Loss of Orphan Nuclear Receptor GCNF Function Disrupts Forebrain Development and the Establishment of the Isthmic Organizer ⁎ Arthur C.-K

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Developmental Biology 293 (2006) 13–24 www.elsevier.com/locate/ydbio

Loss of orphan nuclear receptor GCNF function disrupts forebrain development and the establishment of the isthmic organizer ⁎ Arthur C.-K. Chung a, Xueping Xu a, Karen A. Niederreither a,b, Austin J. Cooney a,

a Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA b Department of Medicine, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Received for publication 28 June 2005; revised 17 November 2005; accepted 6 December 2005 Available online 10 March 2006

Abstract

The isthmic organizer, which is located at the midbrain–hindbrain boundary, is important for midbrain development. The mechanism by which the development of the organizer is initiated and maintained is not well understood. Inactivation of the gene encoding the orphan nuclear receptor, GCNF, diminishes the expression of secreted signaling molecules, Fgf8 and Wnt1, the paired box genes Pax2/5, En1/2, and homeodomain transcription factor Gbx2; all of which are essential for isthmic organizer function. In addition, full neuronal differentiation is not observed in the midbrain region of GCNF−/− embryos. Increased cell death may contribute to the loss of midbrain structure in GCNF−/− embryos. These results indicate that GCNF is required for establishment of the isthmic organizer, thereby regulating the midbrain development. © 2005 Elsevier Inc. All rights reserved.

Keywords: Nuclear receptor; GCNF; Isthmic organizer; Midbrain–hindbrain boundary

Introduction activity during early somitogenesis (Liu and Joyner, 2001b; Urbanek et al., 1997). The isthmic organizer expresses Midbrain development has been extensively investigated, secreted signaling molecules such as Fgf8 and Wnt1 and and many transcription factors and secreted signaling thereby induces the development of the midbrain and molecules have been shown to be involved in regulating its anterior hindbrain (Crossley and Martin, 1995; Liu et al., development and function. Expression of the homeodomain 1999; Martinez et al., 1999; Meyers et al., 1998), Fgf8 transcription factors, Otx2 and Gbx2, regulate anterior– expression in the isthmic organizer terminates around posterior patterning during brain development. Otx2 is embryonic day (E) 13.5, while Wnt1 expression terminates expressed in the presumptive forebrain and midbrain, while at E15.5 (Xu et al., 2000). Although the genes that maintain Gbx2 is expressed in the presumptive hindbrain and spinal the activities of isthmic organizer, such as homeobox genes, cord. Studies reveal that the boundary region between the have been characterized extensively, there is no report of Otx2 and Gbx2 expression domains, which is located at the involvement of members of the nuclear receptor superfamily, midbrain–hindbrain boundary (MHB), constitutes the orga- except Germ Cell Nuclear Factor, GCNF (Song et al., nizing center, called the isthmic organizer (Joyner et al., 2000; 1999). Wurst and Bally-Cuif, 2001). GCNF belongs to the nuclear receptor superfamily which Antagonistic regulation between Otx2 and Gbx2 regulates consists of several ligand-dependent transcription factors that the precise position of the isthmic organizer (Broccoli et al., bind to steroids or non-steroids, such as retinoic acid (Aranda 1999; Millet et al., 1999), while other homeobox genes such and Pascual, 2001; Lee and Lee Kraus, 2001). Some members as Pax2/5 and En1/2 are required for isthmic organizer of this superfamily are orphan receptors that are involved in regulating embryonic development. GCNF has been shown to be essential for normal embryonic development (Chung et al., ⁎ Corresponding author. Fax: +1 713 790 1275. 2001; David et al., 1998; Lan et al., 2002). However, GCNF E-mail address: [email protected] (A.J. Cooney). was initially described to have tissue-specific expression in

