (Life Technologies) as previously that the Lhx1-RNAi eye had one cell layer of presump- described (Kawaue et al. 2012). tive pigmented epithelium in the NR region. Further- more, there were some ectopic pigments in the induced presumptive pigmented epithelium (Fig. 3F, Results arrowheads). Thus, Lhx1-RNAi inhibited the formation of NR. No NR morphogenesis was detectable in the Electroporation of the RNAi vector against Lhx1 into thinner retina of Lhx1-RNAi embryos (Fig. 3). the forebrain region We previously reported that normal Lhx1 and Lhx5 The effect of Lhx1-RNAi on NR formation expression is observed in the developing forebrain from HH stage 8 (Kawaue et al. 2012). We have also Considering that the retina after Lhx1-RNAi resulted in shown that overexpression of Lhx1 or Lhx5 induced morphological abnormalities, that is, loss of retinal the ectopic expression of NR differentiation markers cyto-differentiation, we tested whether Lhx1-RNAi (Kawaue et al. 2012). Therefore, it seemed likely that converted the identity of NR cells to the RPE fate. The the activity of Lhx1 and Lhx5 in the forebrain might antibodies against early neural differentiation markers have a role in promoting NR identity. To address this Sox2 and HuC/D, and an RPE marker Otx2 were possibility, a vector for RNAi against Lhx1 or Lhx5 was used. Strong expression of HuC/D (Fig. 4A,B) or Sox2 introduced into the forebrain region by in ovo electro- protein (Fig. 4E,F) was detected in the NR on the con- poration at HH stage 8 of the chick embryo. We first tralateral side, and Otx2 protein was detected in the performed Lhx1-RNAi by delivering the pRFPRNAi vec- RPE (Fig. 4I,J). In contrast, Lhx1-RNAi retinal cells tor containing the target messenger RNA (mRNA) exhibited intense staining for Otx2 protein in the NR sequences into the chick embryos (n > 30) using a region (Fig. 4K,L), whereas no staining of the NR mar- new pulse-pattern generator (see Materials and meth- ker HuC/D or Sox2 was detected in the same region ods) and a modified electroporation method (Fig. 1). (Fig. 4C,D,G,H).

(A) (D)

Fig. 2. Lhx1-RNA interference (RNAi) (C) inhibits normal eye formation. Lateral (A, B, D, E) and frontal (C) views of the chicken embryonic head at stage 29. (B) (E) (A, B) On the control (contralateral) side, normal eye morphology and pig- mentation are observed. (C–E) Com- pared with the control (left) side, a small eye with high pigmentation is formed on the Lhx1-RNAi (operated) side.

2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists Lhx1 and Lhx5 in retinal development 671

(A) (B) (C)

(D) (E) (F)

Fig. 3. Histology of the eye after Lhx1-RNA interference (RNAi). (A-F) Hematoxylin-eosin staining at stage 29 (96 h post-electroporation). (B, C) Magnified view of the posterior retina on the contralateral side (A). (C) Distinct pigmentation is observed on the basal side of the single-layered retinal pigmented epithelium (RPE) (black arrowheads), but not in the neural retina (NR). (E, F) Magnified view of the pos- terior retina on the Lhx1-RNAi side (D). (D, E) Small eye is observed on the Lhx1-RNAi side, in which a pigmented vesicle forms and nor- mal lens development is perturbed. (F) Intense pigmentation is preserved on the basal side of the RPE (black arrowheads). Ectopic pigmentation is observed in the induced pigmented epithelium (white arrowheads). Scale bar 100 lm.

pigmentation (Fig. 6F; arrowhead). Thus, Lhx5-RNAi Retinal development post Lhx5-RNAi inhibited formation of NR as was seen in Lhx1-RNAi. At 96 h after electroporation, distinct pigmentation was present in the normal embryonic eye. After Lhx5-RNAi, Are the effects of Lhx5-RNAi the same as those of as observed after Lhx1-RNAi, the operated eye was Lhx1-RNAi? small and highly pigmented, when compared with the contralateral eye (Fig. 5). Histological analysis revealed Lhx5-RNAi resulted in defects in the formation of NR that the Lhx5-RNAi eye exhibited a thin optic cup with and loss of cyto-differentiation. As such, we tested pigmentation (Fig. 6). The inner layer of the cup (i-RPE whether the identity of authentic NR cells shifted to in Fig. 6D-F) was a pseudo-multi-layered retina with the RPE cells after Lhx5-RNAi. As mentioned, the

