Developmental Biology 358 (2011) 91–101

Contents lists available at ScienceDirect

Developmental Biology

journal homepage: www.elsevier.com/developmentalbiology

Hes1 regulates the number and anterior–posterior patterning of mesencephalic dopaminergic at the mid/hindbrain boundary (isthmus)

Yoko Kameda a,⁎, Takayoshi Saitoh b, Takao Fujimura c a Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan b Graduate school of Science, Kitasato University, Sagamihara, Kanagawa 252-0374, Japan c Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan article info abstract

Article history: The lack of the Hes1 gene leads to the failure of cranial neurulation due to the premature onset of neural Received for publication 12 January 2011 differentiation. Hes1 homozygous null mutant mice displayed a closure defect, and exencephaly Revised 22 June 2011 was induced at the mid/hindbrain boundary. In the mutant mesencephalon, the roof plate was not formed and Accepted 12 July 2011 therefore the ventricular zone showing cell proliferation was displaced to the brain surface. Furthermore, the Available online 23 July 2011 telencephalon and ventral diencephalon were defective. Despite the severe defects of neurogenesis in null mutants, the mesencephalic dopaminergic (mesDA) neurons were specified at the midline of the ventral Keywords: — fl Hes1 knockout mice mesencephalon in close proximity to two important signal centers oor plate and mid/hindbrain boundary Neural tube closure defect (i.e., the isthmic organizer). Using mesDA neuronal markers, tyrosine hydroxylase (TH) and Pitx3, the Mesencephalic dopaminergic neurons development of mesDA neurons was studied in Hes1 null mice and compared with that in the wild type. At Tyrosine hydroxylase early stages, between embryonic day (E) 11.5 and E12.5, mesDA neurons were more numerous in null Pitx3 mutants than in the wild type. From E13.5 onward, however, the cell number and fiber density of mesDA Serotonergic neurons neurons were decreased in the mutants. Their distribution pattern was also different from that of the wild Isthmic organizer type. In particular, mesDA neurons grew dorsally and invaded the rostral hindbrain. 5-HT neurons were also ectopically located in the mutant . Thus, the loss of Hes1 resulted in disturbances in the inductive and repulsive activities of the isthmic organizer. It is proposed that Hes1 plays a role in regulating the location and density of mesDA neurons. © 2011 Elsevier Inc. All rights reserved.

Introduction (Partanen, 2007). Furthermore, Hes1 is persistently expressed at high levels by isthmic cells (Baek et al., 2006). In Hes1 and Hes3 double Hes genes are basic helix–loop–helix (bHLH) transcription factors mutant mice, isthmic organizer cells prematurely terminate the that are essential effectors of Notch signaling, which regulates the expression of Fgf8 and Wnt1, resulting in early differentiation into maintenance of progenitor cells and the timing of their differentiation neurons (Hirata et al., 2001). Thus, Hes genes are involved in the in various tissues and organs (Kageyama and Ohtsuka, 1999). In the formation of isthmic cells and regulate the development of the nervous system, Hes genes are crucial for generating the correct midbrain and anterior hindbrain. numbers and full diversity of neurons and glial cells by maintaining During development, distinct classes of neurons are specified in neural stem cells until later stages (Hatakeyama and Kageyama, 2006; precise locations along the dorsal–ventral and anterior–posterior axes Hatakeyama et al., 2004). Hes1, a member of the family of Hes genes, of the neural tube. Mesencephalic dopaminergic (mesDA) neurons represses the expression of proneural bHLH factors such as Mash1 and develop in the ventral midbrain in close proximity to two important neurogenin (Ngn) 2, acting as a negative regulator of neuronal signaling centers in the embryonic neural tube — the floor plate and differentiation. Mice deficient in Hes1 display neural hypoplasia, the mid/hindbrain boundary (i.e., the isthmus) (Prakash and Wurst, which is attributed to accelerated neuronal differentiation (Ishibashi 2004). Shh expressed along the ventral neural tube and Fgf8, which is et al., 1995). The defective morphogenesis of the neural tube and locally produced at the mid/hindbrain boundary, create induction exencephaly are caused in Hes1 null mutants. sites for dopaminergic neurons in the midbrain (Ye et al., 1998). Hes1 The isthmic organizer, which is located at the mid/hindbrain expression is detected in mesencephalic floor plate cells at E9.5 and boundary, expresses secreting molecules such as Fgf8 and Wnt1 maintained until E11.5 (Ono et al., 2010). MesDA neurons are anatomically divided into three main sub- groups (see Ang, 2006 for review). Neurons in the substantia nigra ⁎ Corresponding author. Fax: +81 42 778 8441. pars compacta (SNc) mainly project to the dorsolateral striatum and E-mail address: [email protected] (Y. Kameda). form the nigro-striatal pathway. Neurons in the ventral tegmental