0012-1606/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.12.017 14 A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24 germ cells of the adult mouse (Chen et al., 1994; Hirose et al., Whole-mount in situ hybridization (WMISH) and TUNEL assays 1995) and human (Agoulnik et al., 1998; Kapelle et al., 1997; +/− Lei et al., 1997), however, it is also expressed during Embryos were obtained from heterozygote GCNF crosses on embryonic days 8.0–9.5. Embryos were fixed in MEMFA (0.2 M MOPS, pH7.4, 2 mM gastrulation (Chung et al., 2001; David et al., 1998; Heinzer EGTA, 1 mM MgSO4, 3.7% formaldehyde) at 4°C overnight. To examine the et al., 1998; Lei et al., 1997; Susens et al., 1997). In the expression of marker genes, whole-mount in situ hybridization (WMISH) was developing brain, GCNF is expressed in postmitotic neurons used as previously described (Chung et al., 2001). cRNA probes for BF1 of the marginal zone of the neuroepithelium at E15 (Agoulnik (Shimamura and Rubenstein, 1997), En1 (Danielian and McMahon, 1996), En2 et al., 1998; Bauer et al., 1997; Susens et al., 1997). Targeted (Conlon and Rossant, 1992), FGF8 (Crossley and Martin, 1995), Gbx2 (Millet et al., 1999), Otx2 (Broccoli et al., 1999), Sprouty1 (Minowada et al., 1999), deletion of the GCNF gene results in embryonic lethality by Pax2 (Dressler et al., 1990), Pax5 (Schwarz et al., 1997), Wnt1 (McMahon et al., E10.5 with loss of proper repression of the Oct4 gene in 1992) were DIG labeled and used for the WMISH analyses. somatic cells (Chung et al., 2001; Fuhrmann et al., 2001; Lan Whole-mount TUNEL assay was performed as previously described (Qiu et et al., 2002). In addition, the GCNF−/− embryos suffer from al., 1997). Briefly, embryos were first fixed in MEMFA, washed in 50% − generalized defects, such as failure of neural tube closure and methanol, and stored in 90% methanol at 20°C. After serial rehydration to PBT (PBS with 0.1% Tween-20), the embryos were digested in 10 μg/ml proteinase axial rotation. It has been shown that GCNF is a repressor of K in PBT. After rinsing in PBT, the embryos were postfixed in freshly prepared gene transcription in cellular contexts through the binding to 4% paraformaldehyde in PBT. After 20 min in blocking solution (0.1% sodium GCNF DNA response element (DR0), which is a direct repeat borohydride in PBT) at room temperature, the embryos were rinsed with TdT of the sequence AGGTCA separated by a zero-nucleotide buffer (30 mM Tris, pH 7.2, 140 mM Cacodylate, 1 mM CoCl2). Subsequently, μ μ spacer (Cooney et al., 1998; Hummelke et al., 1998; Yan and the embryos were incubated in a reaction mix (20 m DIG-UTP, 20 M dTTP, and 0.3 U/ml Terminal transferase in TdT buffer) at 37°C for 2 h. Embryos were Jetten, 2000). For example, repression of Oct4 is mediated by washed extensively in PBT solution and incubated in blocking solution (2% GCNF binding to a DR0 element in the proximal promoter Blocking reagent (Roche) in PBT.3 [PBS with 0.3% Triton X-100]). Then (Fuhrmann et al., 2001). embryos were incubated with alkaline phosphatase-conjugated anti-digoxigenin Recent in vitro studies in a mouse embryonic carcinoma cell antibody at 4°C overnight. After washing in PBT.5 (PBS with 0.5% Triton X- line demonstrate that GCNF is important for differentiation and 100), the WMISH signals were detected by incubating the embryos in BM purple (Roche) solution in the dark. The color reaction was stopped by adding maturation of neuronal precursor cells (Sattler et al., 2004). 1mM EDTA in PBT.3 solution. GCNF knockdown impairs the differentiation of neuronal precursor cells and delays their maturation, suggesting that Whole-mount immunohistochemistry GCNF may regulate neuronal differentiation in embryos. In Xenopus, GCNF has also been reported to be required for Whole-mount immunohistochemistry with a monoclonal antibody (2H3) different patterns of cell intercalation during neurulation recognizing a 165-kDa neurofilament protein was performed essentially as (Barreto et al., 2003b). GCNF knockdown affects the medial previously described (Qiu et al., 1997). Briefly, embryos were collected in PBS, fixed in methanol:DMSO (4:1) overnight at 4°C. Embryos were then bleached in migration and radial intercalation. These defects may impair the methanol:DMSO:30% H2O2 (4:1:1) overnight at room temperature, rehydrated morphogenetic movements that close the neural tube. In for 30 min each through 70%, 50%, and 25% methanol, and finally PBS. addition, GCNF can synergize with ectopic chordin to induce Embryos were incubated twice in PBSMT (2% instant skim milk powder, 0.1% the En2 expression in Xenopus (Song et al., 1999), which Triton X-100 in PBS) for 1 h at room temperature, then with primary together with results from gain- and loss-of-function experi- monoclonal antibody against alpha-neurofilament (2H3, Developmental Studies – Hybridoma Bank, IL) diluted (1:500) in PBSMT at 4°C overnight. Embryos ments, suggests that GCNF may play a role in the midbrain were washed in PBSMT twice at 4°C and 3 times at room temperature for 1 hindbrain formation (Song et al., 1999). h each, followed by an overnight incubation at 4°C with peroxidase-conjugated − − Here, we report that, in GCNF / mouse embryos, goat anti-mouse IgG (Santa Cruz, CA) diluted (1:1000) in PBSMT. The washes expression of genes important for isthmic organizer function, were similar to washes after the primary antibody one with an additional 20-min including Fgf8, Gbx2, Wnt1, En1/2, and Pax2/5, are signifi- wash in PBT (0.2% BSA, 0.1% Triton X-100 in PBS) at room temperature. For −/− the color reaction, embryos were incubated with 0.3 mg/ml of diaminobenzidine cantly down-regulated. In GCNF embryos, cells in the tetrahydrochloride (Sigma) in PBT for 30–60 min at room temperature and isthmic organizer do not differentiate fully into neurons. visualized by addition of H2O2 to 0.0003% and incubation at room temperature. Significant cell death may be the cause of loss of the midbrain Embryos were then rinsed in PBT to stop the reaction, dehydrated through a structures in GCNF−/− embryos. These results demonstrate that methanol series; 25%, 50%, 70%, 100% for 30 min each, and cleared in benzyl GCNF is required to establish the organizer activity at the alcohol:benzyl benzoate (1:2). isthmus, thereby regulating midbrain development. Results Materials and methods GCNF is expressed in the neuroectoderm during early Generation of GCNF knockout mice and genotypic analysis of somitogenesis embryos GCNF is expressed in wild-type (WT) mouse embryos as Mice deficient in GCNF were generated by gene targeting in embryonic early as E6.5 (Fuhrmann et al., 2001). At the onset of stem cells (ES cells) as previously described (Chung et al., 2001). Timed somitogenesis at E8.0, GCNF expression appeared in the matings of mice heterozygous for the GCNF mutation were initiated to obtain litters at different developmental stages. All studies were performed on a 129Sv/ proliferating neuroepithelium, with a gradient from the C57BL6 genetic background. Yolk sac DNA from each embryo was extracted posterior to anterior ends (Fig. 1A). Its expression has also and genotyped by PCR as previously described (Chung et al., 2001). been reported to be present in the underlying mesoderm A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24 15

Fig. 1. Expression of GCNF in wild-type mouse embryos. Whole-mount in situ hybridization analysis of on WT embryos with GCNF DIG-labeled cRNA probe. (A) GCNF expression in a mouse embryo with three somites at E8.0, (B) GCNF expression in a mouse embryo with six somites at E8.5, (C) GCNF expression in a mouse embryo with 13 somites at E8.75, and (D) GCNF expression in a mouse embryo with 25 somites at E9.5.

