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Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions

Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions RESEARCH ARTICLE 3993 Development 133, 3993-4004 (2006) doi:10.1242/dev.02585 Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions 1, ,† 1, 1 2 3 Tsutomu Hirata * , Masato Nakazawa *, Osamu Muraoka , Rika Nakayama , Yoko Suda and 1,‡ Masahiko Hibi Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice we found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene- mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions. KEY WORDS: Fez (Fezf1) Fez-like (Fezf2), Zinc finger, Forebrain, Telencephalon, Diencephalon, Zona limitans intrathalamica, Thalamus, Prethalamus, Pretectum, Prosomere, Mouse, Transcriptional repressor INTRODUCTION Although the structures of the diencephalic subdivisions become The forebrain of adult mammals is one of the most complicated obvious in mice late in gestation, individual subdivisions can be biological structures; it is essential for higher neural functions, distinguished by their expression of genetic markers at the beginning such as memory, emotion, reasoning and the planning of of forebrain patterning. Members of the Dlx family of genes are coordinated movements. Various models for forebrain subdivisions expressed in the prethalamus, Gbx2 is expressed in the thalamus (neuromeres) have been developed over the past 10 years (Bulfone et al., 1993) and Ebf1 and Lhx1 are expressed in the (reviewed by Kiecker and Lumsden, 2005). Among them, Puelles anterior and posterior pretectum, respectively (Barnes et al., 1994; and Rubenstein and colleagues proposed a neuromeric Garel et al., 1997). Studies of genetically modified mice have organization of the forebrain on the basis of the differential revealed several genes involved in the patterning and/or development expression of neural marker genes and morphological of the diencephalon. These include Pax6, Otx2, Emx2 and Six3 considerations (the so called ‘prosomeric model’) (Puelles and (Kimura et al., 2005; Kurokawa et al., 2004a; Kurokawa et al., Rubenstein, 1993; Puelles and Rubenstein, 2003; Rubenstein et al., 2004b; Lagutin et al., 2003; Stoykova et al., 1996; Suda et al., 2001). 1994; Rubenstein et al., 1998). In the most recent model (Puelles However, it is largely unknown how these genes function in the and Rubenstein, 2003), the forebrain can be divided into rostral formation of the diencephalon subdivisions and what other genes are and caudal regions. The rostral part of the forebrain is the involved in diencephalon patterning. telencephalon and can be divided into several anatomical and The zona limitans intrathalamica (ZLI) is located on the functional territories, including the pallium, subpallium, preoptic boundary between the prethalamus and thalamus (Larsen et al., area and hypothalamus. The caudal part of the forebrain is the 2001; Shimamura et al., 1995). The ZLI marks the interface diencephalon and can be divided into three transverse subdivisions between regions of different ability to respond to inductive signals known as prosomeres: the prethalamus (also called the ventral such as Fgf8 and Shh; for instance, in neural tissue rostral to the thalamus, p3), thalamus (also known as the dorsal thalamus, p2), ZLI, Fgf8 induces the expression of Foxg1 (also called BF-1), but and pretectum (p1). The eminentia thalamic, habenula/epithalamus caudal to the ZLI it induces the expression of En2 (Shimamura and and posterior commissures are located dorsally to the prethalamus, Rubenstein, 1997). The ZLI also has inductive influences on the thalamus and pretectum, respectively. adjacent subdivisions (prethalamus and thalamus). Shh expressed in the ZLI is involved in development of the prethalamus and thalamus in chick and zebrafish embryos; inhibition of Shh Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, signaling represses the expression of the prethalmus and thalamus Kobe 650-0047, Japan. Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan. Vertebrate Body markers (Kiecker and Lumsden, 2004; Scholpp et al., 2006; Vieira Plan Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan. et al., 2005). In chick embryos, the future position of the ZLI is at the abutting expression domains of two homeobox genes, Six3 *These authors contributed equally to this work Present address: Center for Neuroscience Research, Children’s National Medical rostrally and Irx3 caudally (Kobayashi et al., 2002). Six3 and Irx3 Center, Washington DC 20010-2970, USA regulate expression of each other, and the misexpression of Six3 or Author for correspondence (e-mail: [email protected]) Irx3 affects the formation of the prethalamus and thalamus (Braun et al., 2003; Kobayashi et al., 2002). Irx3 expression confers Accepted 11 August 2006 DEVELOPMENT 3994 RESEARCH ARTICLE Development 133 (20) competence to respond to the Fgf8 and Shh inductive signals by Hirata et al., 2006) revealed that the mouse gene reported by Matsuo- Takasaki et al. is more similar to zebrafish fezl than to Xenopus Fez expressing thalamus-specific genes (Kiecker and Lumsden, 2004; (Hashimoto et al., 2000; Matsuo-Takasaki et al., 2000). Therefore, we refer Kobayashi et al., 2002). The expression of Six3 and Irx3 is regulated to AB042399 as mouse Fezl (Hashimoto et al., 2000; Hirata et al., 2004), by Wnt signaling (Braun et al., 2003); Wnt1, Wnt3 and Wnt3a are and to AK014242, which is more similar to Xenopus Fez, as mouse Fez expressed in the dorsal neural tissue caudal to the prospective ZLI +/– +/– and Fez mice were previously described (Hirata et al., 2006). Fezl when the expression of Six3 and Irx3 begins (Braun et al., 2003; +/– +/– and Fezl (Hirata et al., 2006; Hirata et al., 2004). Both the Fez Lagutin et al., 2003; Roelink and Nusse, 1991; Salinas and Nusse, heterozygous mice were originally established in a 129sv genetic 1992). These reports suggest that Wnt signaling controls the rostro- background and backcrossed to the C57BL/6 background for several +/– +/– caudal polarity of the forebrain through the regulation of Six3 and Fezl double heterozygous mice were generated by generations. Fez +/– +/– Irx3, and the mutually repressive interaction of Six3 and Irx3 and Fezl heterozygotes, and were used to generate crossing Fez –/– +/– +/– –/– –/– –/– Fezl , Fez Fezl and Fez Fezl embryos. Mice were housed in controls the position of the ZLI and thus confers differential Fez an environmentally controlled room at the Animal Facility of the Center for competence to respond to the inductive signals to form the Developmental Biology (CDB), RIKEN, under the guidelines for animal prethalamus and thalamus. This hypothesis is based mainly on the experiments from RIKEN CDB. The genotypes of newborn mice and results of misexpression studies and explant assays in the chicken embryos were determined by PCR analysis (Hirata et al., 2006; Hirata et al., embryo, but there is little genetic evidence to support it. Six3- 2004). The primer sequences and PCR conditions are available on request. deficient mice display a strong reduction of the neural tissue rostral Noon of the day on which the vaginal plug was detected was designed as to the ZLI, but still express Shh in the ZLI and rudimentary rostral embryonic day (E) 0.5. tissue (Lagutin et al., 2003), suggesting that other genes may Histological sections and Nissl staining cooperate with or function parallel to Six3 to determine the position Brains or embryos were fixed with Carnoy’s solution at room of the ZLI or control the formation of the rostral diencephalon. temperature overnight. Specimens were dehydrated and embedded in Fez (Fezf1 – Mouse Genome Informatics) and Fez-like (Fezl, paraffin. Serial sections were prepared and stained with 0.1% Cresyl Fezf2 – Mouse Genome Informatics) are closely related genes that Violet (MERCK). encode transcriptional repressors containing six C2H2-type zinc fingers and an Eh1 (Engrailed homology 1) repressor motif, and RNA probes and in situ hybridization were originally isolated as anterior neuroectoderm-specific genes Embryos were fixed overnight at 4°C in 4% paraformaldehyde (PFA) in PBS. Specimens were gradually dehydrated in ethanol/H O and stored in in Xenopus and zebrafish (Hashimoto et al., 2000; Matsuo-Takasaki 2 ethanol at –20°C. The protocol for in situ hybridization was described et al., 2000). Orthologs of these two genes exist in the puffer fish, previously (Hirata et al., 2006; Hirata et al., 2004). Single-stranded zebrafish, mouse and human (Hirata et al., 2006). In mouse and digoxigenin-UTP-labeled (Roche) RNAs were used. In situ signals were zebrafish, Fez and Fezl are expressed in overlapping domains in the detected with an anti-digoxigenin antibody and BM Purple (Roche). For forebrain during development (Hashimoto et al., 2000; Hirata et al., two-color staining of the histological sections in Fig. 6D, Fez and Irx1 probes 2006; Hirata et al., 2004; Matsuo-Takasaki et al., 2000). A number labeled with FITC-UTP and digoxigenin-UTP (Roche), were used of studies have previously investigated the specific roles of Fez and respectively; BM Purple and Fast Red (Roche) were used for staining. For Fezl in neural development in both zebrafish and mice. The Fig. 6H-M, Fez and Fezl probes were labeled with FITC-UTP and Irx1 probe zebrafish mutant too few (tfu) has a fezl gene mutation and displays with digoxigenin-UTP. The hybridized signals for Fez and Fezl were a loss or reduction of dopaminergic neurons in the hypothalamus detected by an alkaline phosphatase-conjugated anti-Fluorescein antibody (Roche) and BM Purple, and those for Irx1 were detected by a peroxidase- (Levkowitz et al., 2003). Fezl-deficient mice show no abnormalities conjugated anti-digoxigenin antibody (Roche) and tyramide signal in the dopaminergic neurons, but do show abnormal formation of amplification system (TSA-Plus Fluorescein System, PerkinElmer). The the subplate neurons and thalamocortical axons, and loss of the precise protocols for in situ hybridization are available on request. The fornix/fimbria system (Hirata et al., 2004). Fezl has also been shown probes were: Dlx1 (Bulfone et al., 1993), Gbx2 (Bulfone et al., 1993), Lhx1 to be required for the development of subcerebral projection (Fujii et al., 1994), Ebf1 (Garel et al., 1997), Sox14 (Hashimoto-Torii et al., neurons in layer V of the neocortex (Chen et al., 2005a; Chen et al., 2003), Emx2 (Yoshida et al., 1997), Pax6 (Walther and Gruss, 1991), Shh 2005b; Molyneaux et al., 2005). Fez-deficient mice display (Echelard et al., 1993), Tcf4 (Ishibashi and McMahon, 2002), Lhx5 abnormal olfactory sensory neuronal projections and olfactory bulb (Nakagawa and O’Leary, 2001), Fgf8 (Crossley and Martin, 1995), Irx1 (a formation (Hirata et al., 2006). The relatively weak forebrain gift of T. Ogura), Wnt3a (Takada et al., 1994), En2 (Joyner and Martin, phenotypes of Fez and Fezl-deficient mice suggest that Fez and Fezl 1987), Foxg1 (Tao and Lai, 1992), Fez (Hirata et al., 2006) and Fezl (Hirata et al., 2004). In situ hybridization images were taken using an AxioPlan2 function redundantly in the patterning and development of the microscope or a SteREO Lumar V12, equipped with an AxioCam CCD forebrain. camera (Zeiss). Figures were assembled using AxioVision version 4.3 and Here, we generated mice deficient in both the Fez and Fezl genes. Adobe Photoshop CS2. The Fez;Fezl-deficient mice showed defects in the rostro-caudal patterning of the diencephalon. We found that Fez and Fezl Generation and genotyping of transgenic mice redundantly control the rostro-caudal patterning of the diencephalon We isolated an approximately 8.2 kbp enhancer/promoter region of Fezl by repressing the caudal diencephalon fate in the prospective from the bacterial artificial chromosome clone containing the coding and non-coding regions of Fezl (Hirata et al., 2004). To make the Fezl prethalamic region, and that ZLI formation depends on Fez/Fezl- enhancer/promoter-driven lacZ (-galactosidase) transgene constructs, the mediated formation of diencephalon subdivisions. 8.2 or 2.7 kbp enhancer/promoter region of Fezl was connected to lacZ cDNA at the position of the translational initiation site of Fezl (Fezl8.2p- MATERIALS AND METHODS lacZ, Fezl2.7p-lacZ). For misexpression of Fezl, its 2.7 kbp enhancer/ Mouse mutants and gene naming promoter was connected to Fezl or Fez cDNA, followed by internal A mouse Fezl cDNA fragment (GenBank Accession Number AI325906) ribosomal entry site (IRES)-Gap43-Venus (Fezl2.7p-Fezl-IRES-Venus and was originally reported as the mouse ortholog of Xenopus Fez (Matsuo- Fezl2.7p-Fez-IRES-Venus) (IRES-Gap43-Venus, a gift of Y. Yoshihara). Takasaki et al., 2000). However, comparing the sequences of zebrafish fez Similarly, Otx2 FM enhancer (1.4 kbp) (Kurokawa et al., 2004a) and the (AB207804) and fezl (AB041824), mouse Fez homolog (AK014242), and promoter of mouse heat shock protein 68 (pHsp68) (Sasaki and Hogan, the full-length clone of mouse Fezl (AB042399) (Hashimoto et al., 2000; 1996) were connected to Fezl cDNA and IRES-Gap43-Venus (OtxFM- DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 3995 Hsp68-FeZLIRES-Venus). The transgene-derived Fezl expression was RESULTS monitored as Venus expression with an epifluorescence microscope Fez and Fezl function redundantly in forebrain AxioPlan2. Genotypes of the transgenic mice were analyzed by PCR with formation 5-AAACCCTGGCGTTACCCAACT-3 and 5-ACGACAGTATCGGCC- To reveal whether Fez and Fezl have redundant roles in forebrain TCAGGA-3 for the lacZ reporter lines, 5-TGTGTCTGCAGAGA- formation, we crossed Fez- and Fezl-deficient heterozygous mice GTGCTGGCCTG-3 and 5-CTGGCTGCTGCTCACCCCAAGCTTT-3 (Hirata et al., 2006; Hirata et al., 2004) and generated mice deficient for the Fezl2.7p-Fezl-IRES-Venus and OtxFM-Hsp68-Fezl-IRES-Venus lines homozygously or heterozygously in the Fez and/or Fezl genes (Fig. and 5-AAAACGTATTTAGCCGAAAGGAAT-3 and 5-ACTTTACACA- –/– +/– Fezl embryos showed a small olfactory bulb at E15.5 1). Fez CGAAGGGTCTGG-3 for the Fezl2.7p-Fez-IRES-Venus lines. Transient –/– +/– –/– embryos do (Hirata et al., 2006). Fez Fezl (Fig. 1F), as Fez transgenic embryos were generated and -galactosidase staining was embryos showed defects in the formation of the dentate gyrus at performed as described previously (Kimura et al., 1997; Kimura et al., –/– –/– Fezl embryos showed more severe 2000). E17.5 (Fig. 1K). Fez –/– +/– +/– –/– –/– –/– +/– +/– Fig. 1. Morphology of the forebrain of wild-type, Fez Fezl , Fez Fezl , Fez Fezl and Fez Fezl mice. Coronal (A-D,I-L) and –/– +/– +/– –/– –/– –/– sagittal (E-H) sections of E15.5 (A-H), E17.5 (I-L) wild-type (A,E,I), Fez Fezl (B,F,J), Fez Fezl (C,G,K), and Fez Fezl (D,H,L) mice. +/– +/– –/– –/– (M-Q) Sagittal sections of E18.5 Fez Fezl (M-O) and Fez Fezl (P,Q) mice at the level where posterior commissures (M,P, indicated by white arrowheads), habenulo-interpeduncular tracts (N,Q, white arrows) or mammillthalamic tracts (O, black arrowhead) were observed. Nissl staining. –/– +/– +/– –/– Fez Fezl mice had a very small olfactory bulb and Fez Fezl showed a reduction of the dentate gyrus (arrow in K,L), compared with wild-type –/– –/– littermates. Fez Fezl mice showed loss of the olfactory bulb (arrow in H), dentate gyrus and the CA3 region of hippocampus (arrow in L), +/– +/– –/– –/– prethalamus, strong reduction of the thalamus (region between arrowheads) and a reduced neocortex. At E18.5, both Fez Fezl and Fez Fezl mice had a posterior commissure and habenulo-interpeduncular tract. The habenulo-interpeduncular tracts were small and abnormally located, due –/– –/– +/– +/– –/– –/– to severe reduction of the thalamus in Fez Fezl mice. Fez Fezl (O), but not Fez Fezl mice, had mammilothalamic tracts. DG, dentate gyrus; hit, habenulo-interpeduncular tract; hip, hippocampus; ht, hypothalamus; ma, mamillary region; mtt, mammilothalamic trancts; ncx, neocortex; OB, olfactory bulb; pc, posterior commissure; th, thalamus; pt, pretectum; pth, prethalamus; s, septum; 3v, third ventricle. Scale bars: 0.5 mm. DEVELOPMENT 3996 RESEARCH ARTICLE Development 133 (20) phenotypes in the olfactory bulb and hippocampus: nearly complete the boundary between the thalamus and pretectum, respectively (Fig. loss of the olfactory bulb, stronger reduction of the CA3 region and 1M,N). However, the habenulo-interpeduncular tracts in the –/– –/– –/– –/– Fezl embryos also Fezl embryos tracts were small and abnormally located (Fig. loss of dentate gyrus in the hippocampus. Fez Fez –/– –/– Fezl showed impaired neocortex formation. In addition, Fez 1Q). This is probably due to the strong reduction of the thalamus. –/– –/– embryos were found to have abnormalities in the formation of Fezl embryos did not have a mammilothalamic tract, The Fez diencephalon, including loss of prethalamus and a strong reduction which is located in between the prethalamus and thalamus (Fig. 1O). –/– –/– Fezl embryos had no of the thalamus in size (Fig. 1H, arrowheads). These diencephalon These data indicate that the Fez –/– +/– +/– –/– Fezl and Fez Fezl defects were not observed in the Fez prethalamus and displayed a strong reduction of thalamus, while embryos (Fig. 1F,G), indicating a redundant role of Fez and Fezl in having a relatively normal development of the pretectum. the formation of diencephalon. Although it is intriguing to study the phenotypes of the olfactory bulb, hippocampus and neocortex in Loss of the prethalamus and expansion of the these mice, we restricted the focus of our study to the role of Fez and caudal diencephalon in Fez, Fezl double mutants Fezl in diencephalon formation. We next examined the Fez;Fezl-deficient mice with various genetic –/– –/– Fezl embryos A series of sagittal sections revealed that the Fez markers at E12.5. Dlx1 is expressed in the ventral telencephalon had posterior commissures and habenulo-interpeduncular tracts at (including the medial and lateral ganglionic eminences), E18.5 (Fig. 1P,Q), located on the dorsal side of the pretectum and on hypothalamus and prethalamus, but not the thalamus (Fig. 2A) –/– –/– Fig. 2. Defects in prethalamus and thalamus development and rostral expansion of the pretectum in Fez Fezl embryos at E12.5. (A-T) Expression of Dlx1 (marker for prethalamus and ganglionic eminence, A-D), Gbx2 (thalamus, E-H), Lhx1 (posterior pretectum and ZLI, I-L), –/– +/– +/– –/– –/– –/– Ebf1 (anterior pretectum, M-P) and Sox14 (rostral domain of thalamus, Q-T) in control, Fez Fezl , Fez Fezl and Fez Fezl embryos was analyzed by in situ hybridization. Sagittal sections with anterior to the left. Dlx1 expression was not detected in the prethalamus but was –/– –/– maintained in the hypothalamus and ganglionic eminence in Fez Fezl embryos (D). Gbx2 expression was strongly reduced and detected in –/– –/– patches (arrows in H) in Fez Fezl embryos. Lhx1 expression was absent in the ZLI, but was not significantly affected in the posterior pretectum of –/– –/– –/– –/– Fez Fezl embryos (I-L). The expression domain of Ebf1 was expanded rostrally in Fez Fezl embryos (P), compared with that in the wild-type, –/– +/– +/– –/– –/– –/– Fez Fezl and Fez Fezl littermates (M-O). The expression of Sox14 was abolished in Fez Fezl embryos (T). apt, anterior pretectum; GE, ganglionic eminence; ht, hypothalamus; ppt, posterior pretectum; pth, prethalamus; th, thalamus. DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 3997 –/– –/– (Bulfone et al., 1993; Stuhmer et al., 2002). In the Fez Fezl embryos (Fig. 2P). Sox14 is expressed in the rostral part of the embryos, Dlx1 expression was maintained in the ganglionic thalamus (Hashimoto-Torii et al., 2003) (Fig. 2Q), and its expression –/– –/– Fezl embryos (Fig. 2T). Neither the eminences and hypothalamus, but was completely absent in the was absent in the Fez –/– +/– +/– –/– Fezl nor the Fez Fezl embryos showed abnormal diencephalon (Fig. 2D). Gbx2 is expressed strongly in the thalamus Fez and weakly in the ganglionic eminences (Fig. 2E) (Bulfone et al., expression of Dlx1, Gbx2, Lim1, Ebf1 or Sox14 at E12.5 (Fig. –/– –/– Fezl embryos, Gbx2 expression in the thalamus 1993). In Fez 2B,C,F,G,J,K,N,O,R,S), further confirming the strictly redundant was markedly reduced and shifted rostrally, but not abrogated (Fig. function of Fez and Fezl in the diencephalon patterning. The data –/– –/– Fezl embryos at E12.5, the prethalamus 2H). Lhx1 is expressed in the posterior pretectum and ZLI (Fig. 2I) indicate that, in the Fez (Barnes et al., 1994; Fujii et al., 1994; Mastick et al., 1997; Suda et did not form, the thalamus formed at a reduced size and the anterior al., 2001). Although its expression in the posterior pretectum was pretectum expanded rostrally, suggesting that Fez and Fezl play an not affected significantly, we found that it was not expressed in the important role in rostro-caudal patterning of the diencephalon. –/– –/– Fezl embryos (Fig. 2L). Ebf1 is expressed in the ZLI in the Fez Although we detected Tuj-1-postive postmitotic neurons in the –/– –/– Fezl embryos (data not shown), the anterior pretectum (Garel et al., 1997; Suda et al., 2001) (Fig. 2M), thalamus at E11.5 in the Fez –/– –/– –/– –/– Fezl Fezl embryos failed to form the rostral part of the thalamus and its expression was expanded rostrally in the Fez Fez Fig. 3. Defects in telencephalon formation and regionalization of the diencephalon in –/– –/– Fez Fezl embryos. Expression of Emx2 (A-D), Pax6 (E-H), Dlx1 (I-L), Shh (M-P), Tcf4 (Q-T) and Lhx5 (U-X) in control (A,E,I,M,Q,U), –/– +/– +/– –/– Fez Fezl (B,F,J,N,R,V), Fez Fezl –/– –/– (C,G,K,O,S,W) and Fez Fezl embryos (D,H,L,P,T,X) at E10.5. (A-T) Whole-mount in situ hybridization and lateral views of anterior neuroectoderm, with anterior to the left. (U-X) Coronary sections of the diencephalon. Emx2 expression in the dorsal telencephalon –/– –/– was reduced in Fez Fezl embryos. Pax6 expression was downregulated in the thalamus at this stage (marked by dots, E,F,G) in control, –/– +/– +/– –/– Fez Fezl and Fez Fezl embryos, but not –/– –/– in Fez Fezl embryos (arrow, H). The expression domain of Pax6 in the dorsal telencephalon was reduced but not significantly affected in the diencephalon except for the thalamus (region between –/– –/– arrowheads) in Fez Fezl embryos. Dlx1 expression was detected in the ganglionic eminence (arrow, I,L) but not in the –/– –/– prethalamus (arrowhead, I,L) in Fez Fezl embryos. Shh expression was detected in the –/– –/– AEP, but not in the ZLI in Fez Fezl embryos (arrow, P), compared with controls (M-O). The domain expressing Tcf4 at a high level (thalamus and pretectum region, marked by –/– –/– dots) was expanded in Fez Fezl embryos (T), compared with controls (Q-S). Lhx5 expression was abolished in the prethalamus of –/– –/– Fez Fezl embryos (X). ht, hypothalamus; pth, prethalamus; th, thalamus. DEVELOPMENT 3998 RESEARCH ARTICLE Development 133 (20) (Sox14-positive region), the formation of which is known to depend on Shh from the ZLI (Hashimoto-Torii et al., 2003). The data indicate that the redundant function of Fez and Fezl also controls the rostro-caudal patterning of the thalamus directly or indirectly. Defects in regionalization of the diencephalon in the Fez, Fezl double mutant We analyzed the Fez;Fezl-deficient mice with genetic markers at E10.5, when forebrain patterning becomes apparent. Emx2 is expressed in the dorsal telencephalon (pallium) (Fig. 3A) (Yoshida et al., 1997), and its expression domain was reduced in the –/– –/– –/– +/– +/– –/– Fezl , but not in the Fez Fezl or Fez Fezl embryos Fez (Fig. 3B-D). This is consistent with the reduction of the neocortex and loss of the hippocampus, as Emx2 (and Emx1) is required for neocortical and hippocampal formation (Pellegrini et al., 1996; Yoshida et al., 1997). Pax6 is initially expressed in the dorsal telencephalon and diencephalon, but its expression in the thalamus is reduced by E10.5 (Fig. 3E) (Mastick et al., 1997; Stoykova et al., 1996; Stoykova and Gruss, 1994; Warren and Price, 1997). The rostral and caudal limits of Pax6 expression in the diencephalon –/– –/– –/– +/– Fezl embryos as in wild type, Fez Fezl were the same in Fez +/– –/– and Fez Fezl embryos (Fig. 3E-H). However, the Pax6 –/– –/– Fezl expression in the thalamus was not reduced in the Fez embryos (Fig. 3H), suggesting that the rostro-caudal patterning, but not the formation of the diencephalon, was affected in the –/– –/– Fezl embryos. The Dlx1 expression in the prethalamus was Fez –/– –/– Fezl embryos (Fig. 3L), indicating already absent at E10.5 in Fez that the prethalamus was not established. Shh is expressed in the anterior entopeduncular area (AEP) of the ventral telencephalon, the basal plate of the entire neuroectoderm (including the hypothalamus) and the ZLI (Fig. 3M) (Echelard et al., 1993; Ericson –/– +/– +/– –/– Fezl and Fez Fezl embryos, et al., 1995) in the wild-type, Fez –/– –/– Fezl embryos but its expression in the ZLI was absent in the Fez (Fig. 3M-P), suggesting that the ZLI was not established in the absence of Fez and Fezl. Tcf4 is expressed at a high level in the thalamus and pretectum (Cho and Dressler, 1998; Korinek et al., –/– +/– +/– –/– Fezl and Fez Fezl embryos, 1998) in the wild type, Fez –/– –/– Fezl whereas the Tcf4-high domain expanded rostrally in the Fez embryos (Fig. 3Q-T). Lhx5 is expressed in the prethalamus in the –/– +/– +/– –/– Fezl and Fez Fezl embryos (Fig. 3U-W) wild type, Fez (Nakagawa and O’Leary, 2001), but was not detected in the Fig. 4. Reduction of rostral diencephalon and expansion of –/– –/– Fezl embryos (Fig. 3X). Taking these data together, loss of –/– –/– Fez caudal diencephalon in Fez Fezl embryos at E9.5. Expression of the prethalamus and expansion of the caudal diencephalon took Emx2 (A,B), Pax6 (C,D), Fgf8 (E,F), Wnt3a (G,H) and En2 (I,J) in control –/– –/– place before E10.5, and the ZLI, the prethalamus-thalamus (A,C,E,G,I) and Fez Fezl embryos (B,D,F,H,J) was analyzed by whole- boundary, was not established in the absence of Fez and Fezl. mount in situ hybridization. Lateral views of anterior neuroectoderm, with anterior to the left. Expression of Emx2 (A) and Pax6 in the dorsal telencephalon (between arrowheads, C) was reduced, but the Pax6 Fez and Fezl are involved in early rostro-caudal expression in the diencephalon (marked by dots) was maintained in forebrain patterning –/– –/– Fez Fezl embryos. Fgf8 expression in the commissural plate and mid- Formation and patterning of the forebrain are regulated by hindbrain boundary was maintained, but that in the dorsal prethalamus transcription factors and inductive signals expressed in specific –/– –/– was absent (arrowheads E,F) in Fez Fezl embryos. The expression rostro-caudal positions in the neuroectoderm at the beginning of domain of Wnt3a was caudal to the ZLI in control embryos (G) and was –/– –/– neurogenesis and neural patterning. We examined gene expression expanded rostrally in Fez Fezl embryos (arrow in H). Expression of –/– –/– –/– –/– Fezl embryos at around E9.5. The in wild type and Fez En2, which marks the midbrain, was not affected in Fez Fezl expression domain of Emx2 and Pax6 in the dorsal telencephalon embryos (I,J). cp, commissural plate; mhb, mid-hindbrain boundary; –/– –/– Fezl embryos at E9.5 (Fig. 4A- was already reduced in the Fez pth, prethalamus. D). Fgf8 was expressed in the commissural plate, the infundibulum of the hypothalamus, the dorsal part of the prethalamus (eminentia Nusse, 1991; Salinas and Nusse, 1992), and the rostral limit of its thalami) and the mid-hindbrain boundary (isthmus) in wild-type –/– –/– expression was shifted rostrally in the Fez Fezl embryos (Fig. embryos (Fig. 4E) (Crossley and Martin, 1995), whereas its 4H). En2 expression in the midbrain was not affected in the expression in the prospective prethalamus was specifically absent in –/– –/– –/– –/– Fezl embryos (Fig. 4F). Wnt3a is expressed in the dorsal Fez Fezl embryos (Fig. 4I,J) (Joyner and Martin, 1987). All these the Fez data indicate that the diencephalon patterning was already affected neural tissue caudal to the prospective ZLI (Fig. 4G) (Roelink and DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 3999 had shifted rostrally at E9.0, suggesting that Fez and Fezl are required to repress the Irx1 expression in the rostral diencephalon. We found that the expression of Six3 was not affected at E8.5 and 9.5 (Fig. 5I,J, data not shown for E8.5), suggesting that the –/– –/– Fezl embryos expansion of the caudal diencephalon in the Fez was independent of Six3. Complementary expression of Fez/Fezl and Irx1 The expression of Fez and Fezl in the forebrain is initiated at E8.0 and 8.5, respectively (Hirata et al., 2006; Hirata et al., 2004) (Fig. 6B). At E12.5, Fez and Fezl are expressed in the pallium, septum, hypothalamus and prethalamus, which are located rostral to the ZLI (Hirata et al., 2004; Hirata et al., 2006). We examined the early expression domains of Fez and Fezl, and compared them with those of Irx1. At E8.5, the expression domain of Fez was slightly wider than that of Fezl (Fig. 6A,B), and the caudal limit of Fez abutted the rostral limit of Irx1 (Fig. 6D). Two-color staining for Fez or Fezl with Irx1 revealed that the expression of Fez and Fezl was strictly rostral to that of Irx1 at E9.5 (Fig. 6H-M). Fez and Fezl repress the caudal diencephalon fate The expression of Fez and Fezl suggests that Fez and Fezl function in the region rostral to the ZLI and repress the caudal diencephalon fate. To address this issue, we misexpressed Fez or Fezl caudal to the ZLI using the Fezl gene enhancer/promoter or the Otx2 forebrain-midbrain (FM) enhancer. We constructed -galactosidase (lacZ) reporter genes in which the lacZ gene was connected to the 8.2 kbp or 2.7 kbp enhancer/promoter region upstream of the translational initiation site of the mouse Fezl gene (Fig. 7A), and examined the lacZ expression in the resulting transgenics (Fig. 7B- D). The 8.2 kbp Fezl enhancer/promoter recapitulated the expression in the forebrain at E8.5 (Fig. 7B). By contrast, the 2.7 kbp Fezl enhancer/promoter (Fezl2.7p) drove the lacZ expression in a wider region at E8.5 than the 8.2 kbp promoter did (Fig. 7C). At E9.5 in transgenic mice with the 2.7 kbp Fezl promoter-lacZ, activity was detected in a wider region than the endogenous lacZ Fezl expression (Fig. 7D), indicating that the enhancer/promoter could drive expression caudal to the ZLI. Using Fezl2.7p, we expressed Fezl or Fez cDNA ectopically in the caudal diencephalon. We monitored the exogenous Fezl or Fez expression with a green fluorescence protein variant, Venus (IRES-Venus) (Nagai et al., –/– –/– Fig. 5. Rostrally expanded expression of Irx1 in Fez Fezl 2002). In embryos with the Fezl2.7p-Fezl-IRES-Venus or Fezl2.7p- embryos. Irx1 and Six3 expression in control (A,C,E,G,I) and Fez-IRES-Venus transgene, the expression of Foxg1 in the –/– –/– Fez Fezl (B,D,F,H,J) embryos at E10.0 (A,B), E9.5 (C,D,I,J), E9.0 (E,F) telencephalon (Tao and Lai, 1992) was not affected, but the and E8.5 (G,H). Lateral views, with anterior to the right. Irx1 –/– –/– expression of Irx1 was reduced and its rostral limit was shifted expression was not significantly different between Fez Fezl and caudally at E9.5 (Fig. 8B,C,S). The rostral limit of Irx1 control embryos at E8.5. A rostral expansion of Irx1 expression was –/– –/– detected at E9.0 in Fez Fezl embryos (arrow). At E9.5 and E10.5, corresponded to the caudal limit of the exogenous Fezl-IRES-Venus the rostral limit of Irx1 expression (arrows) reached the caudal edge of expression in these transgenic embryos (Fig. 8A,R). Similarly, in the telencephalon (marked by dots). Expression of Six3 was not the Fezl2.7p-Fezl-IRES-Venus and Fezl2.7p-Fez-IRES-Venus –/– –/– significantly affected in Fez Fezl embryos at E8.5 (data not shown) transgenic embryos, the Tcf4-high domains, corresponding to the and at E9.5 (I,J). thalamus and pretectum, were strongly reduced at E10.5 (Fig. 8E,F,U). In transgenic embryos strongly expressing Fezl, Dlx1 expression expanded caudally (n=1/2), with Gbx2 expression being when neural patterning began. In chick, ZLI is positioned on the prominently reduced (n=2/3) at E12.5 (Fig. 8,M,N,P,Q). boundary of the expression domains of Six3 and Irx3, and Six3 and Furthermore, the misexpression of Fezl by Fezl2.7p abolished Shh Irx3 can repress the expression of each other, possibly determining expression in the ZLI and ventral diencephalon (n=2/4) or shifted the position of the ZLI (Kobayashi et al., 2002). In mouse, Irx1 and the ZLI-specific Shh expression caudally (n=2/4) at E10.5 (Fig. Irx3 display a similar expression profile in the neuroectoderm, but 8H,I,K). The misexpression of Fez by Fezl2.7p abolished Shh the rostral limit of the Irx1 expression is more rostral than that of expression in the ZLI (data not shown, n=1/1). We also used an Irx3 and is positioned on the ZLI (Bosse et al., 1997; Cohen et al., Otx2FM enhancer, which can drive a transgene in the midbrain, 2000). We examined the expression of Irx1 in wild-type and diencephalon and archicortex (Kurokawa et al., 2004a), to double-mutant embryos (Fig. 5). The rostral limit of Irx1 expression misexpress Fezl. Fezl misexpression under this enhancer also DEVELOPMENT 4000 RESEARCH ARTICLE Development 133 (20) Fig. 6. Complementary expression of Fez/Fezl and Irx1. (A-D) Expression of Fez (A,D), Fezl (B) and Irx1 (C,D) at E8.5. (D) Two- color staining. Fez and Irx1 transcripts were stained with BM Purple and Fast Red; the fluorescence image from the Fast Red was superimposed on the bright-field image. (E-M) Expression of Fez (E), Fezl (F) and Irx1 (G) at E9.5. (H-M) Sagittal sections of E9.5 embryos were hybridized with Fez and Irx1 (H-J), or Fezl and Irx1 probes (K-M). The hybridized signals were stained with BM Purple (Fez and Fezl) and Fluorescein (Irx1). Bright-field images (H,K), fluorescence images (I,L) and the bright-field and fluorescence superimposed images (J,M). suppressed Irx1 expression without affecting Foxg1 expression the wild-type embryos, when the rostro-caudal patterning in the (Fig. 8W). These data indicate that Fez and Fezl can suppress the diencephalon became abnormal (Figs 3, 4). Our data strongly caudal diencephalon fate. suggest that the transformation of the prethalamus into the caudal diencephalon takes place during early neural patterning in the DISCUSSION absence of Fez and Fezl (Fig. 9). Future studies including the Cre- Role of Fez and Fezl in diencephalon patterning loxP-mediated cell-fate mapping of Fez and Fezl-expressing cells –/– –/– –/– +/– The Fez Fezl mutant embryos, but not the Fez Fezl or will definitely clarify this issue. The misexpression of Fez or Fezl +/– –/– Fez Fezl embryos, showed defects in patterning of the suppressed the caudal diencephalon fate and induced the expression diencephalon (Fig. 1), indicating a strictly redundant role for Fez and of Dlx1, which normally marks the prethalamus, in the region caudal Fezl in diencephalon development. This is consistent with the to the ZLI (Fig. 8). All of these data indicate that Fez and Fezl overlapping expression of Fez and Fezl in the prethalamus at later function to repress the caudal diencephalon fate and establish the stages (Hirata et al., 2006; Hirata et al., 2004). Fez and Fezl, prethalamus fate (Fig. 9). Although the caudal diencephalon was respectively, begin expression at E8 and 8.5 (Fig. 6) (Hirata et al., initially expanded, the thalamus eventually became smaller and the 2006; Hirata et al., 2004). We detected a defect in the rostro-caudal rostral part of the thalamus was missing in the Fez;Fezl-deficient polarity of the diencephalon: rostral expansion of the Irx1 expression embryos at E12.5 (Figs 1, 2). The later development of the thalamus at E9.0, indicating that the Fez/Fezl-mediated diencephalon is known to be dependent on inductive signals from the ZLI patterning starts soon after the onset of Fez and Fezl expression. (discussed below). The loss of the ZLI in the Fez;Fezl-deficient Marker analyses showed that the prethalamic region was not embryos secondarily affects the development of the thalamus in established; instead, the caudal diencephalon, which includes the these embryos (Fig. 9). thalamus and the anterior pretectum, expanded rostrally in the Both FEZ and FEZL contain an Eh1 repressor motif, which double-mutant embryos (Figs 3, 4). There are two possible interacts with the Groucho/TLE family of transcriptional co- explanations for the phenotype of Fez;Fezl-deficient embryos: (1) repressors (Bae et al., 2003; Kobayashi et al., 2001; Muhr et al., transformation of the prethalamus into the thalamus; and (2) 2001; Shimizu et al., 2002). The Eh1 repressor motif of zebrafish truncation of the prethalamus and the rostral shift of the caudal Fezl is required for at least part of the Fezl function in this animal neural tissue. We found that the size of the diencephalon was not (Levkowitz et al., 2003), suggesting that FEZ and FEZL function as significantly different in the double-mutant embryos, compared with transcriptional repressors to regulate patterning of the diencephalon. DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 4001 Fig. 7. Enhancer and promoter region of Fezl. (A) Schematic diagram of the Fezl enhancer/promoter and the constructs used for transgenesis. (B-D) Detection of lacZ expression by X-gal staining in Fezl8.2p-lacZ (B) and Fezl2.7p-lacZ (C,D) transgenic mouse embryos, in which lacZ expression was driven by the 8.2 kbp and 2.7 kbp Fezl enhancer/promoter, respectively. (B,C) E8.5 embryos, lateral views with anterior to the top. (D) E9.5 embryo, lateral views of the anterior neuroectoderm. The 8.2 kbp Fezl enhancer/promoter recapitulated the endogenous Fezl expression (rostral to the ZLI), whereas the 2.7 kbp Fezl enhancer/promoter showed a caudally expanded expression of lacZ (arrows, D). In the Fez;Fezl double-mutant embryos, expression domains of the How Fez and Fezl expression is regulated remains unclear. There caudal diencephalic genes, such as Irx1, Wnt3a and Tcf4 (high- are several genes with expression domains that overlap with those expression domain), expanded rostrally, and misexpression of Fez of Fez and Fezl. They include Pax6, Emx1/2, Dlx1/2/5/6 and Otx1/2 or Fezl caudal to the ZLI inhibited the expression of Irx1 and Tcf4 in in addition to Six3 (Bulfone et al., 1993; Oliver et al., 1995; Simeone the caudal diencephalon (Figs 4, 5, 8). Fez and Fezl are expressed in et al., 1992a; Simeone et al., 1993; Simeone et al., 1992b; Stuhmer the region rostral to the rostral limit of Irx1 expression, which marks et al., 2002; Walther and Gruss, 1991). These genes might be the prospective position of the ZLI. Taking these findings together, involved in the regulation of Fez and Fezl expression. The expression we conclude that FEZ and FEZL directly or indirectly repress the of zebrafish fezl and Xenopus Fez is negatively regulated by Wnt caudal diencephalon genes in the rostral diencephalon (Fig. 9). signaling (Hashimoto et al., 2000) (M. Matsuo-Takasaki, personal Identification of target genes for FEZ and FEZL and/or chromatin communication). Thus, the initial expression of Fez and Fezl may be immunoprecipitation assay of FEZ/FEZL-binding genomic controlled by the rostro-caudal polarity information, in which Wnt fragments will help to clarify the precise mechanism by which Fez signaling is strongly involved (Niehrs, 2004). In this context, Fez and and Fezl control the rostro-caudal polarity of diencephalon. Fezl may link the rostro-caudal polarity information to the SIX3 negatively regulates the caudally expressed Wnt1 (Lagutin subdivision formation in the diencephalon. et al., 2003) and functions as a transcriptional repressor (Kobayashi et al., 2001; Lopez-Rios et al., 2003; Zhu et al., 2002). Six3-deficient Role of Fez and Fezl in formation of the ZLI mice show strong reduction of the neural tissue rostral to the ZLI The ZLI position is predicted as abutting the expression domains of (Lagutin et al., 2003). Six3 negatively controls the expression of Irx3 rostral Six3 and caudal Irx3 in chick embryos (Kobayashi et al., in chick (Kobayashi et al., 2002). These reports suggest that the role 2002). We found that it is predicted by the expression of rostrally of Fez and Fezl in diencephalon patterning is similar to that of Six3, expressed Fez and Fezl and caudally expressed Irx1 in mouse at least in part. However, Six3-deficient mice have rudimentary embryos (Fig. 6). Furthermore, the deficiency of both Fez and Fezl tissue rostral to the ZLI, and express Shh in the ZLI (Lagutin et al., led to loss of the ZLI, and misexpression of Fez or Fezl could inhibit 2003), and Fez;Fezl-double mutants display complete loss of the ZLI formation or shift its position (Figs 3, 8). These data indicate prethalamus and ZLI, implying there is a difference between the that Fez and Fezl are involved in the formation and position of the functions of Six3 and Fez/Fezl. We examined the expression of Six3 ZLI. The ZLI initially forms as a wedge-shaped structure on the –/– –/– in the Fez Fezl embryos, but did not observe any significant boundary between the prethalamus and thalamus, which is alteration in the Six3 expression at E8.5 and 9.5 (Fig. 5, data not characterized by a gap in Lfrg expression; subsequently, it collapses shown for E8.5), suggesting that Six3 does not function downstream to a narrow band (Zeltser et al., 2001). It is not clear whether Fez of Fez and Fezl. Rather, Six3 may function upstream of, or in parallel and Fezl are expressed in the prospective ZLI domain, although they with, Fez and Fezl. Future studies examining Fez and Fezl expression are not expressed in the ZLI at E12.5 (Hirata et al., 2006; Hirata et in Six3-deficient embryos and combinatory gene disruption of Six3 al., 2004). Fez and Fezl are expressed in the prethalamus, but they and Fez and/or Fezl will clarify this issue. do not induce Shh expression there. Thus, Fez and Fezl are not DEVELOPMENT 4002 RESEARCH ARTICLE Development 133 (20) Fig. 8. Misexpression of Fez or Fezl affects rostro-caudal polarity in the diencephalon. Misexpression of Fezl (A-Q) or Fez (R-U) by the 2.7 kbp Fezl enhancer/promoter (Fezl2.7p-Fezl- IRES-Venus, Fezl2.7p-Fez-IRES-Venus), or Fezl by the FM enhancer of the Otx2 gene and mouse Hsp68 promoter (Otx2FM-Hsp68-Fezl-IRES-Venus, V,W) affected the diencephalon subdivisions. Exogenous Fezl expression was monitored by the expression of Venus attached to an IRES (Fig. 7) (fluorescence images, A,D,G,J,L,O,R,T,V). E9.5 embryos were analyzed by whole-mount in situ hybridization with Foxg1 and Irx1 (B,C,S,W). E10.5 embryos were analyzed by in situ hybridization with Tcf4 (E,F,U) or Shh (H,I,K). Sagittal sections of E12.5 embryos were analyzed with Dlx1 or Gbx2 probes (M,N,P,Q). (C,F,I,N,Q) Control non-transgenic embryos. Expression of Foxg1 in the telencephalon was not affected (caudal limit marked by arrows), but the rostral limit of Irx1 expression (marked by arrowheads) was shifted caudally in Fezl2.7p-Fezl-IRES-Venus (B), Fezl2.7p-Fez-IRES-Venus (S) and Otx2FM-Hsp68-Fezl-IRES-Venus embryos (W), compared with the control (C). Tcf4-high expression domain in the thalamus and prethalamus was strongly reduced in the Fezl2.7p-Fezl- IRES-Venus (E) and Fezl2.7p-Fez-IRES-Venus (U) embryos. Shh expression in the ZLI (arrows) and ventral diencephalon was reduced (H, n=2/4), or Shh expression in the ZLI was shifted caudally (K, n=2/4) in the Fezl2.7p-Fezl-IRES-Venus embryos, compared with control (I). Dlx1 expression in the prethalamus was expanded caudally when the exogenous Fezl-IRES-Venus was strongly expressed (M, n=1/2). Gbx2 expression in the thalamus was strongly reduced in the Fezl2.7p-Fezl-IRES-Venus embryos (P, n=2/3). DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 4003 Fig. 9. Schematic presentation of a role of Fez and Fezl in diencephalon patterning. Fez and Fezl are expressed in the telencephalon and rostral diencephalon (prospective prethalamus) and function to suppress the formation of the caudal diencephalon, which expresses Irx1 and Wnt3a. In the absence of Fez and Fezl, the rostral diencephalon does not form and instead caudal diencephalon expands rostrally at E9.5. Subsequently at E10.5, the prethalmaus and the ZLI, which is normally located in the interface between the prethalamus and thalamus, do not form in –/– –/– Fez Fezl embryos. The caudal diencephalon, –/– –/– including the thalamus and pretectum, is expanded in Fez Fezl embryos. The formation of thalamus, however, is dependent on inductive signals –/– –/– (e.g. Shh) from the ZLI. In Fez Fezl embryos, the thalamus does not grow properly, but the anterior pretectum remains expanded at E12.5. apt, anterior pretectum; di, diencephalon; pth, prethalamus, ppt, posterior pretectum; tel, telencephalon; th, thalamus. likely to be instructive factors, but rather to function as permissive work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Technology, and by a grant factors. Alternatively, Fez and Fezl may regulate ZLI formation from RIKEN (to M.H.). indirectly and non-cell-autonomously by controlling formation of the prethalamus. Grafting experiments in chick embryos show that References Shh expression is induced in the interface between the prechordal Bae, Y. K., Shimizu, T., Yabe, T., Kim, C. H., Hirata, T., Nojima, H., Muraoka, O., Hirano, T. and Hibi, M. (2003). 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TCF-4 binds beta-catenin and is expressed in distinct regions of the embryonic brain and limbs. Mech. Dev. 77, 9-18. to be a secondary consequence of the loss of the ZLI in the Cohen, D. R., Cheng, C. W., Cheng, S. H. and Hui, C. C. (2000). Expression of –/– –/– –/– –/– Fez Fezl embryos (Fig. 9). Consistent with this, the Fez Fezl two novel mouse Iroquois homeobox genes during neurogenesis. Mech. Dev. embryos showed complete loss of the Sox14 expression (Fig. 2), 91, 317-321. which is dependent on Shh from the ZLI (Hashimoto-Torii et al., Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning 2003). Our findings support the inductive role of the ZLI in thalamus in the developing embryo. Development 121, 439-451. development. Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. 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Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions

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RESEARCH ARTICLE 3993 Development 133, 3993-4004 (2006) doi:10.1242/dev.02585 Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions 1, ,† 1, 1 2 3 Tsutomu Hirata * , Masato Nakazawa *, Osamu Muraoka , Rika Nakayama , Yoko Suda and 1,‡ Masahiko Hibi Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice we found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene- mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions. KEY WORDS: Fez (Fezf1) Fez-like (Fezf2), Zinc finger, Forebrain, Telencephalon, Diencephalon, Zona limitans intrathalamica, Thalamus, Prethalamus, Pretectum, Prosomere, Mouse, Transcriptional repressor INTRODUCTION Although the structures of the diencephalic subdivisions become The forebrain of adult mammals is one of the most complicated obvious in mice late in gestation, individual subdivisions can be biological structures; it is essential for higher neural functions, distinguished by their expression of genetic markers at the beginning such as memory, emotion, reasoning and the planning of of forebrain patterning. Members of the Dlx family of genes are coordinated movements. Various models for forebrain subdivisions expressed in the prethalamus, Gbx2 is expressed in the thalamus (neuromeres) have been developed over the past 10 years (Bulfone et al., 1993) and Ebf1 and Lhx1 are expressed in the (reviewed by Kiecker and Lumsden, 2005). Among them, Puelles anterior and posterior pretectum, respectively (Barnes et al., 1994; and Rubenstein and colleagues proposed a neuromeric Garel et al., 1997). Studies of genetically modified mice have organization of the forebrain on the basis of the differential revealed several genes involved in the patterning and/or development expression of neural marker genes and morphological of the diencephalon. These include Pax6, Otx2, Emx2 and Six3 considerations (the so called ‘prosomeric model’) (Puelles and (Kimura et al., 2005; Kurokawa et al., 2004a; Kurokawa et al., Rubenstein, 1993; Puelles and Rubenstein, 2003; Rubenstein et al., 2004b; Lagutin et al., 2003; Stoykova et al., 1996; Suda et al., 2001). 1994; Rubenstein et al., 1998). In the most recent model (Puelles However, it is largely unknown how these genes function in the and Rubenstein, 2003), the forebrain can be divided into rostral formation of the diencephalon subdivisions and what other genes are and caudal regions. The rostral part of the forebrain is the involved in diencephalon patterning. telencephalon and can be divided into several anatomical and The zona limitans intrathalamica (ZLI) is located on the functional territories, including the pallium, subpallium, preoptic boundary between the prethalamus and thalamus (Larsen et al., area and hypothalamus. The caudal part of the forebrain is the 2001; Shimamura et al., 1995). The ZLI marks the interface diencephalon and can be divided into three transverse subdivisions between regions of different ability to respond to inductive signals known as prosomeres: the prethalamus (also called the ventral such as Fgf8 and Shh; for instance, in neural tissue rostral to the thalamus, p3), thalamus (also known as the dorsal thalamus, p2), ZLI, Fgf8 induces the expression of Foxg1 (also called BF-1), but and pretectum (p1). The eminentia thalamic, habenula/epithalamus caudal to the ZLI it induces the expression of En2 (Shimamura and and posterior commissures are located dorsally to the prethalamus, Rubenstein, 1997). The ZLI also has inductive influences on the thalamus and pretectum, respectively. adjacent subdivisions (prethalamus and thalamus). Shh expressed in the ZLI is involved in development of the prethalamus and thalamus in chick and zebrafish embryos; inhibition of Shh Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, signaling represses the expression of the prethalmus and thalamus Kobe 650-0047, Japan. Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan. Vertebrate Body markers (Kiecker and Lumsden, 2004; Scholpp et al., 2006; Vieira Plan Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan. et al., 2005). In chick embryos, the future position of the ZLI is at the abutting expression domains of two homeobox genes, Six3 *These authors contributed equally to this work Present address: Center for Neuroscience Research, Children’s National Medical rostrally and Irx3 caudally (Kobayashi et al., 2002). Six3 and Irx3 Center, Washington DC 20010-2970, USA regulate expression of each other, and the misexpression of Six3 or Author for correspondence (e-mail: [email protected]) Irx3 affects the formation of the prethalamus and thalamus (Braun et al., 2003; Kobayashi et al., 2002). Irx3 expression confers Accepted 11 August 2006 DEVELOPMENT 3994 RESEARCH ARTICLE Development 133 (20) competence to respond to the Fgf8 and Shh inductive signals by Hirata et al., 2006) revealed that the mouse gene reported by Matsuo- Takasaki et al. is more similar to zebrafish fezl than to Xenopus Fez expressing thalamus-specific genes (Kiecker and Lumsden, 2004; (Hashimoto et al., 2000; Matsuo-Takasaki et al., 2000). Therefore, we refer Kobayashi et al., 2002). The expression of Six3 and Irx3 is regulated to AB042399 as mouse Fezl (Hashimoto et al., 2000; Hirata et al., 2004), by Wnt signaling (Braun et al., 2003); Wnt1, Wnt3 and Wnt3a are and to AK014242, which is more similar to Xenopus Fez, as mouse Fez expressed in the dorsal neural tissue caudal to the prospective ZLI +/– +/– and Fez mice were previously described (Hirata et al., 2006). Fezl when the expression of Six3 and Irx3 begins (Braun et al., 2003; +/– +/– and Fezl (Hirata et al., 2006; Hirata et al., 2004). Both the Fez Lagutin et al., 2003; Roelink and Nusse, 1991; Salinas and Nusse, heterozygous mice were originally established in a 129sv genetic 1992). These reports suggest that Wnt signaling controls the rostro- background and backcrossed to the C57BL/6 background for several +/– +/– caudal polarity of the forebrain through the regulation of Six3 and Fezl double heterozygous mice were generated by generations. Fez +/– +/– Irx3, and the mutually repressive interaction of Six3 and Irx3 and Fezl heterozygotes, and were used to generate crossing Fez –/– +/– +/– –/– –/– –/– Fezl , Fez Fezl and Fez Fezl embryos. Mice were housed in controls the position of the ZLI and thus confers differential Fez an environmentally controlled room at the Animal Facility of the Center for competence to respond to the inductive signals to form the Developmental Biology (CDB), RIKEN, under the guidelines for animal prethalamus and thalamus. This hypothesis is based mainly on the experiments from RIKEN CDB. The genotypes of newborn mice and results of misexpression studies and explant assays in the chicken embryos were determined by PCR analysis (Hirata et al., 2006; Hirata et al., embryo, but there is little genetic evidence to support it. Six3- 2004). The primer sequences and PCR conditions are available on request. deficient mice display a strong reduction of the neural tissue rostral Noon of the day on which the vaginal plug was detected was designed as to the ZLI, but still express Shh in the ZLI and rudimentary rostral embryonic day (E) 0.5. tissue (Lagutin et al., 2003), suggesting that other genes may Histological sections and Nissl staining cooperate with or function parallel to Six3 to determine the position Brains or embryos were fixed with Carnoy’s solution at room of the ZLI or control the formation of the rostral diencephalon. temperature overnight. Specimens were dehydrated and embedded in Fez (Fezf1 – Mouse Genome Informatics) and Fez-like (Fezl, paraffin. Serial sections were prepared and stained with 0.1% Cresyl Fezf2 – Mouse Genome Informatics) are closely related genes that Violet (MERCK). encode transcriptional repressors containing six C2H2-type zinc fingers and an Eh1 (Engrailed homology 1) repressor motif, and RNA probes and in situ hybridization were originally isolated as anterior neuroectoderm-specific genes Embryos were fixed overnight at 4°C in 4% paraformaldehyde (PFA) in PBS. Specimens were gradually dehydrated in ethanol/H O and stored in in Xenopus and zebrafish (Hashimoto et al., 2000; Matsuo-Takasaki 2 ethanol at –20°C. The protocol for in situ hybridization was described et al., 2000). Orthologs of these two genes exist in the puffer fish, previously (Hirata et al., 2006; Hirata et al., 2004). Single-stranded zebrafish, mouse and human (Hirata et al., 2006). In mouse and digoxigenin-UTP-labeled (Roche) RNAs were used. In situ signals were zebrafish, Fez and Fezl are expressed in overlapping domains in the detected with an anti-digoxigenin antibody and BM Purple (Roche). For forebrain during development (Hashimoto et al., 2000; Hirata et al., two-color staining of the histological sections in Fig. 6D, Fez and Irx1 probes 2006; Hirata et al., 2004; Matsuo-Takasaki et al., 2000). A number labeled with FITC-UTP and digoxigenin-UTP (Roche), were used of studies have previously investigated the specific roles of Fez and respectively; BM Purple and Fast Red (Roche) were used for staining. For Fezl in neural development in both zebrafish and mice. The Fig. 6H-M, Fez and Fezl probes were labeled with FITC-UTP and Irx1 probe zebrafish mutant too few (tfu) has a fezl gene mutation and displays with digoxigenin-UTP. The hybridized signals for Fez and Fezl were a loss or reduction of dopaminergic neurons in the hypothalamus detected by an alkaline phosphatase-conjugated anti-Fluorescein antibody (Roche) and BM Purple, and those for Irx1 were detected by a peroxidase- (Levkowitz et al., 2003). Fezl-deficient mice show no abnormalities conjugated anti-digoxigenin antibody (Roche) and tyramide signal in the dopaminergic neurons, but do show abnormal formation of amplification system (TSA-Plus Fluorescein System, PerkinElmer). The the subplate neurons and thalamocortical axons, and loss of the precise protocols for in situ hybridization are available on request. The fornix/fimbria system (Hirata et al., 2004). Fezl has also been shown probes were: Dlx1 (Bulfone et al., 1993), Gbx2 (Bulfone et al., 1993), Lhx1 to be required for the development of subcerebral projection (Fujii et al., 1994), Ebf1 (Garel et al., 1997), Sox14 (Hashimoto-Torii et al., neurons in layer V of the neocortex (Chen et al., 2005a; Chen et al., 2003), Emx2 (Yoshida et al., 1997), Pax6 (Walther and Gruss, 1991), Shh 2005b; Molyneaux et al., 2005). Fez-deficient mice display (Echelard et al., 1993), Tcf4 (Ishibashi and McMahon, 2002), Lhx5 abnormal olfactory sensory neuronal projections and olfactory bulb (Nakagawa and O’Leary, 2001), Fgf8 (Crossley and Martin, 1995), Irx1 (a formation (Hirata et al., 2006). The relatively weak forebrain gift of T. Ogura), Wnt3a (Takada et al., 1994), En2 (Joyner and Martin, phenotypes of Fez and Fezl-deficient mice suggest that Fez and Fezl 1987), Foxg1 (Tao and Lai, 1992), Fez (Hirata et al., 2006) and Fezl (Hirata et al., 2004). In situ hybridization images were taken using an AxioPlan2 function redundantly in the patterning and development of the microscope or a SteREO Lumar V12, equipped with an AxioCam CCD forebrain. camera (Zeiss). Figures were assembled using AxioVision version 4.3 and Here, we generated mice deficient in both the Fez and Fezl genes. Adobe Photoshop CS2. The Fez;Fezl-deficient mice showed defects in the rostro-caudal patterning of the diencephalon. We found that Fez and Fezl Generation and genotyping of transgenic mice redundantly control the rostro-caudal patterning of the diencephalon We isolated an approximately 8.2 kbp enhancer/promoter region of Fezl by repressing the caudal diencephalon fate in the prospective from the bacterial artificial chromosome clone containing the coding and non-coding regions of Fezl (Hirata et al., 2004). To make the Fezl prethalamic region, and that ZLI formation depends on Fez/Fezl- enhancer/promoter-driven lacZ (-galactosidase) transgene constructs, the mediated formation of diencephalon subdivisions. 8.2 or 2.7 kbp enhancer/promoter region of Fezl was connected to lacZ cDNA at the position of the translational initiation site of Fezl (Fezl8.2p- MATERIALS AND METHODS lacZ, Fezl2.7p-lacZ). For misexpression of Fezl, its 2.7 kbp enhancer/ Mouse mutants and gene naming promoter was connected to Fezl or Fez cDNA, followed by internal A mouse Fezl cDNA fragment (GenBank Accession Number AI325906) ribosomal entry site (IRES)-Gap43-Venus (Fezl2.7p-Fezl-IRES-Venus and was originally reported as the mouse ortholog of Xenopus Fez (Matsuo- Fezl2.7p-Fez-IRES-Venus) (IRES-Gap43-Venus, a gift of Y. Yoshihara). Takasaki et al., 2000). However, comparing the sequences of zebrafish fez Similarly, Otx2 FM enhancer (1.4 kbp) (Kurokawa et al., 2004a) and the (AB207804) and fezl (AB041824), mouse Fez homolog (AK014242), and promoter of mouse heat shock protein 68 (pHsp68) (Sasaki and Hogan, the full-length clone of mouse Fezl (AB042399) (Hashimoto et al., 2000; 1996) were connected to Fezl cDNA and IRES-Gap43-Venus (OtxFM- DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 3995 Hsp68-FeZLIRES-Venus). The transgene-derived Fezl expression was RESULTS monitored as Venus expression with an epifluorescence microscope Fez and Fezl function redundantly in forebrain AxioPlan2. Genotypes of the transgenic mice were analyzed by PCR with formation 5-AAACCCTGGCGTTACCCAACT-3 and 5-ACGACAGTATCGGCC- To reveal whether Fez and Fezl have redundant roles in forebrain TCAGGA-3 for the lacZ reporter lines, 5-TGTGTCTGCAGAGA- formation, we crossed Fez- and Fezl-deficient heterozygous mice GTGCTGGCCTG-3 and 5-CTGGCTGCTGCTCACCCCAAGCTTT-3 (Hirata et al., 2006; Hirata et al., 2004) and generated mice deficient for the Fezl2.7p-Fezl-IRES-Venus and OtxFM-Hsp68-Fezl-IRES-Venus lines homozygously or heterozygously in the Fez and/or Fezl genes (Fig. and 5-AAAACGTATTTAGCCGAAAGGAAT-3 and 5-ACTTTACACA- –/– +/– Fezl embryos showed a small olfactory bulb at E15.5 1). Fez CGAAGGGTCTGG-3 for the Fezl2.7p-Fez-IRES-Venus lines. Transient –/– +/– –/– embryos do (Hirata et al., 2006). Fez Fezl (Fig. 1F), as Fez transgenic embryos were generated and -galactosidase staining was embryos showed defects in the formation of the dentate gyrus at performed as described previously (Kimura et al., 1997; Kimura et al., –/– –/– Fezl embryos showed more severe 2000). E17.5 (Fig. 1K). Fez –/– +/– +/– –/– –/– –/– +/– +/– Fig. 1. Morphology of the forebrain of wild-type, Fez Fezl , Fez Fezl , Fez Fezl and Fez Fezl mice. Coronal (A-D,I-L) and –/– +/– +/– –/– –/– –/– sagittal (E-H) sections of E15.5 (A-H), E17.5 (I-L) wild-type (A,E,I), Fez Fezl (B,F,J), Fez Fezl (C,G,K), and Fez Fezl (D,H,L) mice. +/– +/– –/– –/– (M-Q) Sagittal sections of E18.5 Fez Fezl (M-O) and Fez Fezl (P,Q) mice at the level where posterior commissures (M,P, indicated by white arrowheads), habenulo-interpeduncular tracts (N,Q, white arrows) or mammillthalamic tracts (O, black arrowhead) were observed. Nissl staining. –/– +/– +/– –/– Fez Fezl mice had a very small olfactory bulb and Fez Fezl showed a reduction of the dentate gyrus (arrow in K,L), compared with wild-type –/– –/– littermates. Fez Fezl mice showed loss of the olfactory bulb (arrow in H), dentate gyrus and the CA3 region of hippocampus (arrow in L), +/– +/– –/– –/– prethalamus, strong reduction of the thalamus (region between arrowheads) and a reduced neocortex. At E18.5, both Fez Fezl and Fez Fezl mice had a posterior commissure and habenulo-interpeduncular tract. The habenulo-interpeduncular tracts were small and abnormally located, due –/– –/– +/– +/– –/– –/– to severe reduction of the thalamus in Fez Fezl mice. Fez Fezl (O), but not Fez Fezl mice, had mammilothalamic tracts. DG, dentate gyrus; hit, habenulo-interpeduncular tract; hip, hippocampus; ht, hypothalamus; ma, mamillary region; mtt, mammilothalamic trancts; ncx, neocortex; OB, olfactory bulb; pc, posterior commissure; th, thalamus; pt, pretectum; pth, prethalamus; s, septum; 3v, third ventricle. Scale bars: 0.5 mm. DEVELOPMENT 3996 RESEARCH ARTICLE Development 133 (20) phenotypes in the olfactory bulb and hippocampus: nearly complete the boundary between the thalamus and pretectum, respectively (Fig. loss of the olfactory bulb, stronger reduction of the CA3 region and 1M,N). However, the habenulo-interpeduncular tracts in the –/– –/– –/– –/– Fezl embryos also Fezl embryos tracts were small and abnormally located (Fig. loss of dentate gyrus in the hippocampus. Fez Fez –/– –/– Fezl showed impaired neocortex formation. In addition, Fez 1Q). This is probably due to the strong reduction of the thalamus. –/– –/– embryos were found to have abnormalities in the formation of Fezl embryos did not have a mammilothalamic tract, The Fez diencephalon, including loss of prethalamus and a strong reduction which is located in between the prethalamus and thalamus (Fig. 1O). –/– –/– Fezl embryos had no of the thalamus in size (Fig. 1H, arrowheads). These diencephalon These data indicate that the Fez –/– +/– +/– –/– Fezl and Fez Fezl defects were not observed in the Fez prethalamus and displayed a strong reduction of thalamus, while embryos (Fig. 1F,G), indicating a redundant role of Fez and Fezl in having a relatively normal development of the pretectum. the formation of diencephalon. Although it is intriguing to study the phenotypes of the olfactory bulb, hippocampus and neocortex in Loss of the prethalamus and expansion of the these mice, we restricted the focus of our study to the role of Fez and caudal diencephalon in Fez, Fezl double mutants Fezl in diencephalon formation. We next examined the Fez;Fezl-deficient mice with various genetic –/– –/– Fezl embryos A series of sagittal sections revealed that the Fez markers at E12.5. Dlx1 is expressed in the ventral telencephalon had posterior commissures and habenulo-interpeduncular tracts at (including the medial and lateral ganglionic eminences), E18.5 (Fig. 1P,Q), located on the dorsal side of the pretectum and on hypothalamus and prethalamus, but not the thalamus (Fig. 2A) –/– –/– Fig. 2. Defects in prethalamus and thalamus development and rostral expansion of the pretectum in Fez Fezl embryos at E12.5. (A-T) Expression of Dlx1 (marker for prethalamus and ganglionic eminence, A-D), Gbx2 (thalamus, E-H), Lhx1 (posterior pretectum and ZLI, I-L), –/– +/– +/– –/– –/– –/– Ebf1 (anterior pretectum, M-P) and Sox14 (rostral domain of thalamus, Q-T) in control, Fez Fezl , Fez Fezl and Fez Fezl embryos was analyzed by in situ hybridization. Sagittal sections with anterior to the left. Dlx1 expression was not detected in the prethalamus but was –/– –/– maintained in the hypothalamus and ganglionic eminence in Fez Fezl embryos (D). Gbx2 expression was strongly reduced and detected in –/– –/– patches (arrows in H) in Fez Fezl embryos. Lhx1 expression was absent in the ZLI, but was not significantly affected in the posterior pretectum of –/– –/– –/– –/– Fez Fezl embryos (I-L). The expression domain of Ebf1 was expanded rostrally in Fez Fezl embryos (P), compared with that in the wild-type, –/– +/– +/– –/– –/– –/– Fez Fezl and Fez Fezl littermates (M-O). The expression of Sox14 was abolished in Fez Fezl embryos (T). apt, anterior pretectum; GE, ganglionic eminence; ht, hypothalamus; ppt, posterior pretectum; pth, prethalamus; th, thalamus. DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 3997 –/– –/– (Bulfone et al., 1993; Stuhmer et al., 2002). In the Fez Fezl embryos (Fig. 2P). Sox14 is expressed in the rostral part of the embryos, Dlx1 expression was maintained in the ganglionic thalamus (Hashimoto-Torii et al., 2003) (Fig. 2Q), and its expression –/– –/– Fezl embryos (Fig. 2T). Neither the eminences and hypothalamus, but was completely absent in the was absent in the Fez –/– +/– +/– –/– Fezl nor the Fez Fezl embryos showed abnormal diencephalon (Fig. 2D). Gbx2 is expressed strongly in the thalamus Fez and weakly in the ganglionic eminences (Fig. 2E) (Bulfone et al., expression of Dlx1, Gbx2, Lim1, Ebf1 or Sox14 at E12.5 (Fig. –/– –/– Fezl embryos, Gbx2 expression in the thalamus 1993). In Fez 2B,C,F,G,J,K,N,O,R,S), further confirming the strictly redundant was markedly reduced and shifted rostrally, but not abrogated (Fig. function of Fez and Fezl in the diencephalon patterning. The data –/– –/– Fezl embryos at E12.5, the prethalamus 2H). Lhx1 is expressed in the posterior pretectum and ZLI (Fig. 2I) indicate that, in the Fez (Barnes et al., 1994; Fujii et al., 1994; Mastick et al., 1997; Suda et did not form, the thalamus formed at a reduced size and the anterior al., 2001). Although its expression in the posterior pretectum was pretectum expanded rostrally, suggesting that Fez and Fezl play an not affected significantly, we found that it was not expressed in the important role in rostro-caudal patterning of the diencephalon. –/– –/– Fezl embryos (Fig. 2L). Ebf1 is expressed in the ZLI in the Fez Although we detected Tuj-1-postive postmitotic neurons in the –/– –/– Fezl embryos (data not shown), the anterior pretectum (Garel et al., 1997; Suda et al., 2001) (Fig. 2M), thalamus at E11.5 in the Fez –/– –/– –/– –/– Fezl Fezl embryos failed to form the rostral part of the thalamus and its expression was expanded rostrally in the Fez Fez Fig. 3. Defects in telencephalon formation and regionalization of the diencephalon in –/– –/– Fez Fezl embryos. Expression of Emx2 (A-D), Pax6 (E-H), Dlx1 (I-L), Shh (M-P), Tcf4 (Q-T) and Lhx5 (U-X) in control (A,E,I,M,Q,U), –/– +/– +/– –/– Fez Fezl (B,F,J,N,R,V), Fez Fezl –/– –/– (C,G,K,O,S,W) and Fez Fezl embryos (D,H,L,P,T,X) at E10.5. (A-T) Whole-mount in situ hybridization and lateral views of anterior neuroectoderm, with anterior to the left. (U-X) Coronary sections of the diencephalon. Emx2 expression in the dorsal telencephalon –/– –/– was reduced in Fez Fezl embryos. Pax6 expression was downregulated in the thalamus at this stage (marked by dots, E,F,G) in control, –/– +/– +/– –/– Fez Fezl and Fez Fezl embryos, but not –/– –/– in Fez Fezl embryos (arrow, H). The expression domain of Pax6 in the dorsal telencephalon was reduced but not significantly affected in the diencephalon except for the thalamus (region between –/– –/– arrowheads) in Fez Fezl embryos. Dlx1 expression was detected in the ganglionic eminence (arrow, I,L) but not in the –/– –/– prethalamus (arrowhead, I,L) in Fez Fezl embryos. Shh expression was detected in the –/– –/– AEP, but not in the ZLI in Fez Fezl embryos (arrow, P), compared with controls (M-O). The domain expressing Tcf4 at a high level (thalamus and pretectum region, marked by –/– –/– dots) was expanded in Fez Fezl embryos (T), compared with controls (Q-S). Lhx5 expression was abolished in the prethalamus of –/– –/– Fez Fezl embryos (X). ht, hypothalamus; pth, prethalamus; th, thalamus. DEVELOPMENT 3998 RESEARCH ARTICLE Development 133 (20) (Sox14-positive region), the formation of which is known to depend on Shh from the ZLI (Hashimoto-Torii et al., 2003). The data indicate that the redundant function of Fez and Fezl also controls the rostro-caudal patterning of the thalamus directly or indirectly. Defects in regionalization of the diencephalon in the Fez, Fezl double mutant We analyzed the Fez;Fezl-deficient mice with genetic markers at E10.5, when forebrain patterning becomes apparent. Emx2 is expressed in the dorsal telencephalon (pallium) (Fig. 3A) (Yoshida et al., 1997), and its expression domain was reduced in the –/– –/– –/– +/– +/– –/– Fezl , but not in the Fez Fezl or Fez Fezl embryos Fez (Fig. 3B-D). This is consistent with the reduction of the neocortex and loss of the hippocampus, as Emx2 (and Emx1) is required for neocortical and hippocampal formation (Pellegrini et al., 1996; Yoshida et al., 1997). Pax6 is initially expressed in the dorsal telencephalon and diencephalon, but its expression in the thalamus is reduced by E10.5 (Fig. 3E) (Mastick et al., 1997; Stoykova et al., 1996; Stoykova and Gruss, 1994; Warren and Price, 1997). The rostral and caudal limits of Pax6 expression in the diencephalon –/– –/– –/– +/– Fezl embryos as in wild type, Fez Fezl were the same in Fez +/– –/– and Fez Fezl embryos (Fig. 3E-H). However, the Pax6 –/– –/– Fezl expression in the thalamus was not reduced in the Fez embryos (Fig. 3H), suggesting that the rostro-caudal patterning, but not the formation of the diencephalon, was affected in the –/– –/– Fezl embryos. The Dlx1 expression in the prethalamus was Fez –/– –/– Fezl embryos (Fig. 3L), indicating already absent at E10.5 in Fez that the prethalamus was not established. Shh is expressed in the anterior entopeduncular area (AEP) of the ventral telencephalon, the basal plate of the entire neuroectoderm (including the hypothalamus) and the ZLI (Fig. 3M) (Echelard et al., 1993; Ericson –/– +/– +/– –/– Fezl and Fez Fezl embryos, et al., 1995) in the wild-type, Fez –/– –/– Fezl embryos but its expression in the ZLI was absent in the Fez (Fig. 3M-P), suggesting that the ZLI was not established in the absence of Fez and Fezl. Tcf4 is expressed at a high level in the thalamus and pretectum (Cho and Dressler, 1998; Korinek et al., –/– +/– +/– –/– Fezl and Fez Fezl embryos, 1998) in the wild type, Fez –/– –/– Fezl whereas the Tcf4-high domain expanded rostrally in the Fez embryos (Fig. 3Q-T). Lhx5 is expressed in the prethalamus in the –/– +/– +/– –/– Fezl and Fez Fezl embryos (Fig. 3U-W) wild type, Fez (Nakagawa and O’Leary, 2001), but was not detected in the Fig. 4. Reduction of rostral diencephalon and expansion of –/– –/– Fezl embryos (Fig. 3X). Taking these data together, loss of –/– –/– Fez caudal diencephalon in Fez Fezl embryos at E9.5. Expression of the prethalamus and expansion of the caudal diencephalon took Emx2 (A,B), Pax6 (C,D), Fgf8 (E,F), Wnt3a (G,H) and En2 (I,J) in control –/– –/– place before E10.5, and the ZLI, the prethalamus-thalamus (A,C,E,G,I) and Fez Fezl embryos (B,D,F,H,J) was analyzed by whole- boundary, was not established in the absence of Fez and Fezl. mount in situ hybridization. Lateral views of anterior neuroectoderm, with anterior to the left. Expression of Emx2 (A) and Pax6 in the dorsal telencephalon (between arrowheads, C) was reduced, but the Pax6 Fez and Fezl are involved in early rostro-caudal expression in the diencephalon (marked by dots) was maintained in forebrain patterning –/– –/– Fez Fezl embryos. Fgf8 expression in the commissural plate and mid- Formation and patterning of the forebrain are regulated by hindbrain boundary was maintained, but that in the dorsal prethalamus transcription factors and inductive signals expressed in specific –/– –/– was absent (arrowheads E,F) in Fez Fezl embryos. The expression rostro-caudal positions in the neuroectoderm at the beginning of domain of Wnt3a was caudal to the ZLI in control embryos (G) and was –/– –/– neurogenesis and neural patterning. We examined gene expression expanded rostrally in Fez Fezl embryos (arrow in H). Expression of –/– –/– –/– –/– Fezl embryos at around E9.5. The in wild type and Fez En2, which marks the midbrain, was not affected in Fez Fezl expression domain of Emx2 and Pax6 in the dorsal telencephalon embryos (I,J). cp, commissural plate; mhb, mid-hindbrain boundary; –/– –/– Fezl embryos at E9.5 (Fig. 4A- was already reduced in the Fez pth, prethalamus. D). Fgf8 was expressed in the commissural plate, the infundibulum of the hypothalamus, the dorsal part of the prethalamus (eminentia Nusse, 1991; Salinas and Nusse, 1992), and the rostral limit of its thalami) and the mid-hindbrain boundary (isthmus) in wild-type –/– –/– expression was shifted rostrally in the Fez Fezl embryos (Fig. embryos (Fig. 4E) (Crossley and Martin, 1995), whereas its 4H). En2 expression in the midbrain was not affected in the expression in the prospective prethalamus was specifically absent in –/– –/– –/– –/– Fezl embryos (Fig. 4F). Wnt3a is expressed in the dorsal Fez Fezl embryos (Fig. 4I,J) (Joyner and Martin, 1987). All these the Fez data indicate that the diencephalon patterning was already affected neural tissue caudal to the prospective ZLI (Fig. 4G) (Roelink and DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 3999 had shifted rostrally at E9.0, suggesting that Fez and Fezl are required to repress the Irx1 expression in the rostral diencephalon. We found that the expression of Six3 was not affected at E8.5 and 9.5 (Fig. 5I,J, data not shown for E8.5), suggesting that the –/– –/– Fezl embryos expansion of the caudal diencephalon in the Fez was independent of Six3. Complementary expression of Fez/Fezl and Irx1 The expression of Fez and Fezl in the forebrain is initiated at E8.0 and 8.5, respectively (Hirata et al., 2006; Hirata et al., 2004) (Fig. 6B). At E12.5, Fez and Fezl are expressed in the pallium, septum, hypothalamus and prethalamus, which are located rostral to the ZLI (Hirata et al., 2004; Hirata et al., 2006). We examined the early expression domains of Fez and Fezl, and compared them with those of Irx1. At E8.5, the expression domain of Fez was slightly wider than that of Fezl (Fig. 6A,B), and the caudal limit of Fez abutted the rostral limit of Irx1 (Fig. 6D). Two-color staining for Fez or Fezl with Irx1 revealed that the expression of Fez and Fezl was strictly rostral to that of Irx1 at E9.5 (Fig. 6H-M). Fez and Fezl repress the caudal diencephalon fate The expression of Fez and Fezl suggests that Fez and Fezl function in the region rostral to the ZLI and repress the caudal diencephalon fate. To address this issue, we misexpressed Fez or Fezl caudal to the ZLI using the Fezl gene enhancer/promoter or the Otx2 forebrain-midbrain (FM) enhancer. We constructed -galactosidase (lacZ) reporter genes in which the lacZ gene was connected to the 8.2 kbp or 2.7 kbp enhancer/promoter region upstream of the translational initiation site of the mouse Fezl gene (Fig. 7A), and examined the lacZ expression in the resulting transgenics (Fig. 7B- D). The 8.2 kbp Fezl enhancer/promoter recapitulated the expression in the forebrain at E8.5 (Fig. 7B). By contrast, the 2.7 kbp Fezl enhancer/promoter (Fezl2.7p) drove the lacZ expression in a wider region at E8.5 than the 8.2 kbp promoter did (Fig. 7C). At E9.5 in transgenic mice with the 2.7 kbp Fezl promoter-lacZ, activity was detected in a wider region than the endogenous lacZ Fezl expression (Fig. 7D), indicating that the enhancer/promoter could drive expression caudal to the ZLI. Using Fezl2.7p, we expressed Fezl or Fez cDNA ectopically in the caudal diencephalon. We monitored the exogenous Fezl or Fez expression with a green fluorescence protein variant, Venus (IRES-Venus) (Nagai et al., –/– –/– Fig. 5. Rostrally expanded expression of Irx1 in Fez Fezl 2002). In embryos with the Fezl2.7p-Fezl-IRES-Venus or Fezl2.7p- embryos. Irx1 and Six3 expression in control (A,C,E,G,I) and Fez-IRES-Venus transgene, the expression of Foxg1 in the –/– –/– Fez Fezl (B,D,F,H,J) embryos at E10.0 (A,B), E9.5 (C,D,I,J), E9.0 (E,F) telencephalon (Tao and Lai, 1992) was not affected, but the and E8.5 (G,H). Lateral views, with anterior to the right. Irx1 –/– –/– expression of Irx1 was reduced and its rostral limit was shifted expression was not significantly different between Fez Fezl and caudally at E9.5 (Fig. 8B,C,S). The rostral limit of Irx1 control embryos at E8.5. A rostral expansion of Irx1 expression was –/– –/– detected at E9.0 in Fez Fezl embryos (arrow). At E9.5 and E10.5, corresponded to the caudal limit of the exogenous Fezl-IRES-Venus the rostral limit of Irx1 expression (arrows) reached the caudal edge of expression in these transgenic embryos (Fig. 8A,R). Similarly, in the telencephalon (marked by dots). Expression of Six3 was not the Fezl2.7p-Fezl-IRES-Venus and Fezl2.7p-Fez-IRES-Venus –/– –/– significantly affected in Fez Fezl embryos at E8.5 (data not shown) transgenic embryos, the Tcf4-high domains, corresponding to the and at E9.5 (I,J). thalamus and pretectum, were strongly reduced at E10.5 (Fig. 8E,F,U). In transgenic embryos strongly expressing Fezl, Dlx1 expression expanded caudally (n=1/2), with Gbx2 expression being when neural patterning began. In chick, ZLI is positioned on the prominently reduced (n=2/3) at E12.5 (Fig. 8,M,N,P,Q). boundary of the expression domains of Six3 and Irx3, and Six3 and Furthermore, the misexpression of Fezl by Fezl2.7p abolished Shh Irx3 can repress the expression of each other, possibly determining expression in the ZLI and ventral diencephalon (n=2/4) or shifted the position of the ZLI (Kobayashi et al., 2002). In mouse, Irx1 and the ZLI-specific Shh expression caudally (n=2/4) at E10.5 (Fig. Irx3 display a similar expression profile in the neuroectoderm, but 8H,I,K). The misexpression of Fez by Fezl2.7p abolished Shh the rostral limit of the Irx1 expression is more rostral than that of expression in the ZLI (data not shown, n=1/1). We also used an Irx3 and is positioned on the ZLI (Bosse et al., 1997; Cohen et al., Otx2FM enhancer, which can drive a transgene in the midbrain, 2000). We examined the expression of Irx1 in wild-type and diencephalon and archicortex (Kurokawa et al., 2004a), to double-mutant embryos (Fig. 5). The rostral limit of Irx1 expression misexpress Fezl. Fezl misexpression under this enhancer also DEVELOPMENT 4000 RESEARCH ARTICLE Development 133 (20) Fig. 6. Complementary expression of Fez/Fezl and Irx1. (A-D) Expression of Fez (A,D), Fezl (B) and Irx1 (C,D) at E8.5. (D) Two- color staining. Fez and Irx1 transcripts were stained with BM Purple and Fast Red; the fluorescence image from the Fast Red was superimposed on the bright-field image. (E-M) Expression of Fez (E), Fezl (F) and Irx1 (G) at E9.5. (H-M) Sagittal sections of E9.5 embryos were hybridized with Fez and Irx1 (H-J), or Fezl and Irx1 probes (K-M). The hybridized signals were stained with BM Purple (Fez and Fezl) and Fluorescein (Irx1). Bright-field images (H,K), fluorescence images (I,L) and the bright-field and fluorescence superimposed images (J,M). suppressed Irx1 expression without affecting Foxg1 expression the wild-type embryos, when the rostro-caudal patterning in the (Fig. 8W). These data indicate that Fez and Fezl can suppress the diencephalon became abnormal (Figs 3, 4). Our data strongly caudal diencephalon fate. suggest that the transformation of the prethalamus into the caudal diencephalon takes place during early neural patterning in the DISCUSSION absence of Fez and Fezl (Fig. 9). Future studies including the Cre- Role of Fez and Fezl in diencephalon patterning loxP-mediated cell-fate mapping of Fez and Fezl-expressing cells –/– –/– –/– +/– The Fez Fezl mutant embryos, but not the Fez Fezl or will definitely clarify this issue. The misexpression of Fez or Fezl +/– –/– Fez Fezl embryos, showed defects in patterning of the suppressed the caudal diencephalon fate and induced the expression diencephalon (Fig. 1), indicating a strictly redundant role for Fez and of Dlx1, which normally marks the prethalamus, in the region caudal Fezl in diencephalon development. This is consistent with the to the ZLI (Fig. 8). All of these data indicate that Fez and Fezl overlapping expression of Fez and Fezl in the prethalamus at later function to repress the caudal diencephalon fate and establish the stages (Hirata et al., 2006; Hirata et al., 2004). Fez and Fezl, prethalamus fate (Fig. 9). Although the caudal diencephalon was respectively, begin expression at E8 and 8.5 (Fig. 6) (Hirata et al., initially expanded, the thalamus eventually became smaller and the 2006; Hirata et al., 2004). We detected a defect in the rostro-caudal rostral part of the thalamus was missing in the Fez;Fezl-deficient polarity of the diencephalon: rostral expansion of the Irx1 expression embryos at E12.5 (Figs 1, 2). The later development of the thalamus at E9.0, indicating that the Fez/Fezl-mediated diencephalon is known to be dependent on inductive signals from the ZLI patterning starts soon after the onset of Fez and Fezl expression. (discussed below). The loss of the ZLI in the Fez;Fezl-deficient Marker analyses showed that the prethalamic region was not embryos secondarily affects the development of the thalamus in established; instead, the caudal diencephalon, which includes the these embryos (Fig. 9). thalamus and the anterior pretectum, expanded rostrally in the Both FEZ and FEZL contain an Eh1 repressor motif, which double-mutant embryos (Figs 3, 4). There are two possible interacts with the Groucho/TLE family of transcriptional co- explanations for the phenotype of Fez;Fezl-deficient embryos: (1) repressors (Bae et al., 2003; Kobayashi et al., 2001; Muhr et al., transformation of the prethalamus into the thalamus; and (2) 2001; Shimizu et al., 2002). The Eh1 repressor motif of zebrafish truncation of the prethalamus and the rostral shift of the caudal Fezl is required for at least part of the Fezl function in this animal neural tissue. We found that the size of the diencephalon was not (Levkowitz et al., 2003), suggesting that FEZ and FEZL function as significantly different in the double-mutant embryos, compared with transcriptional repressors to regulate patterning of the diencephalon. DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 4001 Fig. 7. Enhancer and promoter region of Fezl. (A) Schematic diagram of the Fezl enhancer/promoter and the constructs used for transgenesis. (B-D) Detection of lacZ expression by X-gal staining in Fezl8.2p-lacZ (B) and Fezl2.7p-lacZ (C,D) transgenic mouse embryos, in which lacZ expression was driven by the 8.2 kbp and 2.7 kbp Fezl enhancer/promoter, respectively. (B,C) E8.5 embryos, lateral views with anterior to the top. (D) E9.5 embryo, lateral views of the anterior neuroectoderm. The 8.2 kbp Fezl enhancer/promoter recapitulated the endogenous Fezl expression (rostral to the ZLI), whereas the 2.7 kbp Fezl enhancer/promoter showed a caudally expanded expression of lacZ (arrows, D). In the Fez;Fezl double-mutant embryos, expression domains of the How Fez and Fezl expression is regulated remains unclear. There caudal diencephalic genes, such as Irx1, Wnt3a and Tcf4 (high- are several genes with expression domains that overlap with those expression domain), expanded rostrally, and misexpression of Fez of Fez and Fezl. They include Pax6, Emx1/2, Dlx1/2/5/6 and Otx1/2 or Fezl caudal to the ZLI inhibited the expression of Irx1 and Tcf4 in in addition to Six3 (Bulfone et al., 1993; Oliver et al., 1995; Simeone the caudal diencephalon (Figs 4, 5, 8). Fez and Fezl are expressed in et al., 1992a; Simeone et al., 1993; Simeone et al., 1992b; Stuhmer the region rostral to the rostral limit of Irx1 expression, which marks et al., 2002; Walther and Gruss, 1991). These genes might be the prospective position of the ZLI. Taking these findings together, involved in the regulation of Fez and Fezl expression. The expression we conclude that FEZ and FEZL directly or indirectly repress the of zebrafish fezl and Xenopus Fez is negatively regulated by Wnt caudal diencephalon genes in the rostral diencephalon (Fig. 9). signaling (Hashimoto et al., 2000) (M. Matsuo-Takasaki, personal Identification of target genes for FEZ and FEZL and/or chromatin communication). Thus, the initial expression of Fez and Fezl may be immunoprecipitation assay of FEZ/FEZL-binding genomic controlled by the rostro-caudal polarity information, in which Wnt fragments will help to clarify the precise mechanism by which Fez signaling is strongly involved (Niehrs, 2004). In this context, Fez and and Fezl control the rostro-caudal polarity of diencephalon. Fezl may link the rostro-caudal polarity information to the SIX3 negatively regulates the caudally expressed Wnt1 (Lagutin subdivision formation in the diencephalon. et al., 2003) and functions as a transcriptional repressor (Kobayashi et al., 2001; Lopez-Rios et al., 2003; Zhu et al., 2002). Six3-deficient Role of Fez and Fezl in formation of the ZLI mice show strong reduction of the neural tissue rostral to the ZLI The ZLI position is predicted as abutting the expression domains of (Lagutin et al., 2003). Six3 negatively controls the expression of Irx3 rostral Six3 and caudal Irx3 in chick embryos (Kobayashi et al., in chick (Kobayashi et al., 2002). These reports suggest that the role 2002). We found that it is predicted by the expression of rostrally of Fez and Fezl in diencephalon patterning is similar to that of Six3, expressed Fez and Fezl and caudally expressed Irx1 in mouse at least in part. However, Six3-deficient mice have rudimentary embryos (Fig. 6). Furthermore, the deficiency of both Fez and Fezl tissue rostral to the ZLI, and express Shh in the ZLI (Lagutin et al., led to loss of the ZLI, and misexpression of Fez or Fezl could inhibit 2003), and Fez;Fezl-double mutants display complete loss of the ZLI formation or shift its position (Figs 3, 8). These data indicate prethalamus and ZLI, implying there is a difference between the that Fez and Fezl are involved in the formation and position of the functions of Six3 and Fez/Fezl. We examined the expression of Six3 ZLI. The ZLI initially forms as a wedge-shaped structure on the –/– –/– in the Fez Fezl embryos, but did not observe any significant boundary between the prethalamus and thalamus, which is alteration in the Six3 expression at E8.5 and 9.5 (Fig. 5, data not characterized by a gap in Lfrg expression; subsequently, it collapses shown for E8.5), suggesting that Six3 does not function downstream to a narrow band (Zeltser et al., 2001). It is not clear whether Fez of Fez and Fezl. Rather, Six3 may function upstream of, or in parallel and Fezl are expressed in the prospective ZLI domain, although they with, Fez and Fezl. Future studies examining Fez and Fezl expression are not expressed in the ZLI at E12.5 (Hirata et al., 2006; Hirata et in Six3-deficient embryos and combinatory gene disruption of Six3 al., 2004). Fez and Fezl are expressed in the prethalamus, but they and Fez and/or Fezl will clarify this issue. do not induce Shh expression there. Thus, Fez and Fezl are not DEVELOPMENT 4002 RESEARCH ARTICLE Development 133 (20) Fig. 8. Misexpression of Fez or Fezl affects rostro-caudal polarity in the diencephalon. Misexpression of Fezl (A-Q) or Fez (R-U) by the 2.7 kbp Fezl enhancer/promoter (Fezl2.7p-Fezl- IRES-Venus, Fezl2.7p-Fez-IRES-Venus), or Fezl by the FM enhancer of the Otx2 gene and mouse Hsp68 promoter (Otx2FM-Hsp68-Fezl-IRES-Venus, V,W) affected the diencephalon subdivisions. Exogenous Fezl expression was monitored by the expression of Venus attached to an IRES (Fig. 7) (fluorescence images, A,D,G,J,L,O,R,T,V). E9.5 embryos were analyzed by whole-mount in situ hybridization with Foxg1 and Irx1 (B,C,S,W). E10.5 embryos were analyzed by in situ hybridization with Tcf4 (E,F,U) or Shh (H,I,K). Sagittal sections of E12.5 embryos were analyzed with Dlx1 or Gbx2 probes (M,N,P,Q). (C,F,I,N,Q) Control non-transgenic embryos. Expression of Foxg1 in the telencephalon was not affected (caudal limit marked by arrows), but the rostral limit of Irx1 expression (marked by arrowheads) was shifted caudally in Fezl2.7p-Fezl-IRES-Venus (B), Fezl2.7p-Fez-IRES-Venus (S) and Otx2FM-Hsp68-Fezl-IRES-Venus embryos (W), compared with the control (C). Tcf4-high expression domain in the thalamus and prethalamus was strongly reduced in the Fezl2.7p-Fezl- IRES-Venus (E) and Fezl2.7p-Fez-IRES-Venus (U) embryos. Shh expression in the ZLI (arrows) and ventral diencephalon was reduced (H, n=2/4), or Shh expression in the ZLI was shifted caudally (K, n=2/4) in the Fezl2.7p-Fezl-IRES-Venus embryos, compared with control (I). Dlx1 expression in the prethalamus was expanded caudally when the exogenous Fezl-IRES-Venus was strongly expressed (M, n=1/2). Gbx2 expression in the thalamus was strongly reduced in the Fezl2.7p-Fezl-IRES-Venus embryos (P, n=2/3). DEVELOPMENT Fez and Fezl in forebrain formation RESEARCH ARTICLE 4003 Fig. 9. Schematic presentation of a role of Fez and Fezl in diencephalon patterning. Fez and Fezl are expressed in the telencephalon and rostral diencephalon (prospective prethalamus) and function to suppress the formation of the caudal diencephalon, which expresses Irx1 and Wnt3a. In the absence of Fez and Fezl, the rostral diencephalon does not form and instead caudal diencephalon expands rostrally at E9.5. Subsequently at E10.5, the prethalmaus and the ZLI, which is normally located in the interface between the prethalamus and thalamus, do not form in –/– –/– Fez Fezl embryos. The caudal diencephalon, –/– –/– including the thalamus and pretectum, is expanded in Fez Fezl embryos. The formation of thalamus, however, is dependent on inductive signals –/– –/– (e.g. Shh) from the ZLI. In Fez Fezl embryos, the thalamus does not grow properly, but the anterior pretectum remains expanded at E12.5. apt, anterior pretectum; di, diencephalon; pth, prethalamus, ppt, posterior pretectum; tel, telencephalon; th, thalamus. likely to be instructive factors, but rather to function as permissive work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Technology, and by a grant factors. Alternatively, Fez and Fezl may regulate ZLI formation from RIKEN (to M.H.). indirectly and non-cell-autonomously by controlling formation of the prethalamus. Grafting experiments in chick embryos show that References Shh expression is induced in the interface between the prechordal Bae, Y. K., Shimizu, T., Yabe, T., Kim, C. H., Hirata, T., Nojima, H., Muraoka, O., Hirano, T. and Hibi, M. (2003). 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Journal

Development – The Company of Biologists

Published: Oct 15, 2006

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