0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2011.07.016 92 Y. Kameda et al. / Developmental Biology 358 (2011) 91–101 area (VTA) and the retrorubal field of the midbrain project to the The specimens were fixed in Bouin's solution or 8% paraformal- ventromedial striatum, as part of the mesocortical limbic system. The dehyde (PFA) in phosphate buffer (PB) overnight, embedded in degeneration of the nigro-striatal system induces the Parkinson's paraffin and then serially sectioned along the frontal and sagittal disease, one of the most common human neurological disorders. planes at a thickness of 5 μm. For frozen sections, the specimens were Dysregulation of DA transmission in the limbic system is linked to fixed in 4% PFA for 2 h. Selected sections were stained with psychiatric and affective disorders. The factors involved in the hematoxylin–eosin to determine the morphological orientation. generation, differentiation and survival of mesDA neurons during development have been studied extensively owing to their involve- Immunohistochemistry ment in neurological disorders. The generation of mesDA neurons from neural progenitor cells is regulated by the combined expressions Immunohistochemical staining was performed using the of certain transcription factors including Otx2, Foxa1, Foxa2, Lmx1a, streptavidin–biotin-peroxidase method, as previously described Lmx1b, Engrailed 1 (En1), Engrailed 2 (En2), Msx1, Msx2, Ngn2 and (Kameda, 1995). The following primary antibodies were employed: Mash1 (Ang, 2006; Ferri et al., 2007; Prakash and Wurst, 2006; Smits rabbit polyclonal antibodies to tyrosine hydroxylase (TH)(Chemicon, El et al., 2006). Loss-of-function studies in mice have revealed roles for Segundo, CA; 1:100 dilution), Pitx3 (Invitrogen, Camarillo, CA; 1:50 Nurr1 (Zetterström et al., 1997), Lmx1b (Smidt et al., 2000), Pitx3 dilution), 5-HT () (Chemicon; 1:1000 dilution), Otx2 (Abcam, (Smidt et al., 2004), En1/2 (Simon et al., 2001), and Foxa1/Foxa2 ab21990, 1:200 dilution), protein gene product (PGP) 9.5, (Dako, (Ferri et al., 2007) in the late differentiation and maintenance of these Carpinteria, CA; 1:200 dilution), and the monoclonal antibody TuJ1 neurons. The Pitx3 gene, belonging to the Pitx subfamily of paired-like that recognizes the -specific class III β-tubulin isotype (Berkeley homeodomain proteins, is required for the terminal differentiation of antibody company, Richmond, CA; 1:500 dilution) and the Mash1 the SNc subpopulation of mesDA neurons (Smidt et al., 2004). Ngn2 monoclonal antibody (BD Biosciences; 1:100 dilution). Furthermore, has also been shown to be the key proneural gene regulating the proliferating cells were examined using the proliferating cell nuclear neurogenesis of mesDA progenitors in the midbrain (Kele et al., 2006). antigen (PCNA) monoclonal antibody (Dako, pre-diluted) and an A closure defect of the cranial neural tube is a characteristic antiserum against phospho-Histone H3 (Ser10) (Upstate Biotechnology, phenotype of Hes1 null mutants. Exencephaly and the loss of ventricles Temecula, CA; Santa Cruz Biotechnology; 1:100 dilution), a mitosis are seen in null mutant embryos (Akimoto et al., 2010a,b). Thus, marker. abnormalities in the mesencephalon, diencephalon and telencephalon are induced in Hes1 null mutants. At present, however, little is known Quantitative analysis about the development of mesDA neurons in Hes1 null mutants in which midbrain morphogenesis is defective. In this study, the phenotypes of To compare the numbers of TH-immunoreactive cell bodies and mesDA neurons in Hes1 null mutant mice were analyzed using two Pitx3-positive nuclei of mesDA neurons in each genotype, the ventral markers for mesDA neurons — tyrosine hydroxylase (TH) and Pitx3. In mesencephalic area including mesDA neurons in the frontal sections addition, the distribution of serotonergic neurons was examined in the was photographed in Hes1 null mutants and littermate wild types at mutants. E12.5, 13.5 and E15.5. Images of photographed TH- and Pitx3-positive neurons were digitized using a digital camera system (Olympus, DP70, Materials and methods Tokyo) attached to an Olympus AX80 microscope. Morphometric quantification was performed using WinRoof Image software (version Animals 5.5; Mitani Corp., Fukui, Japan). The numbers of TH-immunoreactive cells and Pitx3-positive nuclei were assessed automatically by color Wild-type, heterozygous, and homozygous Hes1 mice were binarization, as described previously (Chisaka and Kameda, 2005). obtained from intercrosses of Hes1+/− mice, which were maintained Three sections were analyzed bilaterally for each animal, and the on a CD1 background (Ishibashi et al., 1995). The Hes1+/− mouse was highest value obtained was utilized to estimate the number of DA supplied by Prof. R. Kageyama. E0.5 was designated as noon on the neurons for every single animal. At least four animals per genotype day that a copulation plug was identified. Mouse genotypes were were analyzed at each stage for quantification. Results were expressed obtained by PCR analysis using previously described primers as means±SEM. Differences were assessed by a Student's t-test and (Ishibashi et al., 1995). considered statistically significant at Pb0.05.

Fig. 1. A and B. Comparison of the head of wild-type (+/+, A) and homozygous Hes1 null mutant (−/−, B) littermate mice at E15.5. The null mutant embryo exhibits the phenotype of exencephaly (arrow). Y. Kameda et al. / Developmental Biology 358 (2011) 91–101 93