(A) (B) (C) (D)

(E) (F) (G) (H) Fig. 4. Immunostaining for retinal dif- ferentiation markers of a stage 29 eye after Lhx1-RNA interference (RNAi). (A, B, E, F, I, J) Contralateral control eye. (C, D, G, H, K, L) After Lhx1-RNAi. An RNA-binding protein HuC/D (A-D) and Sox2 (E-H) are used as neural retina (I) (J) (K) (L) (NR) differentiation markers. (I-L) Otx2 is used as a retinal pigmented epithe- lium (RPE) differentiation marker. Sig- nals are shown in green and nuclei are stained with 4´,6-diamidino-2-phenylin- dole (DAPI) (blue). Scale bar 100 lm.

2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists 672 J. Inoue et al.

(A) (D)

(C) Fig. 5. Lhx5-RNA interference (RNAi) causes small eye formation. Lateral (A, (B) (E) B, D, E) and frontal (C) views of the chicken embryonic head at stage 29. (A, B) On the control (contralateral) side, normal eye morphology and pig- mentation are present. (C-E) A small eye with high pigmentation is present on the Lhx5-RNAi (operated) side.

phenotype after Lhx5-RNAi exhibited a small eye on Discussion the operated side. We performed TUNEL staining to determine whether apoptosis was induced by the Lhx1 and Lhx5 are LIM-homeodomain (LIM-HD) RNAi or electroporation, resulting in a small eye or dis- transcription factors (Taira et al. 1994; Shawlot & ruption of eye development (Mende et al. 2008). We Behringer 1995; Hobert & Westphal 2000; Poche et al. confirmed that no apoptosis was evident in the RNAi 2007; Suga et al. 2009). We have previously showed OV, while it was observed in the hindbrain region that the LIN-11 class LIM-HD factors, Lhx1 and Lhx5 where apoptosis normally occurs (Fig. S1). can induce the expression of NR differentiation mark- We next examined the embryos by comparing mar- ers and induce nascent OV cells to form the NR ker expression between the operated side and contra- (Kawaue et al. 2012). In this study, after Lhx1-RNAi or lateral side. Sox2, HuC/D and Otx2 were used. On the Lhx5-RNAi, small eye phenotypes can be observed on operated side, retinal cells of the Lhx5-RNAi embryo the operated side. In addition, these small eyes are exhibited intense staining for Otx2 in the peripheral NR highly pigmented when compared with the contralat- (Fig. 7I–L). In contrast, no staining for Sox2 or HuC/D eral eye. Histological analysis revealed that the NR was observed in the same regions (Fig. 7A–H). region after Lhx1-RNAi exhibited a single layer of a

(A) (B) (C)

(D) (E) (F)

Fig. 6. Histology of the eye after Lhx5-RNA interference (RNAi). (A-F) Hematoxylin-eosin staining at stage 29 (96 h post-electroporation). (B, C) Magnified view of the posterior retina on the contralateral side (A). (C) Pigmentation is observed on the basal side of the single-lay- ered RPE (black arrowheads), but not in the NR. (E, F) Magnified view of the posterior retina on the Lhx5-RNAi side (D). (D, E) Small eye is observed on the Lhx5-RNAi side, and the eye exhibits a narrow cup-shape. (F) Ectopic pigmentation is observed in the induced pigmented epithelium (white arrowheads). i-RPE, induced retinal pigment epithelium; LE, lens epithelium; NR, neural retina; RPE, retinal pigment epithelium. Scale bar100 lm.