(Susens et al., 1997). GCNF continued to be expressed in the anterior neuroepithelium at E8.5 in embryos with six somites (Fig. 1B). Subsequently, GCNF expression decreased in the anterior after E8.75 when the somite number was greater than 12 (Fig. 1C). By E9.5, in embryos with 25 somites, GCNF expression was markedly reduced in the forebrain and midbrain regions as determined by whole-mount in situ hybridization assay (Fig. 1D).

Loss of GCNF function down-regulates the expression of Gbx2, Fgf8, BF1, and Spry1 in the forebrain region, but not Otx2

The earliest anterior neuroectoderm marker gene that is expressed is Otx2, a homeodomain transcription factor, which is expressed in the visceral endoderm and in the epiblast prior Fig. 2. Expression of Otx2 and Gbx2 in GCNF+/+ and GCNF−/− embryos during to the onset of gastrulation (Broccoli et al., 1999). After early mouse embryonic development. (A, B) WMISH analysis of Otx2 gastrulation, Otx2 expression becomes progressively restricted expression in embryos with one to two somites at E8.0. (C, D) WMISH to the anterior region of the embryo (Fig. 2A). During analysis of Otx2 expression at E8.5 in embryos with six somites. Similar Otx2 somitogenesis at E8.5 and E9.5 (with six and 25 somites, expression patterns were observed in the anterior region of GCNF+/+ and −/− respectively), the Otx2 expression domain defines the GCNF embryos at E8.0 and E8.5. (E, F) WMISH analysis of Otx2 expression at E9.5 in WT embryos with 25 somites and in GCNF−/− embryos with 13 prospective forebrain and midbrain territory (Figs. 2C, E). In −/− −/− somites. Otx2 expression domain of GCNF embryos extended to the first GCNF embryos, Otx2 was also expressed in the anterior branchial arch (black arrows). (G, H) WMISH analysis of Gbx2 expression in region of the embryos during the onset of somitogenesis (Fig. embryos with one to two somites at E8.0. (I, J) WMISH analysis of Gbx2 2B). At E8.5 in embryos with six somites, Otx2 is expressed expression at E8.5 in embryos with eight somites. Gbx2 expression at the MHB −/− in the forebrain region of GCNF−/− embryos (Fig. 2D). At was not detected in GCNF embryos at E8.5. (K, L) WMISH analysis of Gbx2 expression at E9.5 in WT embryos with 25 somites and in GCNF−/− embryos E9.5 in embryos with 13 somites, Otx2 expression extended −/− with 13 somites. No Gbx2 expression was observed in the midbrain region of caudally and reached the first branchial arch in GCNF GCNF−/− embryos. White arrows indicate the position of MHB. Insets are dorsal embryos (Black arrow in Fig. 2F). views of the midbrain region of the embryos. 16 A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24

vesicles, is expressed in the ANR of mouse embryos at E8.5 (Shimamura and Rubenstein, 1997)(Figs. 4A, C). Recombinant Fgf8 protein is capable of inducing BF1 expression, thus Fgf8 regulates the development of anterolateral neural plate derivatives (Shimamura and Rubenstein, 1997). Since Fgf8 expression is down-regulated in the ANR of GCNF−/− embryos, we examined the expression of BF1.InWTembryos,BF1 expression is maintained in the ANR from E8.5 to E9.5 in embryos with six and 25 somites, respectively (Figs. 4A, C). In GCNF−/− embryos, BF1 transcript was weakly expressed in the ANR at E8.5 in embryos with six somites (Fig. 4B) and absent at E9.5 in embryos with 13 somites (Fig. 4D). Sprouty expression is also dependent on Fgf signaling and Sprouty can antagonize Fgf function (Minowada et al., 1999). Thus, Sprouty plays an essential role in feedback loop of Fgf Fig. 3. Expression of Fgf8 in GCNF+/+ and GCNF−/− embryos during early signaling (Sivak et al., 2005). Sprouty1 expression is induced by mouse embryonic development. (A, B) WMISH analysis of Fgf8 expression at Fgf8, and it is expressed in the MHB during early somitogenesis E8.5 in embryos with six somites. (C, D) WMISH analysis of Fgf8 expression at E9.5 in WT embryos with 25 somites and in GCNF−/− embryos with 13 somites. Fgf8 expression was significantly reduced in the midbrain (white arrows) and ANR (black arrows) region of GCNF−/− embryos. White arrows indicate the position of the MHB. Insets are dorsal views of the midbrain region of the embryos.