Results Development of mesDA neurons in Hes1 null mutant mice

Neural tube closure defect in Hes1 null mutant mice The development of mesDA neurons in Hes1 null mutant mice in comparison with that in wild-type mice was analyzed by immuno- In wild-type embryos, the neural tube was formed by the proper cytochemistry with an antibody to tyrosine hydroxylase (TH), the elevation and fusion of the dorsal neuroepithelial folds. In homozy- rate-limiting enzyme in DA synthesis. The antibody to Pitx3 which gous Hes1 null mutant mice, the neuroepithelium remained to be was coexpressed with TH in mesDA neurons was also used for everted, resulting in a closure defect of the cranial neural tube. analysis. Therefore, exencephaly occurred at the forebrain and midbrain In wild-type mice at E11.5, mesDA neurons expressing TH boundary (Figs. 1A and B) and dramatic brain malformations were emerged in the basal midline region of the developing ventral induced; the forebrain did not develop properly, the ventral mesencephalon (Fig. 2E). The wild-type midbrain wall consisted of diencephalon collapsed in most cases, and the dorsal midbrain did two well-developed layers — the ventricular zone and the outer layer not form. In spite of the defect in midbrain morphogenesis, mesDA (Fig. 2A). The ventricular zone displayed the proliferating cells that neurons formed in most of the Hes1 mutants. were immunoreactive for PCNA (Fig. 2B). Furthermore, Mash1, a

Fig. 2. A–J. Serial frontal sections of the midbrain of wild-type (+/+, A–E) and Hes1 null mutant (−/−,F–J) mice, respectively, at E11.5. The sections were stained with hematoxylin– eosin (A, F) or immunostained with the PCNA (B, G), TuJ1 (C, H), phospho-Histon H3 (D, I), and TH (E, J) antibodies. In the null mutant showing a neural tube closure defect, the midbrain is expanded laterally and its proliferation zone is located at the brain surface. TH-immunoreactive mesDA neurons (arrow) are more numerous in the null mutant than in the wild type. NT, neural tube. Higher magnification of boxed area in A and F is shown in D and E, I and J, respectively. Scale bars, 280 μm in panels A–C and F–H, and 170 μm in panels D, E, I, and J. 94 Y. Kameda et al. / Developmental Biology 358 (2011) 91–101 marker for proliferating neural progenitors, was localized to the proliferation zone lay on the surface of the midbrain. TH- and Pitx3- ventricular zone (not shown). Cells immunoreactive for phospho- positive mesDA neurons were located underneath the midline surface Histone H3, a mitosis marker, were concentrated at the surface of the region in the mutant mesencephalon which appeared to be partially ventricular zone (Fig. 2D). Markers for postmitotic neurons such as disrupted (Fig. 2J, data not shown). TuJ1- and PGP 9.5-positive cells TuJ1 and PGP 9.5 were confined to the outer layer where TH-positive also lined up under the surface zone (Fig. 2H). mesDA neurons were located (Fig. 2C). In the absence of the Hes1 In wild-type embryos at E12.5, mesDA neurons immunoreactive gene, neural differentiation occurred precociously, resulting in the for TH extended their processes vertically near the ventricular zone accelerated generation of mesDA neurons. The mesDA neurons at (Figs. 3A and C). TH-positive fibers were relatively tightly fasciculated E11.5 were more numerous in null mutants than in wild-type in the developing midbrain at this stage. The localization of Pitx3 embryos (Fig. 2J). The ventricular cavity did not form in null mutants immunoreactivity was restricted to the nuclei of mesDA neurons owing to the neural tube closure defect. The mesencephalon without (Fig. 3D). In Hes1 null mutants at E12.5, TH- and Pitx3-positive the cavity was broadened laterally and thinned (Fig. 2F). The PCNA- neurons were widely distributed in the midline area of the thinned and H3-positive proliferating cells were found at the surface of the midbrain (Figs. 3E, H and I). The surface zone exhibiting PCNA- and mutant midbrain (Figs. 2G and I). Furthermore, Mash1 was expressed Mash1-positive proliferating cells remained thin (Figs. 3F and G), in in the surface zone (not shown). Thus, in Hes1 null mutants, the contrast to the thickening ventricular zone of wild-type embryos

Fig. 3. A–I. Serial frontal sections of the midbrain of wild-type (+/+, A–D) and Hes1 null mutant (−/−,E–I) mice, respectively, at E12.5. The sections were stained with hematoxylin–eosin (A, E) or immunostained with the Mash1 (B, G), PCNA (F), TH (C, H), and Pitx3 (D, I) antibodies. In contrast to the thick neuroepithelium in the wild type, it is thin in the null mutant. In the wild type, TH- and Pitx3-positive mesDA neurons are distributed perpendicularly, whereas in the null mutant, they are extended transversely. NT, neural tube. Higher magnification of boxed area in A and E is shown in C and D, H and I, respectively. Scale bars, 280 μm in panels A, B, E–G, and 170 μm in panels C, D, H, and I. Y. Kameda et al. / Developmental Biology 358 (2011) 91–101 95

(Fig. 3B). Consistent with exencephaly, the cephalic flexure space from those in wild-type embryos. In a few cases, the medial forebrain underlying the midbrain was markedly increased in the mutants bundle and fibers projecting to the striatum ran along the brain compared to wild-type embryos (Figs. 4A–D). The midbrain was surface, i.e., the fibers did not descend downward (Figs. 7E–G). elongated along the expanding cephalic flexure space, and therefore the distribution of mesDA neurons was also extended in the anterior– Quantification of TH- and Pitx3-positive mesDA neurons posterior direction in null mutants (Fig. 4D). In wild-type embryos at E13.5, the mesencephalic wall became The numbers of TH-immunoreactive cell bodies and Pitx3-positive thick, especially in the ventral part (Fig. 5A). TH- and Pitx3-positive nuclei in the midbrain were measured in frontal sections of wild-type DA neurons in the ventral tegmental area (VTA), which were oriented and Hes1 null mutant embryos at E12.5, E13.5 and E15.5 (Figs. 8A and B). perpendicular to the ventricular surface, were well-developed in the At E12.5, the number of TH-immunoreactive neurons was significantly ventral midline (Figs. 5B and C). In addition, mesDA neurons began to higher in the mutants (400±21, Pb0.001) than in wild-type embryos accumulate in a more lateral position, forming the substantia nigra (206±17). However, at E13.5, the number of TH-immunoreactive compacta (SNc) (Fig. 5B). In Hes1 null mutants at E13.5, the neurons in mutant mice was reduced by 47% of that in wild-type mesencephalic wall was still thin, resulting in the short perpendicular embryos (393±42 in the mutants; 830±53 in the wild-type embryo, extension of TH- and Pitx3-positive neurons (Figs. 5D–F). Further- Pb0.001), and at E15.5 by 56% (677±28 in the mutants; 1217±31 in more, mesDA neurons were sparsely distributed in mutants compared the wild-type embryo, Pb0.001). Thus, the lack of the Hes1 gene caused to wild types. a conspicuous reduction in TH-immunoreactive neurons in the In wild-type mice at E15.5, the distribution patterns of TH- and midbrain at E13.5 and E15.5. Pitx3-positive cells in the SNc and VTA were similar to those in mature At E12.5, the number of Pitx3-expressing nuclei was more animals (Figs. 6A and B). In addition, axons of the medial forebrain numerous in null mutants (328±81) than that in wild-type embryos bundle (mfb) which was recognizable as fibers running rostrocaudally (258± 27), although a significant difference was not observed in the lateral margin of the hypothalamus became obvious, and then (P=0.15). At E13.5, however, the number of Pitx3-positive nuclei the TH-fibers extended to the striatum (Figs. 7A and B). In Hes1 null was significantly lower in null mutants (483±54, Pb0.0001), mutants at E15.5, the phenotypes of mesDA neurons varied from compared with that in wild-type embryos (648±42). Similarly, at individual to individual, because the thickness of the midbrain wall E15.5, the number of Pitx3-positive nuclei was lower in null mutants was variable and the diencephalon and telencephalon were defective (730±190, Pb0.05) than that in wild-type embryos (935±84). to different degrees in each animal. In most cases, mesDA neurons were loosely distributed in the surface region of the thin midbrain Defective isthmic organizer in Hes1 null mutants (Figs. 6D and E). In cases in which part of the ventral diencephalon remained, the medial forebrain bundle, extending downward toward In wild-type mice at later stages of fetal development, the tightly the hypothalamus, was detected (Figs. 7C and D). Thus, the nigro- fasciculated mesDA neurons were located rostrally close to the mid/ striatal projection remained partially in the less-affected mutant mice. hindbrain boundary (Figs. 9A and B). In null mutants, mesDA neurons In general, however, mutant projection pathways severely deviated were loosely bundled and their distribution was not restricted by the

Fig. 4. A–D. Serial sagittal sections of the midbrain of wild-type (+/+, A, B) and Hes1 null mutant (−/−, C, D) mice, respectively, at E12.5. The sections were stained with hematoxylin–eosin (A, C) or immunostained with the TH antibody (B, D). In the null mutant, the ventricles (V) are absent and the cephalic flexure space (CFS) is markedly enlarged. The distribution area of TH-immunoreactive mesDA neurons (arrow) is elongated in the exencephalic brain tissue of the null mutant. Arrowhead indicates the midbrain and hindbrain boundary. H, hypophysis; NC, nasal cavity; and OC, oral cavity. Higher magnification of boxed area in A and C is shown in B and D, respectively. Scale bars, 500 μm in panels A and C, and 280 μm in panels B and D. 96 Y. Kameda et al. / Developmental Biology 358 (2011) 91–101

Fig. 5. A–F. Serial frontal sections of the midbrain (MB) of wild-type (+/+, A–C) and Hes1 null mutant (−/−,D–F) mice, respectively, at E13.5. The sections were stained with hematoxylin–eosin (A, D) or immunostained with the TH (B, E), and Pitx3 (C, F) antibodies. In the wild-type mouse, mesDA neurons exhibit intense immunoreactivities for TH and Pitx3, and the SNc is distinguishable from the VTA. In the null mutant, the mesDA neurons immunoreactive for TH and Pitx3 are distributed sparsely at the brain surface. HB, Hind brain; and NT, neural tube. Higher magnification of boxed area in A and D is shown in B and C, E and F, respectively. Scale bars, 280 μm in panels A and D, and 200 μm in panels B, C, E, and F. boundary (Figs. 9C and D). Thus, the distribution of mesDA neurons in Discussion the mutants was significantly broader than that in wild-type embryos. Although some mesDA axons extended caudally even in wild-type mice, Adeficiency in the Hes1 gene results in the failure of cranial a substantial number of the axon bundles grew dorsally and invaded the neurulation due to the premature onset of neural differentiation hindbrain in null mutants (Fig. 9D). In heterozygous Hes1 mice with (Ishibashi et al., 1995). The accelerated neuronal differentiation and closed neural tube, i.e., showing normal brain structures, many TH- concomitant depletion of neural precursor cells induced the charac- immunoreactive neurons were distributed in the rostral hindbrain teristic phenotypes in the brains of Hes1 null mutant mice. The mutant (Fig. 9F). Pitx3-positive nuclei also extended into the hindbrain (Fig. 9E). embryos exhibited a closure defect of the cranial neural tube, leading 5-HT neurons are present as a series of cell groups in the wild-type to exencephaly. Furthermore, the skull vault was missing, uncovered hindbrain, which can be separated into rostral and caudal divisions, a brain tissue was degenerative, and the ventricles were absent or vast majority of which are located within the Raphe nuclei (Fig. 6C). In defective. Closure of the neural tube is essential for normal Hes1 null mutants, ectopic serotonergic neurons were detected in the development of the brain (Copp et al., 2003). The closure defect midbrain (Fig. 6F). causes the disruption of the dorsal–ventral patterning of the neural tube. In the midbrain of Hes1 null mutants, the roof plate was lacking and the floor and basal plates expanded laterally and underwent Otx2 expression in Hes1 null mutant mice thinning. In particular, only the ventral midbrain formed and the proliferation zone corresponding to the ventricular zone of wild types In wild-type mice at E11.5, Otx2 expression was restricted to the was localized at the brain surface. Thus, midbrain morphogenesis was forebrain and midbrain, i.e., the brain regions rostral to the isthmic abnormal in Hes1 null mutant embryos. organizer. At E12.5, however, Otx2 was also expressed in the MesDA neurons develop in the ventral midbrain in close proximity hindbrain (Figs. 10A and C). In Hes1 null mutants at E11.5 and to two important signaling centers in the embryonic neural tube — the E12.5, the expression patterns of Otx2 were similar to those in wild- floor plate and the isthmic organizer (Prakash and Wurst, 2004; Smidt type embryos (Figs. 10B and D). From E13.5 onward, in wild-type and Burbach, 2007). Shh is normally expressed in the floor plate of embryos, the concentrated expression of Otx2 was detected in the Hes1 null mutants at E9.5 (Baek et al., 2006). Wnt1 and Fgf8 are also mesDA neurons (Figs. 10E–G). However, in null mutants, Otx2 was expressed normally in the mutant isthmic organizer at E10.5 when expressed in only a few mesDA neurons immunoreactive for TH and specification of the midbrain and anterior hindbrain occurs (Hirata Pitx3 (Figs. 10H–J). et al., 2001). Thus, the early expression of transcription factors that are Y. Kameda et al. / Developmental Biology 358 (2011) 91–101 97

Fig. 6. A–F. Frontal sections of the midbrain of wild-type (+/+, A–C) and Hes1 null mutant (−/−,D–F) mice at E15.5. The sections were immunostained with the TH (A, D), Pitx3 (B, E), and 5-HT (C, F) antibodies. TH- and Pitx3-immunoreactive mesDA neurons are condensed in the ventral midbrain in the wild type. However, they are sparsely and widely distributed at the brain surface in the null mutant. In the wild type, the distribution of 5-HT-immunoreactive neurons is restricted to the hindbrain (HB). In the null mutant, however, 5-HT neurons (arrows) invade the midbrain (MB). Scale bars, 280 μm.

required for the production of mesDA progenitors may be induced in structure, many TH-immunoreactive neurons were distributed in the Hes1 null mutant embryos. rostral hindbrain and Pitx3-positive nuclei were also detected in the Despite the severe defects in neurogenesis, mesDA neurons were hindbrain. Thus, the ectopic expression of DA neurons is due to the generated in the midbrain of Hes1 null mutant mice. At E11.5 and E12.5, loss of Hes1 and not epiphenomenal to other gross changes in brain mesDA neurons immunoreactive for TH and Pitx3 were more numerous structure. It is proposed that Hes1 is involved in regulating the in the mutants than in the wild types. However, the proliferation zone of number and location of mesDA neurons during development. Like the mutant midbrain, which was located at the brain surface, was DA neurons, 5-HT neurons arise from ventral neuroepithelial already thin at E12.5. In the thin midbrain of null mutants, mesDA progenitors in proximity to the floor plate (Goridis and Rohrer, neurons expanded laterally but could not develop vertically; a dorsally 2002). The rostral cell group is located just caudal to the isthmus. truncated VTA formed. Thus, the distribution patterns of mesDA The development of 5-HT neurons, like that of mesDA neurons, neurons in the mutant midbrain were different from those in wild depends on the previous patterning of the neuroepithelium by Shh types. In wild-type embryos, mesDA neurons immunoreactive for both signaling and, for the rostral cells, on FGF signaling. The ectopic TH and Pitx3 progressively increased in number with age. In Hes1 null expression of 5-HT neurons was detected in the midbrain of Hes1 mutants, however, there was a significant decrease in the mesDA null mutants. Thus, in the absence of the Hes1 gene, the isthmic neurons at E13.5 and E15.5, compared with the respective wild types. In organizer is not fully functional. In Sp8-deficient mouse embryos in particular, there was a conspicuous reduction in TH-immunoreactive which overgrowth of the mid/hindbrain is induced, a posterior shift neurons. The decrease of mesDA neurons may be attributable to the of the isthmic organizer occurs and mesDA neurons expand in the decrease of neuronal progenitors in Hes1 null mice with a very thin rostral hindbrain (Griesel et al., 2006). midbrain neuroepithelium. The position of the isthmic organizer determines the location and In wild-type embryos, mesDA neurons were located rostrally in size of mesDA neurons and hindbrain serotonergic cell populations close contact with the mid/hindbrain boundary. In Hes1 null mutant (Brodski et al., 2003). At the onset of somitogenesis, Otx2 is expressed embryos, however, TH-positive neurons were widely distributed in directly rostral to the isthmic organizer, whereas Gbx2 is expressed the midbrain and many TH neurons grew caudally and invaded the directly caudal to it. The expression border between Otx2 and Gbx2 rostral hindbrain. Even in heterozygote mice exhibiting normal brain defines the position of the isthmic organizer (Puelles et al., 2003; 98 Y. Kameda et al. / Developmental Biology 358 (2011) 91–101

Fig. 7. A–G. Serial sagittal sections of the brain in a wild-type mouse (+/+, A, B) and Hes1 null mutant mice (−/−,C,D;E–G), respectively, at E15.5. The sections were stained with hematoxylin–eosin (A, C, E) or immunostained with the TH (B, D, F), and Pitx3 antibodies (G). In the wild type, mesDA neurons extend the medial forebrain bundle (mfb) and then extend fibers to the striatum (S). In null mutants, the medial forebrain bundle and fibers to the striatum are detected, although their projection pathways deviate from those of wild types. Pitx3 immunoreactivity (arrow) is localized in mesDA neurons only. CB, ; DC, diencephalon; H, hypophysis; MB, midbrain; MC, metencephalon; TC, telencephalon; and VDC, ventral diencephalon. Scale bars, 700 μm in panels A, C, and E, 500 μm in panels B, and D, and 350 μm in panels F, and G.

Fig. 8. A and B. Quantification of (A) TH- and (B) Pitx3-immunoreactive mesDA neurons in wild-type (□) and Hes1 null mutant (■) mice (at least n=4 per genotype at E12.5, E13.5, and E15.5, respectively). Data represent means±SE. *Pb0.05, ***Pb0.001, and N.S. (no significant difference) versus wild-type, as assessed by Student's t-test. Y. Kameda et al. / Developmental Biology 358 (2011) 91–101 99

Fig. 9. A–F. Serial sagittal (A–F) sections of the brain in wild-type (+/+, A, B), homozygous (−/−, C, D), and heterozygous (+/−,E,F)Hes1 mice, respectively, at E15.5 (A–D) and E13.5 (E, F). The sections were stained with hematoxylin–eosin (A, C) or immunostained with TH (B, D, F) or Pitx3 (E) antibody. MesDA neurons of wild types are located rostrally close to the mid/hindbrain boundary (arrow). In the null mutant, TH-immunoreactive neurons are widely distributed in the hindbrain (HB), indicating a defective isthmic organizer. In the heterozygote mouse exhibiting normal brain structure, both TH- and Pitx3(arrowhead)-immunoreactive neurons also invade the hindbrain. IV, fourth ventricle; CFS, cephalic flexure space; H, hypophysis; LV, lateral ventricle; MB, midbrain; NC, nasal cavity; and VDC, ventral diencephalon. Scale bars, 700 μm in panels A and C, 280 μm in panels B and D, 140 μm in panel E, and 180 μm in panel F.

Vernay et al., 2005). Otx2 is required for the establishment of the 2009). In Hes1 null mutants, later developmental functions such as mesencephalic field and controls proliferation and differentiation of axon growth and projections to targets were partly retained when mesDA progenitors (Omodei et al., 2008). In mouse embryos at mid- defects in the diencephalon and telencephalon were moderate. late gestation, Otx2 expression is restricted to a relevant part of the However, their pathways deviated from those in wild types. Thus, VTA and excluded from the SNc (Di Salvio et al., 2010). In Hes1 null Hes1 located in the isthmic organizer appears to regulate the rostrally mutants at E9.5 and E10.5, Otx2 and Gbx2 are normally expressed in a directed growth of axons from mesDA neurons and to delineate the complementary pattern along the anterior–posterior axis and the rostral nonpermissive/repulsive territory of 5-HT neurons. The position of the isthmic organizer does not deviate from that of wild abnormal axon projections of mesDA neurons may be caused by types (Hirata et al., 2001). Thus, the roles of Otx2 and Gbx2 in early malformations of the diencephalon and telencephalon, in addition to embryogenesis to define the border between adjacent compartments defects in the isthmic organizer and axon guidance signaling. appear to be maintained in Hes1 null mutants. At E12.5, Otx2 was Pitx3 is known to be essential for the final step of the expressed in the hindbrain in addition to the midbrain and forebrain differentiation of mesDA neurons. The Pitx3 gene is induced at the in each genotype. At later stages of development, in wild-type mice, same developmental stage as TH, at E11.5, in the mouse midbrain and the Otx2 expression was condensed in the mesDA neurons. In null continues to be expressed in adulthood (Smidt et al., 2004). Mutations mutants, however, Otx2 was expressed in only a few mesDA neurons in the Pitx3 gene have been found in the spontaneous mouse mutant immunoreactive for TH or Pitx3. aphakia, which is characterized by small eyes without a lens (Rieger In wild-type mice, mesDA neurons extend their axons rostrally to et al., 2001). In aphakia mutant mice, the formation of the SNc subfield innervate diencephalic and telencephalic targets. Fgf8 derived from at the onset of DA neuronal differentiation is defective and projections the isthmic organizer regulates the growth polarity of mesDA axons to the caudate putamen are selectively lost (Smidt et al., 2004). Lmx1b along the rostrocaudal axis by inducing sema3F (Yamauchi et al., −/− mice fail to induce Pitx3 in TH-positive DA neurons (Smidt et al., 100 Y. Kameda et al. / Developmental Biology 358 (2011) 91–101

Fig. 10. A–J. Sagittal (A, B) or serial frontal sections (E–G, H–J) of the brain in wild-type (+/+, A, E–G) and Hes1 null mutant (−/−,B,H–J) mice, respectively, at E12.5 (A, B) and E14.5 (E–G, H–J). Higher magnification of boxed area in A and B is shown in C and D, respectively. The sections were immunostained with Otx2 (A, B, G, J), TH (C, D, E, H) or Pitx3 (F, I) antibody. At E12.5, expression pattern of Otx2 in the null mutant is similar to that in the wild type. At E14.5, Otx2 expression is concentrated in the mesDA neurons in the wild type. In the null mutant, however, only a few Otx2-immunoreactive nuclei are localized in the mesDA neurons. Arrow indicates the mid/hindbrain boundary. CFS, cephalic flexure space; HB, hindbrain; and MB, midbrain. Scale bars, 300 μm in panels A–D, and 200 μm in panels E–J.

2000). Thus, the subtypes may be present in mesDA neurons. In Hes1 Hes1 maintains neuronal precursors by repressing the expression of null mutants, Pitx3 was coexpressed with TH in mesDA neurons proneural genes such as Mash1 and Ngn2. In the midbrain of Hes1 null during development. In parallel with the decrease in TH immunore- mutants, the Mash1-immunoreactive proliferation zone was thin, activity, the number of Pitx3-positive nuclei was reduced in null compared with that in wild-type embryos. It remains to be analyzed mutants at E13.5 and E15.5. Pitx3-positive cells, however, were less whether Hes1 mainly regulates Ngn2 expression during the neuro- affected by the absence of the Hes1 gene than TH-immunoreactive genesis of mesDA progenitors. neurons. Proneural bHLH genes such as Mash1 and Ngn2 are expressed Acknowledgments within the ventral zone of the mouse midbrain (Kele et al., 2006). Ngn2 is important for the directing the fate of mesDA progenitor cells We thank Prof. Ryoichiro Kageyama (Institute for Virus Research, (Andersson et al., 2006). The loss of Ngn2 impairs the differentiation Kyoto University) for the gift of the Hes1 mouselineandMissNoriko of mesDA neurons (Andersson et al., 2006; Kele et al., 2006). On the Nemoto and Mr. Tokio Katoh (Research Center for Biological Imaging, other hand, the loss of Mash1 normally has no detectable effect on DA Kitasato University School of Medicine) for their excellent technical neuron development, although it partially rescues the generation of contributions. This work was supported in part by a grant (no. 21590221) mesDA neuron precursors in the absence of Ngn2 (Kele et al., 2006). from the Ministry of Education of Japan to Y.K. Y. Kameda et al. / Developmental Biology 358 (2011) 91–101 101

References Kameda, Y., 1995. Evidence to support the distal vagal ganglion as the origin of C cells of the ultimobranchial gland in the chick. J. Comp. Neurol. 359, 1–14. Akimoto, M., Nishimaki, T., Arai, Y., Uchinuma, E., Yamauchi, H., Kameda, Y., 2010a. Hes1 Kele, J., Simplicio, N., Ferri, A.L.M., Mira, H., Guillemot, F., Arenas, E., Ang, S.L., 2006. regulates formations of the hypophyseal pars tuberalis and the hypothalamus. Cell Neurogenin 2 is required for the development of ventral midbrain dopaminergic Tissue Res. 340, 509–521. neurons. Development 133, 495–505. Akimoto, M., Kameda, Y., Arai, Y., Miura, M., Nishimaki, T., Takeda, A., Uchinuma, E., Omodei, D., Acampora, D., Mancuso, P., Prakash, N., Di Giovannantonio, L.G., Wurst, W., 2010b. Hes1 is required for the development of craniofacial structures derived from Simeone, A., 2008. Anterior–posterior graded response to Otx2 controls proliferation ectomesenchymal neural crest cells. J. Craniofac. Surg. 21, 1443–1449. and differentiation of dopaminergic progenitors in the ventral mesencephalon. Andersson, E., Jensen, J.B., Parmar, M., Guillemot, F., Björklund, A., 2006. Development of Development 135, 3459–3470. the mesencephalic dopaminergic neuron system is compromised in the absence of Ono, Y., Nakatani, T., Minaki, Y., Kumai, M., 2010. The basic helix–loop–helix neurogenin 2. Development 133, 507–516. Nato3 controls neurogenic activity in mesencephalic floor Ang, S.L., 2006. Transcriptional control of midbrain dopaminergic neuron development. plate cells. Development 137, 1897–1906. Development 133, 3499–3506. Partanen, J., 2007. FGF signaling pathways in development of the midbrain and anterior Baek, J.H., Hatakeyama, J., Sakamoto, S., Ohtsuka, T., Kageyama, R., 2006. Persistent and hindbrain. J. Neurochem. 101, 1185–1193. high levels of Hes1 expression regulate boundary formation in the developing Prakash, N., Wurst, W., 2004. Specification of midbrain territory. Cell Tissue Res. 318, . Development 133, 2467–2476. 5–14. Brodski, C., Weisenhorn, D.M.V., Signore, M., Sillaber, I., Oesterheld, M., Broccoli, V., Prakash, N., Wurst, W., 2006. Genetic networks controlling the development of Acampora, D., Simeone, A., Wurst, W., 2003. Location and size of dopaminergic and midbrain dopaminergic neurons. J. Physiol. 575, 403–410. serotonergic cell populations are controlled by the position of the midbrain–hindbrain Puelles, E., Acampora, D., Lacroix, E., Signore, M., Annino, A., Tuoto, F., Filosa, S., Corte, G., organizer. J. Neurosci. 23, 4199–4207. Wurst, W., Ang, S.L., Simeone, A., 2003. Otx dose-dependent integrated control of Chisaka, O., Kameda, Y., 2005. Hoxa3 regulates the proliferation and differentiation of antero-posterior and dorso-ventral patterning of midbrain. Nat. Neurosci. 6, the third pharyngeal arch mesenchyme in mice. Cell Tissue Res. 320, 77–89. 453–460. Copp, A.J., Nicholas, N.D.E., Murdoch, J.N., 2003. The genetic basis of mammalian Rieger, D.K., Reichenberger, E., McLean, W., Sidow, A., Olsen, B.R., 2001. A double-deletion neurulation. Nat. Rev. Genet. 4, 784–793. mutation in the Pitx3 gene causes arrested lens development in aphakia mice. Di Salvio, M., Di Giovannantonio, L.G., Omodei, D., Acampora, D., Simeone, A., 2010. Genomics 72, 61–72. Otx2 expression is restricted to dopaminergic neurons of the ventral tegmental Simon, H.H., Saueressig, H., Wurst, W., Goulding, M.D., O'Leary, D.D.M., 2001. Fate of area in the adult brain. Int. J. Dev. Biol. 54, 939–945. midbrain dopaminergic neurons controlled by the engrailed genes. J. Neurosci. 21, Ferri, A.L.M., Lin, W., Mavromatakis, Y.E., Wang, J.C., Sasaki, H., Whitsett, J.A., Ang, S.L., 3126–3134. 2007. Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron Smidt, M.P., Asbreuk, C.H.J., Cox, J.J., Chen, H., Johnson, R.L., Burbach, J.P.H., 2000. A development in a dosage-dependent manner. Development 134, 2761–2769. second independent pathway for development of mesencephalic dopaminergic Griesel, G., Treichel, D., Collombat, P., Krull, J., Zembrzycki, A., van den Akker, W.M.R., neurons requires Lmx1b. Nat. Neurosci. 3, 337–341. Gruss, P., Simeone, A., Mansouri, A., 2006. Sp8 controls the anteroposterior Smidt, M.P., Smits, S.M., Bouwmeester, H., Hamers, F.P.K., van der Linden, A.J.A., patterning at the midbrain–hindbrain border. Development 133, 1779–1787. Hellemons, A.J.C.G.M., Graw, J., Burbach, J.P.H., 2004. Early developmental failure of Goridis, C., Rohrer, H., 2002. Specification of catecholaminergic and serotonergic substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3. neurons. Nat. Rev. 3, 531–541. Development 131, 1145–1155. Hatakeyama, J., Bessho, Y., Katoh, K., Ookawara, S., Fujioka, M., Guillemot, F., Kageyama, R., Smidt, M.P., Burbach, J.P.H., 2007. How to make a mesodiencephalic dopaminergic 2004. Hes genes regulate size, shape and histogenesis of the nervous system by control neuron. Nat. Rev. 8, 21–32. of the timing of neural stem cell differentiation. Development 131, 5539–5550. Smits,S.M.,Burbach,J.P.H.,Smidt,M.P.,2006.Developmentaloriginandfateof Hatakeyama, J., Kageyama, R., 2006. Notch1 expression is spatiotemporally correlated meso-diencephalic dopamine neurons. Prog. Neurobiol. 78, 1–16. with neurogenesis and negatively regulated by Notch1-independent Hes genes in Vernay, B., Koch, M., Vaccarino, F., Briscoe, J., Simeone, A., Kageyama, R., Ang, S.L., 2005. the developing nervous system. Cereb. Cortex 16 (Suppl 1), 132–137. Otx2 regulates subtype specification and neurogenesis in the midbrain. J. Neurosci. Hirata, H., Tomita, K., Bessho, Y., Kageyama, R., 2001. Hes1 and Hes3 regulate 25, 4856–4867. maintenance of the isthmic organizer and development of the mid/hindbrain. Yamauchi, K., Mizushima, S., Tamada, A., Yamamoto, N., Takashima, S., Murakami, F., EMBO J. 20, 4454–4466. 2009. FGF8 signaling regulates growth of midbrain dopaminergic axons by Ishibashi, M., Ang, S.L., Shiota, K., Nakanishi, S., Kageyama, R., Guillemot, F., 1995. inducing semaphorin 3F. J. Neurosci. 29, 4044–4055. Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) Ye, W., Shimamura, K.I., Rubenstein, J.L., Hynes, M.A., Rosenthal, A., 1998. FGF and Shh leads to up-regulation of neural helix–loop–helix factors, premature neurogenesis, signals control dopaminergic and serotonergic cell fate in the anterior neural plate. and severe neural tube defects. Genes Dev. 9, 3136–3148. Cell 93, 755–766. Kageyama, R., Ohtsuka, T., 1999. The Notch-Hes pathway in mammalian neural Zetterström, R.H., Solomin, L., Jansson, L., Hoffer, B.J., Olson, L., Perimann, T., 1997. development. Cell Res. 9, 179–188. Dopamine neuron agenesis in Nurr1-deficient mice. Science 276, 248–250.