2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists Lhx1 and Lhx5 in retinal development 673

(A) (B) (C) (D)

(E) (F) (G) (H)

Fig. 7. Immunostaining for retinal dif- ferentiation markers of a stage 29 eye after Lhx5-RNA interference (RNAi). Signals are shown in green and nuclei are stained with DAPI (blue). (A, B, E, F, I, J) Contralateral control eye. (C, D, (I) (J) (K) (L) G, H, K, L) After Lhx5-RNAi. HuC/D (A– D) and Sox2 (E–H) are used as NR dif- ferentiation markers. (I–L) Otx2 is used as a retinal pigment epithelium (RPE) differentiation marker. Scale bar 100 lm. pigmented retina, which is different from the normal Lhx5 patterns the NR before OV development NR. In contrast, the NR region after Lhx5-RNAi still exhibited a pseudo-multi-layered retina, with pigmenta- The LIM-homeobox gene Lhx5 play roles in specifying tion. We tested whether the Lhx1 or Lhx5 RNAi con- the forebrain architecture (Sheng et al. 1997) and can verted the identity of NR cells to the RPE fate, using mediate neuronal differentiation (Zhao et al. 1999). the neuronal differentiation markers Sox2 and HuC/D Therefore, we wished to determine whether Lhx5 plays (Marusich et al. 1994; Hyer et al. 1998; Taranova et al. a role in NR differentiation. In previous reports, we 2006;Ishii et al. 2009), and an RPE differentiation mar- described the expression pattern of Lhx5 (Kawaue ker Otx2 (Crossley et al. 2001; Martınez-Morales et al. et al. 2012). Based on this data, we performed Lhx5- 2003; Nishihara et al. 2012). In RNAi-treated eyes, nei- RNAi by electroporation under the same conditions ther Sox2 nor HuC/D protein was detected in the with Lhx1-RNAi. At 96 h after electroporation, the authentic NR region. Furthermore, Lhx1-RNAi induced majority of the embryos exhibited small eyes with the expression of Otx2. Compared with Lhx1-RNAi, excessive pigmentation, as is found in Lhx1-RNAi; minor Otx2 protein was observed in the NR region however, the morphology and characteristics of the after Lhx5-RNAi. small eye were different from that of Lhx1-RNAi embryos. In the NR region, Sox2 and HuC/D protein disappeared, but the retina did not become a single The earlier manipulation of embryos, a new layer of pigmented epithelium; however, a pseudo- electroporator and effective electrode placement multi-layered retina was seen in the neuroepithelium. enhanced the efficiency of in ovo RNAi Moreover, ectopic Otx2 protein was only detected in Based on the onset and domains of Lhx1 and Lhx5 the periphery of the retina. We think that this is expression patterns (Kawaue et al. 2012), we deter- because the expression domain of Lhx5 is larger than mined that placing the anode diagonally to the cathode that of Lhx1. Therefore, Lhx5-RNAi may not confer a and performing electroporation as early as HH stage 8 complete silencing effect as Lhx1-RNAi and it is likely was optimum for suppressing the expression of Lhx1 that the partially reduced expression of Lhx5 may not or Lhx5 (Fig. 1). In addition, a modern electroporator have fully induced NR differentiation. Instead, the with multi-pulse patterns allowed the more effective residual mRNA of Lhx5 may antagonize the action of introduction of DNA into the chicken embryonic cells. the extraocular mesenchyme that patterns the RPE This shows that the expression of Lhx1 in the prospec- (Fuhrmann et al. 2000). Although Lhx5-RNAi can be tive forebrain region may control NR differentiation at further optimized, this is the first report describing how HH stage 8. However, the expression of Lhx1 post HH the normal expression level of Lhx5 in the forebrain is stage 9 has less impact on NR differentiation. required for NR development of the optic primordium.

2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists 674 J. Inoue et al.