Gbx2, another homeobox-containing transcription factor, is expressed throughout the posterior of WT embryo at E7.5 (Millet et al., 1999). After gastrulation, Gbx2 expression becomes progressively restricted to the posterior region of the head-fold (Fig. 2G). At E8.5, in embryos with eight somites, Gbx2 is expressed in the prospective hindbrain (rhombomere 1) (white arrow in Fig. 2I) and presomitic mesoderm (PSM) (Fig. 2I) (Garda et al., 2001). At E9.5, in embryos with 25 somites, anterior Gbx2 expression is restricted to the MHB (white arrow in Fig. 2K). In GCNF−/− embryos, similar posterior Gbx2 expression was observed once somitogenesis initiated (Fig. 2H). At E8.5, in GCNF−/− embryos with six somites, anterior Gbx2 expression was not detected in the MHB (white arrow in Fig. 2J). Gbx2 expression was not detected in the MHB, but it was weakly expressed along the anterior neuroepithelium at E9.5 in GCNF−/− embryos with 13 somites (Fig. 2L). The boundary of the Otx2 and Gbx2 expression defines the position of the MHB. Since Gbx2 expression was lost in the MHB, it suggested that there might be a defect in the development of isthmic organizer in GCNF−/− embryos. At the three somite stage, a secreted member of the fibroblast growth factor (Fgf) family, Fgf8, is expressed, initially, in a broad domain confined to the rostral part of the Gbx2-positive − − territory (Crossley and Martin, 1995). At E8.5 in embryos with Fig. 4. Expression of BF1, and Sprouty1 in GCNF+/+ and GCNF / embryos six somites, Fgf8 was expressed in the prospective midbrain during early mouse embryonic development. (A, B) WMISH analysis of BF1 expression at E8.5 in embryos with six somites. (C, D) WMISH analysis of BF1 region and in the anterior neural ridge as expected (ANR) (Fig. expression at E9.5 in WT embryos with 25 somites and in GCNF−/− embryos 3A). Fgf8 is then expressed in the MHB and ANR at E9.5 in with 13 somites. Weak BF1 expression was detected in the anterior region of −/− − − − − embryos with 25 somites (Fig. 3C). In GCNF embryos, Fgf8 GCNF / embryos at E8.5, and its expression is absent at E9.5 in GCNF / expression was weakly expressed in the head-fold, and its embryos. (E, F) WMISH analysis of Sprouty1 expression at E8.5 in WT embryos −/− expression was significantly reduced in the ANR at both E8.5 with eight somites and in GCNF embryos with six somites. (G, H) WMISH analysis of Sprouty1 expression at E9.5 in WT embryos with 25 somites and in and E9.5 (six and 13 somites, respectively) (Figs. 3B, D). GCNF−/− embryos with 13 somites. Sprouty1 expression was not observed in the BF1, a gene encoding a transcription factor required for midbrain region of GCNF−/− embryos at E9.5. Black arrows indicate the anterior regionalization and growth of the telencephalic and optic neural ridge. White arrows indicate the position of the MHB. A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24 17 in embryos with eight and 25 somites (Minowada et al., 1999) (Figs. 4E, G). In GCNF−/− embryos, Sprouty1 expression in the MHB was undetectable at E8.5 and E9.5 in embryos with six and 13 somites, respectively (Figs. 4F, H). Clearly, Fgf8 signaling in the anterior of GCNF−/− embryos was severely impaired.

The isthmic organizer is not established properly in GCNF−/− embryos

To examine whether the isthmic organizer was affected in the GCNF−/− embryos, we investigated the expression of genes that are important for isthmic organizer function. The expression of the isthmus-specific paired box transcription factor, Pax2, is initiated at the presomitic head-fold stage around the Otx2/Gbx2 boundary, and it is the first gene expressed in the MHB and the tip of forebrain during early somitogenesis (Dressler et al., 1990). At E8.5 in embryos with four somites and E8.75 in embryos with 13 somites, Pax2 was expressed in the prospective MHB (Figs. 5A, C). Pax2 expression was maintained at the MHB at E9.5 in embryos with 30 somites (Fig. 5E). In GCNF−/− embryos, Pax2 was weakly expressed at the onset of somitogenesis at E8.5 when the somite number was four (Fig. 5B), however, its expression was undetectable in the MHB in embryos with somite numbers greater than five (Figs. 5D, F). These results suggest that the development of isthmic organizer was already affected in the GCNF−/− embryos by E8.5. Once somitogenesis initiates, expression of the homeodomain transcription factor engrailed 1 (En1)andthe secreted glycoprotein signaling molecule Wnt1 are simulta- neously initiated within the Pax2 expression domain (Danie- lian and McMahon, 1996; McMahon et al., 1992). In mouse embryos with somite numbers between 8 to 13, En1 and Wnt1 were co-expressed across the Otx2/Gbx2 border in the midbrain and hindbrain (Figs. 5G, I). Later at E9.5 in embryos with 28 somites, Wnt1 expression is largely confined to the posterior Otx2-positive domain and is restricted to a narrow ring encircling the neural tube rostral to the isthmic constriction and the dorsal and ventral midline of the mesencephalon (Fig. 5K). In GCNF−/− embryos that had greater than five somites, En1 and Wnt1 were weakly expressed (Figs. 5H, J). At E9.5 in GCNF−/− embryos with 13 somites, Wnt1 was expressed along the ridge of neuroectoderm, but its characteristic ring-like expression around the MHB was not observed (Fig. 5L). Fig. 5. Expression of Pax2, En1, and Wnt1 in GCNF+/+ and GCNF−/− embryos At the three somite stage, Fgf8 is expressed in a region during early mouse embryonic development. (A, B) WMISH analysis of Pax2 confined to the rostral part of the Gbx2-positive territory expression at E8.5 in embryos with four somites. (C, D) WMISH analysis of (Crossley and Martin, 1995). At E8.5 in embryos with six Pax2 expression at E8.75 in embryos with 13 somites. Weak Pax2 expression −/− somites, Fgf8 expression was found in the prospective MHB was observed in the anterior region of GCNF embryos at E8.0 and E8.5. (E, F) WMISH analysis of Pax2 expression at E9.5 in WT embryos with 30 somites (Fig. 3A). Fgf8 was then expressed in the MHB at E9.5 in −/− −/− and in GCNF embryos with 13 somites. Pax2 expression was undetectable in embryos with 25 somites (Fig. 3C). In GCNF embryos at GCNF−/− embryos at E9.5. (G, H) WMISH analysis of En1 expression at E8.75 both E8.5 with six somites and at E9.5 with 25 somites, in WT embryos with 13 somites and in GCNF−/− embryos with 10 somites. En1 − − Fgf8 expression was barely detectable in the midbrain region was weakly expressed in GCNF / embryos at E8.75. (I, J) WMISH analysis of (Figs. 3B, D). Wnt1 expression at E8.5 in embryos with eight somites. (K, L) WMISH analysis of Wnt1 expression at E9.5 in WT embryos with 30 somites and in GCNF−/− At the five somite stage, transcription of the another paired embryos with 13 somites. No Wnt1 expression was observed in the midbrain box gene Pax5 and the homeobox gene engrailed 2 (En2)is region of GCNF−/− embryos at E9.5. White arrows indicate the position of initiated in a broad region overlapping the Otx2/Gbx2 boundary MHB. Insets are dorsal views of the midbrain region of the embryos. 18 A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24

Significant cell death and loss of neuronal differentiation in the midbrain region of GCNF−/− embryos

Loss of Fgf8 expression causes significant cell death in the midbrain, leading to complete loss of this structure (Chi et al., 2003). The loss of Wnt1 and Pax2/5 expression in the isthmic organizer of GCNF−/− embryos could be due to apoptosis of the organizer cells. To examine this possibility, a whole-mount TUNEL assay was performed on WT and GCNF−/− embryos. This assay demonstrated that there was significant difference in cell death between the WT and GCNF−/− embryos at E9.5 (Figs. 7A–D), thus suggesting the possibility that cell death may contribute to the loss of the midbrain development in the GCNF mutants. To determine whether the many molecular alterations detected in GCNF−/− embryos affect cranial nerve patterning, E9.5 embryos were immunostained with an anti-neurofilament antibody. Normally, cranial nerves III and IV are derived from the midbrain and the isthmic organizer at E9.5 in WT embryos with 30 somites (Fig. 7E). Whole-mount staining with antibody to neurofilament revealed that the cranial nerves normally connected with the midbrain (such as III and IV) did not develop well at E9.5 in GCNF−/− embryos with 13 somites (Fig. 7F). These results are consistent with the severely impaired midbrain development in GCNF−/− embryos. We conclude that

Fig. 6. Expression of Pax5 and En2 in GCNF+/+ and GCNF−/− embryos during early mouse embryonic development. (A, B) WMISH analysis of Pax5 expression at E8.5 in WT embryos with seven somites and in GCNF−/− embryos with six somites. (C, D) WMISH analysis of Pax5 expression at E9.5 in WT embryos with 30 somites and in GCNF−/− embryos with 13 somites. Pax5 expression was significantly reduced in GCNF−/− embryos. (E, F) WMISH analysis of En2 expression at E8.5 in WT embryos with seven somites and in GCNF−/− embryos with six somites. (G, H) WMISH analysis of En2 expression at E9.5 in WT embryos with 30 somites and in GCNF−/− embryos with 13 somites. No En2 expression was detected in the anterior region of GCNF−/− embryos at E8.5 and E9.5. White arrows indicate the position of MHB. Insets are dorsal views of the midbrain region of the embryos.

(Conlon and Rossant, 1992; Schwarz et al., 1997). Pax5 and En2 were expressed in the prospective MHB at E8.5 in embryos with seven somites (Figs. 6A, E). Subsequently, the expression domains of both these marker genes are refined to highly ordered pattern in the MHB at E9.5 in embryos with 30 somites (Figs. 6C, G). In GCNF−/− embryos, Pax5 mRNA was not detected in the prospective MBH region during early somitogenesis when the somite number was six (Fig. 6B). Weak Pax5 expression was observed at E.9.5 in GCNF−/− embryos with 13 somites in a ridge of neuroectoderm, while the medial Pax5 +/+ −/− Fig. 7. Analysis of apoptosis and cranial nerve development in GCNF and expression was lost (Fig. 6D). In GCNF embryos at the six GCNF−/− embryos. (A–D) Whole-mount TUNEL assays in WT embryos with somite stage, En2 was barely detectable in the midbrain region 30 somites and in GCNF−/− embryos with 13 somites at E9.5. (A, B) Lateral (Fig. 6F). Subsequently, En2 transcript was undetectable in the views. (C, D) Dorsal views. Significant increase in cell death was observed in −/− GCNF−/− embryos with 13 somites at E9.5 (Fig. 6H). Loss of the GCNF embryos compared to WT embryos. (E, F) Whole-mount staining with −/− antibody against alpha-neurofilament in WT embryos with 30 somites and in expression of midbrain marker genes in the GCNF embryos GCNF−/− embryos with 13 somites at E9.5. Note that the cranial nerves suggests that GCNF may play an important role of midbrain connected to the midbrain (III and IV) were underdeveloped in GCNF−/− formation. embryos at E9.5. A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24 19

Fig. 8. Schematic representation of the expression of the marker genes of the forebrain and isthmic organizer observed in GCNF−/− embryos relative to wild-type embryos. (A, B) In WT embryos, Otx2 is expressed in the rostral neural tube and Gbx2 is expressed in the posterior neural tube overlapping at the prospective MHB (pMHB) (expression is only shown unilaterally for clarity). Although similar expression patterns are observed in GCNF−/− embryos, Gbx2 expression is significantly down-regulated. Although the defects of neuronal differentiation in the midbrain are depicted at E9.5, the expressions of the MHB marker genes, which was analyzed at E8.5 (prior to the appearance of these defects), are significantly reduced in GCNF−/− embryos. The gene expression patterns are shown as bars and displayed positionally (anterior–posterior) relevant to the cartoon depicting a dorsal view of the developing brain with superimposed Otx2 and Gbx2 expression domain. (C, D) There are two possible mechanisms of how GCNF affects the expression of the midbrain marker genes. GCNF represses the expression of a down stream repressor, which antagonizes the expression of a transcriptional activator that normally regulates the expression of midbrain marker genes (Model I in C). Loss of GCNF function leads to up-regulation of this repressor which in turn leads to the repression of the activator diminishing the expression of midbrain marker genes (Model I in D). Alternatively, GCNF, an orphan receptor, may bind a ligand, which transforms it into a transactivator that directly regulates the expression of midbrain marker genes (Model II in C). Loss of GCNF expression leads to diminished expression of midbrain marker genes (Model II in D). the molecular defects in GCNF−/− embryos lead to abnormal ment. GCNF is expressed as early as the egg cylinder stage patterning of their cranial nerve derivatives. (Chung et al., 2001). After gastrulation, GCNF expression is detected in the head-fold and throughout the primitive streak. A Discussion posterior–anterior gradient of GCNF expression develops by the neural stage (Chung et al., 2001). By the late neural stage, The expression pattern of GCNF during early mouse GCNF expression is markedly reduced (Susens et al., 1997). At embryonic development suggests a role in neuronal develop- E15, GCNF is expressed in the postmitotic neurons of the 20 A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24 marginal zone of the neuroepithelium (Bauer et al., 1997). posteriorly, indicating that an early defect in Gbx2 mutants is a Inactivation of GCNF gene results in a failure of neural tube transformation of r1–3 into a midbrain fate. Thus, loss of Gbx2 closure. (Chung et al., 2001; Fuhrmann et al., 2001; Lan et al., expression in the GCNF−/− embryos may also affect hindbrain 2002). In addition, GCNF is required to regulate the development. differentiation and maturation of neuronal precursor cells (Sattler et al., 2004). In Xenopus, GCNF is important for cell Loss of GCNF function may affect normal midbrain intercalation during neurulation (Barreto et al., 2003b). In development addition, GCNF can synergize with ectopic chordin to induce the En2 expression in Xenopus (Song et al., 1999). Since GCNF−/− embryos die at E10, it is unclear whether the In this study, we investigated brain development in GCNF−/− midbrain is completely deleted in these embryos. But compared embryos and observed that expression of Otx2, Gbx2, En1/2, to the previous studies of midbrain development, it is clear that and Pax2/5 was significantly altered in the midbrain–hindbrain the midbrain is underdeveloped in GCNF−/− embryos. Deletion region. These results indicate that the isthmic organizer is not of portions of the midbrain is found in Wnt1 (McMahon and established by E9.5 in the absence of GCNF. At E9.5, both Bradley, 1990), En1/2 (Joyner, 1996), and Pax2 (Bouchard et Wnt1 and Fgf8 expression disappears in the region where the al., 2000) null mutant mouse embryos. In zebrafish embryos, isthmic organizer should form in the GCNF−/− embryos, thus, loss of a Pax2 ortholog and ace,anFgf8 hypomorph, also GCNF may play an important role in either establishing or causes deletion of the midbrain to the cerebellum (Brand et al., maintaining midbrain formation and signaling. 1996; Lun and Brand, 1998; Reifers et al., 1998). Studies in Fgf8 hypomorphs also demonstrate that a substantial portion of Loss of GCNF function affects normal forebrain development the midbrain, as well as the isthmus and cerebellum, are deleted (Meyers et al., 1998). Recent studies employing a conditional The anterior neural ridge (ANR) is a specific structure at the inactivation of Fgf8 expression in the mes/met region junction of anterior neural plate and the non-neural ectoderm demonstrated a complete deletion of the midbrain and (Eagleson et al., 1995). Fgf8 is expressed along the apex of cerebellum in these mouse embryos (Chi et al., 2003). Since ANR, and alteration of Fgf8 levels can affect the forebrain the expression of these important midbrain marker genes, Fgf8, development by affecting cell survival (Storm et al., 2003). Wnt1, Pax2/5,andEn1/2, are significantly reduced or After neural tube closure, Fgf8 expression is localized in the eliminated in GCNF−/− embryos (summarized in Fig. 8), region of the rostral midline of the telencephalon. Inactivation midbrain development is significantly hindered. Thus, GCNF of Fgf8 expression in this region increases apoptosis and plays a critical role in midbrain development, potentially at the reduces Spry1 expression (Storm et al., 2003). These results are level of transcriptional regulation. similar to what we observed in GCNF−/− embryos. In addition, Although a detailed mechanism of how GCNF affects the ANR expression of Fgf8 has been shown to be vital for the expression of the midbrain marker genes is still unclear, there induction and maintenance of BF1 (Shimamura and Ruben- are two possibilities (Figs. 8C and D). In model I, GCNF, a stein, 1997). Loss of BF1 expression induces hypoplasia of the known transcriptional repressor, normally represses the expres- cerebral hemispheres. Thus, the ANR expression of Fgf8 sion of a down stream repressor, which antagonizes the regulates growth of the telencephalon. In a similar manner, the expression of a transcriptional activator that normally regulates isthmus also expresses Fgf8 and controls midbrain patterning, the expression of midbrain marker genes (Model I in Fig. 8C). the ANR has also been proposed to be another local organizer Loss of GCNF function leads to up-regulation of the repressor, for telencephalon development (Storm et al., 2003). which in turn leads to the repression of the midbrain marker Forebrain development is controlled by factors, such as genes, such as En1/2, Fgf8, Pax2/5, and Wnt1 (Model I in Fig. Otx2. Otx2 expression is required during gastrulation in the 8D). Alternatively, GCNF, an orphan receptor, may bind a anterior visceral endoderm for induction of head tissue anterior ligand, which transforms it into a transactivator that directly to r3 (Li and Joyner, 2001). If Otx2 expression is altered, the regulates the expression of midbrain marker genes (Model II in forebrain and midbrain will not be specified normally (Liu and Fig. 8C). Loss of GCNF expression leads to diminished Joyner, 2001a). Marker gene analysis showed that the domains expression of midbrain marker genes (Model II in Fig. 8D). We of expression of Wnt1, Fgf8, En1, and Pax2 are shifted to the cannot rule out that in addition to these direct roles in anterior of the neural tube from early somite stages, and their transcriptional regulation, loss of GCNF function may alter expression patterns overlap. It appears that the forebrain and cell lineage properties in the brain. In the absence of GCNF, the midbrain are initially specified and then forebrain cells die, and precursor cells of the midbrain may alter their cell fate rendering the midbrain may be transformed into more posterior tissue. In them unable to properly express midbrain markers. contrast, Gbx2 null mutants lack anterior hindbrain tissue Retinoic acid (RA) plays an important role in hindbrain derived from r1–3(Li et al., 2002), whereas GCNF−/− embryos development (Gavalas, 2002). RA can alter the expression of do have anterior hindbrain rhombomeres (date not shown). At the marker genes during development of the MHB (Avan- early somite stages, the domain of Otx2 is expanded into the taggiato et al., 1996). It has been proposed that GCNF may presumptive metencephalon in Gbx2 mutants, before a be involved in RA signaling (Barreto et al., 2003a; Heinzer et morphological defect is obvious (Millet et al., 1999). Fgf8 al., 1998). Addition of RA to P19 embryonic carcinoma cells and Wnt1 expression overlap and are also shifted and expanded GCNF expression (Bauer et al., 1997; Fuhrmann et al., 2001). A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24 21

Studies in Xenopus also suggest that knockdown of GCNF Spry1 are undetectable in the midbrain region. This difference expression can alter Cyp26A1 expression, which in turn can be explained by the different levels of Fgf8 transcript in the affects RA signaling (Barreto et al., 2003a). However, when embryos at the onset of midbrain development. In the Fgf8 we compared the phenotypic alteration of the gain- and loss- MHB KO embryos, the level of Fgf8 expression, before the of-function mutants of RA signaling (Raldh2 and Cyp26A1 Fgf8 gene is inactivated at E8.5, may be sufficient to initiate and mutants) with GCNF−/− embryos, their results differed from maintain the expression of Pax2, En1, and Spry2 in contrast to those in the GCNF−/− embryos in important ways (Abu-Abed what is observed in the GCNF−/− embryos. et al., 2001; Niederreither et al., 1999, 2000; Sakai et al., 2001). En2, Fgf8, and Gbx2 expression patterns are not Differences between xGCNF and mGCNF altered in Raldh2−/−embryos and nor is En2 expression altered in Cyp26A1−/−embryos (Abu-Abed et al., 2001; The results of this study highlight some interesting Niederreither et al., 2000). These two results suggest that similarities and differences between the function of GCNF in absence or alteration of RA does not affect midbrain amphibians and mammals. First, GCNF is important for both development or at least not to a degree observed in the amphibian and mammalian midbrain development. In Xenopus, GCNF−/− embryos. In addition, we did not detect any GCNF synergizes with ectopic chordin to induce En2 difference in endogenous RA signaling in the MHB using expression in animal cap assays (Song et al., 1999). In Keller RARE-lacZ transgenic reporter mice crossed with GCNF−/− explants, which rely on endogenous factors for neural embryos (data not shown). Although loss of GCNF function induction, GCNF can induce En2 expression (Song et al., does affect RA signaling during posterior hindbrain develop- 1999). Injection of a dominant negative form of GCNF is ment (data no shown), the loss of expression of isthmic capable of reducing En2 expression (Song et al., 1999). In our marker genes is unlikely to be directly due to the alteration of study, loss of GCNF function significantly reduces expression RA signaling in the MHB. of important regulatory genes in midbrain development. Taken together, results from these two animal models demonstrate that Comparison of phenotypes in GCNF−/− embryos with previous GCNF plays an evolutionarily conserved role in regulating studies of Fgf8 mutant embryos midbrain development. In contrast to the results in this study, the GCNF knockdown Since Fgf8 expression in the MHB is significantly reduced in experiments in Xenopus demonstrates different results with GCNF−/− embryos, loss of GCNF function may show similar respect to the regulation of gene expression in the forebrain and phenotypes to Fgf8 hypomorphs or the Fgf8 MHB conditional midbrain regions. In Xenopus, both Otx2 and Gbx2 are knockout (MHB KO) embryos. There are several similarities expressed, but their expression domains are shifted caudally, between GCNF−/− embryos and Fgf8-deficient embryos. First, suggesting a repositioning of the MHB (Barreto et al., 2003a). Fgf8 is required to maintain Wnt1 and Gbx2 expression in the Similarly, En2 and Pax2 are expressed in the midbrain region, rostral metencephalon (Garda et al., 2001; Liu et al., 1999; and their expression domains are also shifted caudally after Martinez et al., 1999). Therefore, in Fgf8 hypomorphs and Fgf8 GCNF knockdown (Barreto et al., 2003a). These results suggest MHB KO embryos, expression of Wnt1 and Gbx2 in the MHB an expansion of anterior structures of the forebrain and midbrain is undetectable (Chi et al., 2003; Meyers et al., 1998). Gbx2 with simultaneous loss of posterior structures, such as anterior expression in the MHB determines the posterior limit of Otx2 hindbrain. In mice, loss of function of GCNF reduces the expression (Li and Joyner, 2001; Li et al., 2002). Similarly in expression of En2, Gbx2, and eliminates expression of Pax2 in Fgf8 MHB KO embryos, Gbx2 expression in rhombomere 1 is the MHB. These differences may be due to differences in absent, and Otx2 expression extends caudally (Chi et al., 2003). expression of marker genes or important regulatory factors Interestingly, in E9.5 GCNF−/− embryos, Otx2 expression is during embryonic development in the two species (Chung et al., extended caudally while Gbx2 expression is down-regulated in 2001) or differences in experimental design. the anterior hindbrain. This defect is probably the result of down-regulation of Fgf8 expression, since Fgf8 is required to Comparison of GCNF−/− mouse embryos with the zebrafish maintain Gbx2 expression (Liu et al., 1999; Martinez et al., spiel ohne grenzen (spg) mutant embryos 1999); in turn Gbx2 is required to repress Otx2 expression in the hindbrain (Wassarman et al., 1997; Millet et al., 1999). Recent studies of zebrafish mutant embryos reveal that the Alternatively, it is also possible that deregulation of Otx2 and pou2 gene, which is the zebrafish ortholog of mouse Oct4, Gbx2 expression leads to disruption of the isthmic organizer and which is a GCNF target gene in mice, and human Pou5f1, plays loss of Fgf8 expression in GCNF−/− mice. However, since this an important role in midbrain development (Belting et al., 2001; deregulation occurs only after the disappearance of Fgf8 Fuhrmann et al., 2001; Reim and Brand, 2002). Spg mutant expression after E8.5, it is likely that the disappearance of embryos, which have lesions in the pou2 gene, display a unique Fgf8 expression leads to the loss of Otx2 and Gbx2 expression. phenotype in midbrain and hindbrain development (Belting et On the other hand, expressions of Pax2, En1, and Spry2, al., 2001), and ubiquitous Oct4 expression in transgenic mice which are thought to be regulated by Fgf8 (Wurst and Bally- suggests that the mammalian Oct4 gene may also play a role in Cuif, 2001), are not significantly reduced in Fgf8 MHB KO brain development (Ramos-Mejia et al., 2005). In mice, Oct4 is embryos. In GCNF−/− embryos, expressions of Pax2, En1, and transiently expressed in the neuroepithelium of the head-fold 22 A.C.-K. Chung et al. / Developmental Biology 293 (2006) 13–24 just before somitogenesis initiates (Fuhrmann et al., 2001; Reim markers is altered (Ramos-Mejia et al., 2005). Levels of Pax2 and Brand, 2002). At the onset of somitogenesis, Oct4 and En2 expression are transiently augmented at E8.5 but return expression is down-regulated in somatic cells and is restricted to normal at E9.5. At E8.5, Fgf8 signal was reduced in the to primordial germ cells (Fuhrmann et al., 2001). We have branchial arches and presomitic mesoderm. A broader and less- clearly demonstrated that GCNF is able to repress Oct4 confined domain of Fgf8 expression is found in the MHB and expression and loss of GCNF expression leads to derepression ANR of one-third of the transgenic embryos. At E9.5, Fgf8 of Oct4 expression in the head-fold and posterior regions at expression in the MHB is no longer affected. An increase of cell E8.5 and E9.5 and in GCNF−/− ES cells (Fuhrmann et al., 2001; death in the anterior neuroepithelium is observed at E9.5, Gu et al., 2005). It raises the question as to whether derepression suggesting that this increased cell death may be responsible for of Oct4 expression contributes to the midbrain defects in the reduction in brain size that was observed (Ramos-Mejia et GCNF−/− embryos. al., 2005). Thus, alteration of Oct4 expression may play a role in In zebrafish spg mutant embryos, the MHB is not disrupting normal mammalian brain development. Ectopic Oct4 established, and the midbrain is reduced in size (Belting et al., expression in GCNF−/− embryos may contribute, to a certain 2001; Reim and Brand, 2002). Despite normal expression of extent, to the midbrain defects but are clearly not the sole source Otx2, Gbx1, and Fgf8 during late gastrulation, the initial of the defects. expression of Gbx2, Pax2.1, and En2, and later expression of GCNF is expressed in the neuroepithelium during early Fgf8 and Wnt1 in the MHB, are reduced (Belting et al., 2001; somitogenesis and in postmitotic neurons of the marginal zone Reim and Brand, 2002). Pax2.1 expression in spg mutant of the neuroepithelium in the developing brain at E15 (Bauer et embryos can be restored by injection of Pou2 or even murine al., 1997; Fuhrmann et al., 2001; Susens et al., 1997), and Oct4 mRNA, suggesting that Oct4 may function in activation of GCNF expression is up-regulated during RA-induced neural Pax2 in normal midbrain development (Belting et al., 2001; differentiation of certain embryonic carcinoma cells and Reim and Brand, 2002). Since the injection of pou2 mRNA in embryonic stem cells (Bauer et al., 1997; Fuhrmann et al., normal or high doses does not induce ectopic expression of 2001; Gu et al., 2005). Thus, GCNF likely plays a role in midbrain markers (Belting et al., 2001; Burgess et al., 2002; neurogenesis and/or neuronal differentiation. In this study, we Reim and Brand, 2002), pou2 is necessary but not sufficient to observed that no mature neurons are generated in the midbrain induce midbrain markers, demonstrating a permissive role for of GCNF−/− embryos at E9.5, and significant cell death is the pou2 gene in midbrain development. observed, suggesting the possibility that once generated In addition, the phenotypes of spg mutants are similar to ace/ midbrain cells die. Recent studies in a mouse embryonic Fgf8 mutants, suggesting that the functions of these genes are carcinoma cell line demonstrate that GCNF expression is closely related (Reim and Brand, 2002). Pou2 is shown to be a critical for aggregation of neuronal cells, differentiation, and factor that mediates the competence to respond to Fgf8. maturation of neuronal precursor cells (Sattler et al., 2004). In Injection of Fgf8 mRNA or implantation of Fgf8 beads in spg the future, it will be important to investigate how loss of GCNF mutants does not cause any effect (Reim and Brand, 2002). function affects neuronal differentiation and the expression of Similarly, injection of pou2 mRNA fails to restore ace's important regulatory factors and marker genes. In conclusion, phenotypes. Synergistic effects on midbrain markers in the spg/ there is a clear disruption of midbrain formation in GCNF−/− ace double mutants suggest that pou2 and Fgf8 act together embryos. It is likely that GCNF will probably play a more (Reim and Brand, 2002). widespread role in neuronal development of central nervous It is quite interesting to compare the phenotypes of system. GCNF−/− mouse embryos and spg zebrafish mutant embryos. Both have a loss or reduced expression of midbrain markers and structures. The major difference between these two Acknowledgments models is the expression of Oct4. In zebrafish spg mutants, Oct4 expression is reduced, while in the GCNF−/− embryos, We thank Drs. A. J. Joyner, G. R. Martin, A.P. McMahon, P. Oct4 is ectopically expressed in somatic cells (Fuhrmann et Gruss, J. Rossant, R. L. Rubenstein, and W. Wurst for the al., 2001). Owing to the permissive role of Oct4 in midbrain plasmids. This work was supported by grants from NIH, development shown in spg mutants, ectopic expression of NIDDK DK073524 to (AJC), and American Heart Association Oct4 in GCNF−/− embryos should not induce ectopic to (ACKC) and 0330144N. expression of midbrain markers, but rather it should maintain the expression of these markers. 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