Concluding remarks Self-organizing optic-cup morphogenesis in three-dimen- sional culture. Nature 472,51–56. In the present study, we have shown that Lhx1 and Fujii, T., Pichel, J. G., Taira, M., Toyama, R., Dawid, I. B. & Lhx5 expression is required to specify the neural retina Westphal, H. 1994. Expression patterns of the murine LIM domain in the optic vesicle. Since Lhx1 is induced by class homeobox gene lim1 in the developing brain and excretory system. Dev. Dyn. 199,73–83. Lhx5 (Kawaue et al. 2012), it is conceivable that the Fuhrmann, S., Levine, E. M. & Reh, T. A. 2000. Extraocular mes- outcome of Lhx5-RNAi treatment is similar to that of enchyme patterns the optic vesicle during early eye develop- Lhx1-RNAi. It is possible that Lhx5 and Lhx1 influence ment in the embryonic chick. Development 127, 4599–4609. NR differentiation by inducing the expression of diffus- Hamburger, V. & Hamilton, H. L. 1992. A series of normal stages ible factors, such as FGF (Vogel-Hopker€ et al. 2000; in the development of the chick embryo. 1951. Dev. Dyn. 195, 231–272. Martinez-Morales et al. 2005; Okamoto et al. 2009) Hobert, O. & Westphal, H. 2000. Functions of LIM-homeobox and Wnt (Holliday et al. 1995). Subsequently, these genes. Trends Genet. 16,75–83. mediators may induce the expression of early neuronal Holliday, M., McMahon, J. A. & McMahon, A. P. 1995. Wnt transcription factors such as Sox2 and Chx10 (Pittack expression patterns in chick embryo nervous system. Mech. – et al. 1997; Zhao et al. 2001; Taranova et al. 2006). Dev. 52,9 25. Horsford, D. J., Nguyen, M. T., Sellar, G. C., Kothary, R., Arnhe- Simultaneously, RPE-specific transcription factors, iter, H. & McInnes, R. R. 2005. Chx10 repression of Mitf is such as Otx2 and Mitf, are induced by TGF-b-like sig- required for the maintenance of mammalian neuroretinal nals from the extraocular mesenchyme. In normal eye identity. Development 132, 177–187. development, Chx10 and Mitf repress each other’s Hyer, J., Mima, T. & Mikawa, T. 1998. FGF1 patterns the optic expression (Fuhrmann et al. 2000). In this report, we vesicle by directing the placement of the neural retina domain. Development 125, 869–877. suggest that Lhx5 is necessary for NR development Ishii, Y., Weinberg, K., Oda-Ishii, I., Coughlin, L. & Mikawa, T. and the expression level of Lhx5 required to induce 2009. Morphogenesis and cytodifferentiation of the avian ret- NR differentiation is higher than that of inhibiting RPE inal pigmented epithelium require downregulation of Group differentiation. However, diffusible mediators that link B1 Sox genes. Development 136, 2579–2589. between Lhx5/Lhx1 transcription factors and NR Kawaue, T., Okamoto, M., Matsuyo, A., Inoue, J., Ueda, Y., To- monari, S., Noji, S. & Ohuchi, H. 2012. Lhx1 in the proximal development are elusive, and these important issues region of the optic vesicle permits neural retina development should be examined in future studies. in the chicken. Biol. Open. 1, 1083–1093. Kurose, H., Bito, T., Adachi, T., Shimizu, M., Noji, S. & Ohuchi, H. 2004. Expression of Fibroblast growth factor 19 (Fgf19) Acknowledgements during chicken embryogenesis and eye development, com- pared with Fgf15 expression in the mouse. Gene Expr. Pat- We thank Thomas Jessell for providing the chicken terns 4, 687–693. Lhx1 complementary DNA. We also thank Stuart A. Kurose, H., Okamoto, M., Shimizu, M., Bito, T., Marcelle, C., Wilson for the generous gift of the pRFPRNAi vector. Noji, S. & Ohuchi, H. 2005. FGF19-FGFR4 signaling elabo- Monoclonal antibodies were obtained from the Devel- rates lens induction with the FGF8-L-Maf cascade in the – opmental Studies Hybridoma Bank developed under chick embryo. Dev. Growth Differ. 47, 213 223. Martinez-Morales, J. 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2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists Lhx1 and Lhx5 in retinal development 675

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2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists