Midbrain Patterning

C H A P T E R

3 Midbrain Patterning Isthmus Organizer, Tectum Regionalization, and Polarity Formation H. Nakamura Tohoku University, Sendai, Japan

O U T L I N E 3.1 Function and Development of the Midbrain 3.1.1 Plan of the Vertebrate Brain 3.1.2 Brief Outline of the Midbrain (Mesencephalon) 3.1.3 Development of the Tectum 3.1.4 Optic Tectum as a Visual Center 3.1.4.1 Retinotectal Projection 3.2 Midbrain Regionalization 3.2.1 Gene Expression around the Midbrain at around Stage 10 3.2.2 Midbrain–Hindbrain Boundary Formation 3.2.3 Diencephalon–Mesencephalon Boundary Formation 3.2.4 Dorsal–Ventral Patterning 3.2.4.1 Dorsal Patterning 3.2.4.2 Ventral Patterning 3.2.4.3 Mesencephalic Dopaminergic Neurons

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3.4 Isthmic Organizer and Tectum Polarity 3.4.1 Rostrocaudal Polarity of the Tectum 3.4.2 Positional Specification of the Tectum 3.4.3 Isthmic Organizer and the Rostrocaudal Polarity of the Tectum

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3.5 Conclusion

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metencephalon and myelencephalon. These secondary brain vesicles form the fundamental brain plan. Analysis of gene expression indicated that the prosencephalon is subdivided into several molecular domains called prosomere (Garcia-Lopez et al., 2009; Rubenstein et al., 1994; Shimamura et al., 1995). Rhombencephalon is subdivided into rhombomeres (Garcia-Lopez et al., 2009; Keynes and Lumsden, 1990; Vaage, 1969). No segmentation is recognized in the mesencephalon. The midbrain is thus bordered by prosomere 1 (p1) and rhombomere 1 (r1) rostrally and caudally, respectively. Recently, it has been proposed that r1 be subdivided into

3.1 FUNCTION AND DEVELOPMENT OF THE MIDBRAIN 3.1.1 Plan of the Vertebrate Brain The fundamentals of the brain plan are in the brain vesicles. Primary brain vesicles that are composed of prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain) are formed in the anterior neural tube (Figure 3.1). The prosencephalon is then subdivided into the telencephalon and diencephalon, and the rhombencephalon is subdivided into the

Patterning and Cell Type Specification in the Developing CNS and PNS: Comprehensive Developmental Neuroscience, Volume 1 http://dx.doi.org/10.1016/B978-0-12-397265-1.00020-4

3.3 Isthmus Organizer 3.3.1 Background 3.3.2 Isthmus Organizing Molecule 3.3.3 Isoforms of Fgf8 3.3.4 Other Fgfs and Fgf Receptors 3.3.5 Transduction of Fgf8 Signal 3.3.6 Negative Regulators for Ras–ERK Signaling Pathway 3.3.7 Differential Patterning of Midbrain, Isthmus, and Cerebellum by Fgf8 Signaling

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FgF8 Tel Pro di

Otx2

En2

Mes

Met

Pax3/7

FgF8 Wnt1

Rhomb

Pax6 En1/Pax2

Gbx2 My

FIGURE 3.1 Brain vesicles. Brain vesicles are the plan of the brain of the vertebrate. At the end of the neurula stage, three brain vesicles are formed at the rostral part of the neural tube: prosencephalon, mesencephalon, and rhombencephalon (primary brain vesicles). Prosencephalon is then subdivided into telencephalon and diencephalon, and rhombencephalon is subdivided into metencephalon and myelencephalon (secondary brain vesicles). Fgf8 is expressed in the anterior neural ridge and midbrain–hindbrain boundary (isthmus), and functions as an organizing molecule. Otx2 is expressed in the prosencephalon and mesencephalon, and makes clear boundary with Gbx2, which is expressed in the rhombencephalon. Pax6 is expressed in the prosencephalon. En1 and Pax2 expression first covers the whole mesencephalon but later is confined to the isthmic region. En2 is expressed in the isthmus and mesencephalon. Wnt1 is expressed in the dorsal midline and at the posterior margin of the mesencephalon. Pax3/7 expression covers primordium of the tectum. pro, prosencephalon; mes, mesencephalon; rhomb, rhombencephalon; tel, telencephalon; di, diencephalon; met, metencephalon; my, myelencephalon.

isthmus proper (r0) and r1 proper (Aroca and Puelles, 2005; Garcia-Lopez et al., 2009). Hence, the midbrain abuts r0 posteriorly.

3.1.2 Brief Outline of the Midbrain (Mesencephalon) Dorsoventrally, the midbrain consists of the tectum and the tegmentum. The tectum contains the optic tectum and torus semicircularis. In mammals, the torus semicircularis bulges dorsally as the inferior colliculus. The tectum receives auditory, visual, and somatosensory systems, and maps of the animal’s sensory space are formed in the tectum (Butler and Hodos, 2005; Terashima, 2011). The optic tectum is a primary visual center of the lower vertebrates and receives topographic input from the retina. In mammals, it is called the superior colliculus. The tectum is involved in integrating the spatial aspects of visual and other sensory inputs and in

generating spatially oriented motor responses (Butler and Hodos, 2005; Terashima, 2011). The torus semicircularis receives ascending auditory projections (Butler and Hodos, 2005). The superior colliculus in mammals and the optic tectum in birds and reptiles consist of a laminar structure. The number of laminae differs in mammals and birds: seven in mammals and 16 in birds (Terashima, 2011). The superficial zone receives visual information from the telencephalon or from the retina. The deep layers receive nonvisual input, that is, somatosensory and auditory input in addition to afferents from the cerebellum, substantia nigra, and extrastriate areas of the cerebral cortex in mammals. Afferent fibers from retinal ganglion cells (RGCs) project to the superficial layer in both birds and mammals in a retinotopic manner. Descending efferent outputs are from the deep laminae of the tectum. They project to the motor neurons of the cervical spinal cord, to the pontine nucleus, and to the reticular formation of the medulla (Terashima, 2011). The latter two fibers make tectobulbar tract. Ascending efferent outputs are from the superficial laminae of the tectum and project to the pretectum and tectum. Information projected to the thalamus is relayed to the cerebrum. The midbrain tegmentum lies ventral to the tectum and abuts the isthmus caudally. It contains oculomotor nerve nuclei and nuclei related to visuospatial functions, motor activities, and ascending feedback for the integration and adjustment of motor activities. It also contains ascending and descending fiber systems. Thus, the midbrain tegmentum is a gateway for incoming sensory information and outgoing motor responses to and from the forebrain (Butler and Hodos, 2005).

3.1.3 Development of the Tectum Alar plate of the midbrain rapidly enlarges to form the optic tectum from E2 (embryonic day 2). BrdU (Bromodeoxyuridine) incorporation showed that alar plate cells incorporate BrdU more actively than basal plate cells (Watanabe and Nakamura, 2000). A rostrocaudal gradient of differentiation exists: walls in the rostral part are thicker than in the caudal, and laminar differentiation in the rostral part precedes that in the caudal (LaVail and Cowan, 1971a,b). In the chick, the tectum rotates about 90 between stages 31 and 38 (E7–E12) so that the rostral pole of the tectum comes to the ventral level of the body axis (Goldberg, 1974; Itasaki and Nakamura, 1992). Until E3.5 (stage 21), the tectal wall consists of a simple pseudostratified neuroepithelium where cells are proliferating (LaVail and Cowan, 1971a; Mey and Thanos, 2000; Puelles and Bendala, 1978). Cells undergo

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mitosis when the nuclei are near the ventricular surface, migrate outward, and perform DNA synthesis near the pial surface. Cell proliferation peaks on E5, then declines while some cells divide until E12 (Mey and Thanos, 2000). The mantle layer is formed after E4 (LaVail and Cowan, 1971a; Mey and Thanos, 2000; Puelles and Bendala, 1978); then large numbers of young neurons migrate from the ventricular zone to the mantle layer, and lamination proceeds. The tectal wall thickens remarkably: 200 mm at E6 to about 1 mm at E14 at the rostral part (Mey and Thanos, 2000). The mode of migration of postmitotic neurons for laminar formation of the optic tectum is different from that in the case of the cerebral cortex where ‘insideout’ type migration takes place (Jacobson, 1991), but tectum laminar formation proceeds in a complex way (LaVail and Cowan, 1971b). LaVail and Cowan (1971b) studied tectum laminar formation by autoradiography and showed that three successive waves form the tectum laminae. The first wave occurs between E3 and E5 and forms deeper layers of the SGC (stratum griseum centrale) and SGP (stratum griseum periventriculare). The second migratory wave lasts between E4 and E7 and forms superficial layers, laminae a–g of the SGFS (stratum griseum et fibrosum suerficiale). The cells of the last wave (E6–E8) produce laminae h–j of the SGFS. Consistent results were obtained by labeling tectal premigratory cells by in ovo electroporation of lacZ expression vector (Figure 3.2(a); Sugiyama and Nakamura, 2003). When electroporation was carried out at E3, labeled cells migrated to deep and superficial layers at E12, that is, the SGC, and SAC and laminae a–d of the FGFS, respectively. When electroporation was carried out at E6, labeled cells migrated to laminae h–j of SGFS. Migration pattern change seemed to occur at E5, so tectal neurons that migrate before E5 were regarded as early migratory cells, and those that migrate after E5 were regarded as late migratory cells. Grg1 and Grg4, the Gro/Grg/TLE family that function as transcription repressors by binding to specific transcription factors, are expressed in the early midbrain (E2) of chick embryos, and Grg4 was shown to be involved in polarity formation of the tectum (Sugiyama et al., 2000). Grg4 expression disappears in E4 midbrain, then reexpressed in the midbrain neuroepithelium at E5. Since migration pattern of tectal neurons changes at E5, when Grg4 expression reappears, it was suspected that Grg4 is involved in regulation of tectal cell migration. Misexpression by Grg4 retrovirus vector by electroporation showed that Grg4 misexpressing cells acquired the property of late migratory cells. Consequently, laminae h–j of SGFS is enlarged and lamina g disappeared in the Grg4 misexpressing region. Clonal labeling or misexpression could be achieved by electroporation with retrovirus plasmid vector on virus-resistant chick

E12 Cells from E3 Cells from E6

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FIGURE 3.2 Laminar formation of the tectum. (a) The cells that migrate after E5 split the layers that are formed by cells migrated before E5. At E8, lamina II could be divided into IIu and IId; IId are formed by late migratory cells and later differentiate into laminae h–j of SGFS. (b, c) Clonal analysis of migration of tectal neurons electroporation of plasmid vector of RCAS-AP (b), and of RCAS-Grg4 (c) on virusresistant embryos at E2 and fixed at E8. Left column: immunohistochemistry for alkaline phosphatase (AP) (b) or in situ hybridization for Grg4 (c). Right column: number of AP-positive or Grg4-expressing cells in each lamina. In virus-resistant embryos, misexpression is limited to the descendents of originally transfected cells. AP-positive cells are present throughout all laminae (b), but Grg4-expressing cells are mainly in lamina IId (c). Scale bars: 100 mm. SO, stratum opticum; SGFS, stratum griseum et fibrosum superficiale; SGC, stratum griseum centrale; SFC, stratum fibrosum centrale; SGP, stratum griseum periventriculare; SFP, stratum fibrosum periventriculare; VL, ventricular layer. Reproduced from Sugiyama S and Nakamura H (2003) The role of Grg4 in tectal laminar formation. Development 130: 451–462.

embryos (Figure 3.2(b)and 3.2(c)). When the tectal premigratory cells were labeled clonally with lacZ, the progeny spanned all the layers of the tectum (Figure 3.2(b)). On the other hand, clonal misexpression of Grg4 in tectal premigratory cells showed that Grg4 misexpressing cells preferentially migrated to the layer that will form laminae h–j of the SGFS (Figure 3.2(c)). Treatment with Grg4 morpholino antisense oligonucleotide or N-terminal region of Grg4, which acts as repressor, exerted opposite effects to misexpression of Grg4, that is, decrease of the layer that will form laminae h–j of the SGFS. Both gain

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and loss of function of Grg4 experiments support the idea that Grg4 instructs the tectal postmitotic cells to follow late migratory pathway.

3.1.4 Optic Tectum as a Visual Center 3.1.4.1 Retinotectal Projection In lower vertebrates, the optic tectum functions as the visual center and receives the projection of the RGCs. The retinotectal projection is organized in a precise retinotopic manner, that is, a point of the retina is projected on a target point of the tectum. The temporal–nasal (posterior–anterior) and ventral–dorsal (lateral–medial) axes of the retina are projected as the rostrocaudal (anterior–posterior) and medial–lateral (dorsal–ventral) axes of the contralateral tectum, respectively (Figure 3.3) (Crossland and Uchwat, 1979). The projection pattern shows that the image on the retina is projected as 180 reversed manner on the tectum. Since the image is reversed by the lens, an upright image may be projected on the tectum (Raper and Tessier-Lavigne, 1999). In chick embryos, whole retinal fibers cross the optic chiasm except for a small population of transient fibers and invade the tectum from the rostral pole of the tectum around E6. At that time, the tectum is covered by fibers of the tectobulbar tract that run dorsoventrally and in the future make the SAC (stratum album cenrale). Two fiber Retina

Tectum D

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systems do not touch because cellular migration displaces tectobulbar fibers beneath the surface before retinal fibers arrive (Mey and Thanos, 2000). The retinal fibers extend caudally in the SO (stratum opticum), the superficial layer of the tectum, and make a right turn to arborize. They make arborizations from E10 (Nakamura and O’Leary, 1989). Temporal retinal fibers make arborizations even outside of their target point during E12– E13; the arborizations outside of the target point are pruned and a tight focus of terminal arborizations made by E15 (Nakamura and O’Leary, 1989). Retinal fibers terminate at b, c, d, and f of the SGFS. They never penetrate lamina g, which may be the barrier for the retinal fibers. Early migratory cells populate lamina g, which is not formed when the postmitotic cells are conferred of late migratory character by Grg4 misexpression. In the tectum where lamina g is disrupted, retinal fibers could invade deep into the tectum (Sugiyama and Nakamura, 2003). Arbors of individual RGC are confined to a single retinorecipient lamina (RRL), so studies have been undertaken to learn the mechanisms by which subsets of RGCs project to the specific RRL (Yamagata et al., 2006). Lamina-specific markers were pursued. The most interesting molecule is cadherin 7, which is expressed in lamina c of the SGFS and RGCs that send dendritic arbors to sublamina 4 of the inner plexiform layer. Yamagata et al. showed that cadherin 7-expressing RGCs terminate at lamina c, which may be explained by homophilic adhesion of cadherin 7. This kind of work has just been started.

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3.2.1 Gene Expression around the Midbrain at around Stage 10

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FIGURE 3.3 Retinotectal projection map and Eph–ephrin system. (a) Temporonasal axis of the retina is projected to rostrocaudal axis on the tectum. In the retina, EphA3 is expressed in a gradient as temporal high and nasal low, and ephrin A2 and A5 are expressed in the tectum in a gradient as nasal high and temporal low. Growth cones of the temporal RGCs are repelled by ephrin A2 and A5 and cannot invade the caudal tectum so that temporal retinal fibers terminate at the rostral tectum. Nasal retinal fibers are not repelled and terminate at the caudal tectum. (b) Dorsoventral axis of the retina is projected to ventrodorsal axis of the tectum. EphB2 and B3 are expressed in a gradient in the retina as ventral high and dorsal low, and ephrin B1 is expressed in the tectum in a gradient as ventral high and dorsal low.

In Figure 3.1, expression of the representative genes around the mesencephalic region at around stage 10 of chick embryos is shown. Transcription factors, En1 and En2, which are the homolog of Drosophila segment polarity gene engrailed, are expressed in the isthmus and the mesencephalon (Matsunaga et al., 2000; Patel et al., 1989). In En1 knockout mice, the tectum and the cerebellum are hypomorphic (Wurst et al., 1994). En1 expression commences earlier than En2 and covers the whole of the mesencephalon (Matsunaga et al., 2000). Then its expression regresses and is confined to the isthmic region. Pax2 and Pax5 belong to the same Pax subfamily and contain paired box, octapeptide, and partial homeobox (Mansouri et al., 1996; Walther and Gruss, 1991). Pax2 begins to be expressed earlier than Pax5 both in mouse and chick embryos (Asano and Gruss, 1992; Okafuji et al., 1999). Pax2 expression

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3.2 MIDBRAIN REGIONALIZATION

in chick embryos commences at the 4-somite stage and covers the whole of the mesencephalon (Matsunaga et al., 2000; Okafuji et al., 1999). Pax5 begins to be expressed at around the 10-somite stage. Its expression is high at the isthmic region (Funahashi et al., 1999). In the forebrain down to the diencephalon, Pax6 is expressed (Walther and Gruss, 1991). Pax6 is not expressed in the midbrain. Pax3/7 is expressed in the whole of the tectal region and is thought to be a marker gene for the tectum (Goulding et al., 1991; Jostes et al., 1990). Otx2 is expressed in the anterior neural tube down to the mhb. Gbx2 is expressed in the rhombencephalon (Bouillet et al., 1995). Its expression makes a sharp border to the Otx2 expression (Hidalgo-Sa´nchez et al., 1999; Katahira et al., 2000). Secreted molecules are also expressed. Fgf8/17/18, which fall in the same Fgf subfamily, are expressed in the isthmus region (Maruoka et al., 1998; Ohuchi et al., 2000; Xu et al., 1999). Fgf8 is expressed in the isthmus, posterior to the midbrain. Fgf17 and 18 are expressed a little wider than Fgf8. Wnt1 is expressed at the dorsal midline and at the posterior border of the midbrain (Wilkinson et al., 1987). Shh is expressed in the floor plate (Tanabe and Jessell, 1996). Ephrin A5 is expressed in the posterior tectum (Drescher et al., 1995). Ephrin A2 is expressed in the tectum anlagen from the early stage in a gradient of posterior high and anterior low (Cheng et al., 1995).

3.2.2 Midbrain–Hindbrain Boundary Formation From a very early stage of development, transcription factors Otx2 and Gbx2 are expressed (Bally-Cuif et al., 1995a,b; Bouillet et al., 1995; Shamim and Mason, 1998; Simeone et al., 1992). Otx2 and Gbx2 are homologs of Drosophila orthodenticle (otd) and unplugged (unpg), respectively. In chick and mouse embryos, Otx2 is expressed at the prosencephalon and the mesencephalon. At the gastrulation stage, Otx2 is expressed rostral to the Hensen’s node (Bally-Cuif et al., 1995a,b; Simeone et al., 1992), and Gbx2 is expressed posterior to it (Bouillet et al., 1995; Shamim and Mason, 1998; von Bubnoff et al., 1995). Then, Otx2 expression becomes confined to the prosencephalon down to the metencephalic region, and Gbx2 is expressed in the metencephalic region (Shamim and Mason, 1998; Simeone et al., 1993). The expression domain of Otx2 and Gbx2 overlaps at the mhb region up to this stage, then Otx2 and Gbx2 are expressed, making a clear border at the midbrain– hindbrain boundary (mhb) (Broccoli et al., 1999; Hidalgo-Sa´nchez et al., 1999; Katahira et al., 2000; Millet et al., 1999; Shamim and Mason, 1998). Millet et al. (1996) showed by transplantation experiments in chick embryos that the posterior border of the optic tectum corresponds not to that of the mesencephalic vesicle but to that of the Otx2 expression. They

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showed that the alar plate of the posterior mesencephalic vesicle that does not express Otx2 differentiated into the cerebellum. The results that caudal part of the mesencephalic vesicle at stage 10 differentiates into the cerebellum had already been shown (Hallonet and Le Douarin, 1993; Martinez and Alvarado-Mallart, 1989). For the role of Otx2 in brain regionalization, gain and loss-of-function studies were carried out. Otx2 null mutant mice lack brain regions rostral to rhombomere 3 (r3) (Acampora et al., 1995; Ang et al., 1996; Matsuo et al., 1995). In Otx2 knock-in mice (Broccoli et al., 1999) in which Otx2 expression was driven by En1 promoter, Otx2 was mainly expressed in the dorsal anterior r1. The mesencephalic inferior colliculi enlarged and extended to the posterior. On the other hand, the anterior part of the cerebellum was missing. In chick, Otx2 misexpression changed the fate of metencephalic alar plate to form the complete tectal structure except for SO, which consists of retinal fibers (Katahira et al., 2000). Because the optic tract is in the diencephalon, the retinal fibers may have all innervated the tectum proper. In the region where Otx2 was misexpressed, Gbx2 expression was repressed (Broccoli et al., 1999; Katahira et al., 2000). Endogenous Fgf8 expression was repressed by Otx2, but in the metencephalon, Fgf8 expression was induced adjacent to Otx2; in chick, Fgf8 was induced at the periphery of Otx2 misexpression; and in mice, Fgf8 expression shifted caudally (Broccoli et al., 1999; Katahira et al., 2000). Misexpression of Gbx2 in the midbrain was carried out in mice and chick. In Gbx2 transgenic mice, in which Gbx2 was misexpressed in the posterior mesencephalon under the control of Wnt1 promoter (Millet et al., 1999), Otx2 expression was repressed by Gbx2, and the mice showed an expanded hindbrain and a reduced midbrain at E9.5. Gbx2 misexpression in chick embryos exerted similar effects: anterior shift of the caudal limit of the tectum (Katahira et al., 2000) and repression of Otx2 by Gbx2. In both chick and mice, Fgf8 expression was induced around the Gbx2 misexpression site in the mesencephalon. The results of Otx2 and Gbx2 misexpression showed that ectopic Fgf8 is induced at the interface of Otx2 and Gbx2 expression domain overlapping to Gbx2 expression (Broccoli et al., 1999; Katahira et al., 2000; Millet et al., 1999). The results that Fgf8 is induced at the interface of Otx2 and Gbx2 expression were also obtained by transplantation experiments (HidalgoSa´nchez et al., 1999; Irving and Mason, 1999). Fgf8 was induced when r1 was juxtaposed with prosomere 1 (diencephalon) or with the mesencephalon. Misexpression studies of Otx2 and Gbx2 indicate that repressive interaction of Otx2 and Gbx2 genes defines the posterior limit of the tectum and that at the interface of their expression domains, Fgf8 expression is induced, overlapping with Gbx2 domain.

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3.2.3 Diencephalon–Mesencephalon Boundary Formation The di/mesencephalic boundary formation was mainly studied in the chick and it was shown that it was set through repressive interaction between Pax6 and En1/Pax2 (Araki and Nakamura, 1999; Matsunaga et al., 2000). Pax6 is expressed in the prosencephalon (Walther and Gruss, 1991), and En1 and Pax2 are expressed in the mesencephalon and in the metencephalon. At first, expression of Pax6 and En1/Pax2 is overlapping at the di/mesencephalic boundary (Matsunaga et al., 2000). By stage 10 in the chick, the expression domain of Pax6 and En1/Pax2 abut at the di/mesencephalic boundary (Matsunaga et al., 2000). As will be mentioned in the following section, En can repress Pax6 expression (Araki and Nakamura, 1999; Liu and Joyner, 2001). Pax2 was also shown to repress Pax6 expression (Okafuji et al., 1999). In double mutant mice of Pax2 and Pax5, Pax6 expression extends posteriorly, and the mesencephalon is deleted (Schwarz et al., 1999). In turn, Pax6 was shown to repress En expression. Misexpression of Pax6 in the mesencephalic region repressed expression of En1 and resulted in the posterior shift of the di/mesencephalic boundary (Matsunaga et al., 2000). While Pax6 represses En/Pax2 expression, the swapping of a part of Pax6 with the VP16 active domain or Eh1 repressive domain indicated that Pax6 works as transcriptional enhancer in the di/mesencephalic region (Matsunaga et al., 2000). Thus, it was assumed that repressive interaction of Pax6 and En/Pax2 may determine the position of the di/mesencephalic boundary.

3.2.4 Dorsal–Ventral Patterning 3.2.4.1 Dorsal Patterning Pax3 and Pax7 are expressed in the alar plate of the mesencephalon (Kawakami et al., 1997; Matsunaga et al., 2001). Misexpression of Pax3/7 in the basal plate of the mesencephalon changed its fate to the tectum (Matsunaga et al., 2001). Misexpression of Pax7 in the diencephalon could induce tectum and torus semicircularis, alar plate derivatives of the mesencephalon. The ectopic tectum in the diencephalon was formed through sequential induction of Fgf8, En2, and Pax3/7. It was suggested that Pax3 and Pax7 following Otx2, En1, and Pax2 expression define the tectum region (Matsunaga et al., 2001). Transplantation experiments support the idea; formation of the tectum always coincided with induction/maintenance of Pax7 (Nomura et al., 1998). TALE-homeodomain protein Meis2 is expressed in the tectal anlage, and gain- and loss-of-function experiment of Meis2 showed that Meis2 is sufficient and necessary for tectal development. Unlike Pax3/7, Meis2 initiates

tectal development without inducing the isthmus organizer, but it interacts directly with Otx2 competing with Grg4 corepressor (Agoston and Schulte, 2009). In the spinal cord, it was shown that bone morphogenetic proteins (Bmps) are secreted from the roof plate and function as dorsalizing signals (Lee et al., 1998; Liem et al., 1997). In the roof plate of the chick mesencephalon, Bmp5, 7, and Bmp receptor 1b (BmpR1b) are expressed (Bobak et al., 2009). Bobak et al. (2009) carried out gain- and loss-of-function experiment of BmpR1b by transfecting dominant negative and constitutive active BmpR1b. Mesencephalic alar plate markers were not affected in their experiment. As simultaneous activation of Bmp signaling and blockage of Shh signaling was not sufficient to induce Meis2 expression, Bobak et al. (2009) concluded that Bmp signaling is less important as a dorsalizing morphogen in the mesencephalon. 3.2.4.2 Ventral Patterning Shh is expressed in the notochord and induces a floor plate in the neural tube. Then its expression is induced in the floor plate cells in the entire CNS. It was suggested that Shh acts as a morphogen to induce ventral cell types in the spinal cord, that is, cells that receive a high Shh signal differentiate into the floor plate, cells that receive a medium signal differentiate into motor neurons, and ones that receive a low Shh signal differentiate into some ventral interneurons (reviewed in Jessell and Sanes, 2000; Tanabe and Jessell, 1996). At the dorsal part of the neural tube and adjacent ectoderm, Bmps, which are homologs of Drosophila decapentaplegic, are expressed. Neural crest cells and roof plate cells are induced by Bmps, and after the neural tube closure, Bmps are expressed in the roof plate cells and responsible for the differentiation of the dorsal cell types (Jessell and Sanes, 2000). In the mesencephalon, the alar plate differentiates into the optic tectum and the basal plate differentiates into the tegmentum. The role of Shh in the dorsoventral regionalization in the mesencephalon has been addressed. Application of the Shh signals at the roof plate region of the mesencephalon by transplanting the floor plate or Shh producing cells induced tegmental tissue near the transplant (Nomura and Fujisawa, 2000). Normally, the dorsal tectum receives ventral retinal fibers. In the tectum, in which Shh is expressed ectopically at the dorsal part of the tectum, the dorsal tectum receives dorsal retinal fibers, which normally terminate at the ventral tectum. These results indicate that Shh functions as a morphogen in the mesencephalon. It was also shown in mice, in which Shh was misexpressed by the Wnt1 regulatory element, that it can induce a floor plate-related gene, HNF3, in the dorsal midline of the mesencephalon (Echelard et al., 1993). Misexpression of Shh in a wide region of the mesencephalon by in ovo electroporation resulted in expansion

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of the tegmental region dorsally, and the very small tectum fused with the other side (nontransfected side) of the tectum (Watanabe and Nakamura, 2000). In the tectum-related genes, En1, En2, Pax2, Pax5, and Fgf8, expressions were repressed by Shh. Pax7, a marker gene for the tectum, was also repressed by Shh. Otx2 expression was indifferent to Shh. On the other hand, ventral markers, such as HNF3 and ptc expression, were induced. Ventral cell types were also induced by Shh. Both Isl-1 and Lim1/2 expression regions extended dorsally. Immunohistochemical staining with SC1 antibody, which stains motor neurons, showed that the region of motor neurons also expanded dorsally, and many ectopic motor neuron fibers emerged from the dorsolateral midbrain. The region of the dopamine neurons and serotonergic neurons also extended dorsally. Shh can force cells to enter the differentiation phase (Watanabe and Nakamura, 2000). At E3.5 of chick embryos, cells in the tectal anlagen incorporate BrdU (Bromodeoxyuridine), which indicates that they proliferate extensively. On the other hand, in the region where Shh is transfected by in ovo electroporation, cells that incorporated BrdU reduced remarkably. This fact may explain why the tectal anlage swells but the tegmental region does not. The result that Shh forces cells to differentiate is very interesting because it has been reported that Shh forces neural precursor cells to stay at the proliferation phase (Rowitch et al., 1999). It was also reported that Shh prevents differentiation of cerebellar granule cell precursors and induces a proliferative response (Wechsler-Reya and Scott, 1999). In conclusion, Shh centralizes the mesencephalon. A strong Shh signal may force dorsal mesencephalic cells to differentiate into the tegmentum or floor plate, and a weak signal or other signals emanating from the tegmentum may confer the ventral characteristics to the tectal cells. 3.2.4.3 Mesencephalic Dopaminergic Neurons Recently, development of dopaminergic neurons has attracted attention in relation to Parkinson’s disease. In the early phase of dopaminergic neuron development, conditional knockout of Otx2 under the control of En1 indicated that Otx2 is crucial for determination of dopaminergic fate (Puelles et al., 2004). Then, Lmx1a and Lmx1b cooperate with Foxa2 to specify dopaminergic neuron fate by repressing red nucleus fate, which is characterized by Sim1-Lhxa and Ngn1 expression (Nakatani et al., 2010). Foxa1 may also contribute to this pathway by upregulating Ngn2 expression, which is expressed in the dopaminergic neuron precursors. Foxa1/2 may play important roles in early, middle and late phases of dopaminergic neuron development (Ferri et al., 2007). In the middle phase, Foxa1/2 induces En1 and Nurr1, and in the late phase, Foxa1/2 induces tyrosine

hydroxylase and aromatic L-amino acid decarboxylase (Ferri et al., 2007). From the analysis of double mutant mice of Foxa1 and Foxa2, more dosage of Foxa gene is required for dopaminergic neurons for later phase development than early phase development.

3.3 ISTHMUS ORGANIZER 3.3.1 Background Transplantation of the brain vesicles between quail and chick embryos revealed that midbrain–hindbrain boundary works as an organizer for the tectum and cerebellum. Alvarado-Mallart and Sotelo (1984) transplanted the mesencephalic alar plate to the diencephalon at stage 10 of Hamburger and Hamilton’s (1951) staging series and showed that the transplant kept its original fate and differentiated into the tectum, which receives optic fibers. The alar plate of the diencephalon changes its fate to differentiate into the tectum when transplanted near the isthmus (Nakamura et al., 1986, 1988). In addition, the fate change occurred only when the transplant was completely integrated into the host (Nakamura and Itasaki, 1992). These results suggest that the isthmus secretes some factors that force the tissue to differentiate to the tectum. Thus, the isthmic region had attracted the attention of researchers. Ectopic transplantation of the isthmus to the diencephalon was carried out, and it was found that the transplant induced En and Wnt-1 expression in the surrounding host tissue (Bally-Cuif and Wassef, 1994; Bally-Cuif et al., 1992; Martinez et al., 1991). The tissue, which was induced to express En, differentiated into tectal tissue. On the other hand, transplantation of the isthmus into the rhombencephalon induced En expression in the surrounding tissue and transformed it into the cerebellar tissue (Martinez et al., 1995). Thus, the isthmus has been accepted as the secondary organizer for the tectum and cerebellum (Marin and Puelles, 1994; Martinez et al., 1995).

3.3.2 Isthmus Organizing Molecule Crossley et al. (1996) implanted an Fgf8-soaked bead into the diencephalon and showed that the diencephalon was transformed into a tectum by Fgf8. En and Wnt-1 expression was induced by the Fgf8 bead. Interestingly, the rostrocaudal polarity of the ectopically developed tectum was reversed. The result suggested that Fgf8 is a key isthmus organizing molecule. Subsequent gainof-function studies of Fgf8 in chick and mice and analyses in mutant zebrafish and mice have all shown that Fgf8 is a signaling molecule that emanated from the isthmus and could induce tectal and cerebellar development (Brand et al., 1996; Crossley et al., 1996; Liu et al., 1999;

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(a)

(b)

Fgf8a

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Among several splice isoforms of Fgf8 (Crossley and Martin, 1995; MacArthur et al., 1995a), Fgf8a and Fgf8b were shown to be expressed in the isthmic region by reverse transcription polymerase chain reaction (RT-PCR) (Sato et al., 2001). The difference between Fgf8a and Fgf8b is very subtle; Fgf8b contains 33 additional base pairs (Crossley and Martin, 1995; MacArthur et al., 1995a). But they exert very different effects on mesencephalon–metencephalon development. Misexpression of Fgf8a by in ovo electroporation did not affect expression of most of the mesencephalon– metencephalon-related genes such as Irx2, Pax2, Otx2, and Gbx2, but induced En1 and En2 in the diencephalons, that is, En1 and En2 expression extended anteriorly to the diencephalic region, where Pax6 expression was repressed (Sato et al., 2001). The diencephalic region where En expression was induced changed the fate to the mesencephalon (Figure 3.4(a)). The alar plate differentiated to the tectum, and in the basal plate, the oculomotor nucleus differentiated in the basal plate, from which additional oculomotor nerve trunks came out. Misexpression in mice under the Wnt1 regulation resulted in similar effects (Lee et al., 1997). Misexpression of Fgf8b in chick embryos by in ovo electroporation exerted drastic effects. The presumptive mesencephalon changed its fate and acquired the characteristics of the metencephalon. The alar plate differentiated as the cerebellum; external granular layer and Purkinje cells were differentiated (Figure 3.4(b)). The oculomotor nucleus disappeared, and its nerve trunk was not formed. Fgf8b repressed Otx2 expression, and Gbx2

Exp

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3.3.3 Isoforms of Fgf8

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Martinez et al., 1999; Meyers et al., 1998; Reifers et al., 1999; Shamim et al., 1999). Wnt1 is expressed in the most caudal part of the mesencephalon abutting the expression of Fgf8 and is one of the candidate molecules of the isthmus organizer. Wnt1knockout mice show defects in the midbrain and hindbrain (McMahon and Bradley, 1990; Thomas and Capecci, 1990). But misexpression of Wnt1 in chick embryos exerted weak effects – increase in uptake of BrdU, weak induction of Fgf8 and En2 (Sugiyama et al., 1998) – and caused enlargement of the tectum and rhombic lip (Matsunaga et al., 2001). Thus, Wnt signaling plays a role in growth acceleration of the tectum. Limx1b is expressed in the mesencephalon overlapping with Wnt1 and can induce Wnt1 expression (Adams et al., 2000). Limx1b repressed Fgf8 expression cell autonomously but induced Fgf8 non-cell autonomously via Wnt1. Thus, it was suggested that interaction among Fgf8, Limx1b, and Wnt1 contributes to patterning the Fgf8 expression (Figure 3.1).

DN-Ras

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Tect Ect

FIGURE 3.4 Organizing activity of Fgf8. (a, b) Misexpression of Fgf8a changes the fate of the diencephalon to mesencephalon. Righthand side is the experimental side, and the tectum (tect) is enlarged in expense of the diencephalon (di). (b) is a coronal section of (a). (c, d) Misexpression of Fgf8b changes the fate of the mesencephalon to the metencephalon. In place of the tectum, cerebellum differentiated ectopically in the mesencephalon (cer ect) where external granular layer and Purkinje cells differentiated. (d) is a sagittal section indicated on (c). (e, f) Disruption of Ras–ERK signaling by the dominant negative form of Ras resulted in tectum (tect ect) differentiation in place of the cerebellum. (f) is a transverse section indicated on (e). tel, telencephalon; tect, tectum; cont, control side; exp, experimental side; cer ext, ectopic cerebellum in the mesencephalon; cer, cerebellum proper; tect ect, ectopic tectum in the metecephalon (reproduced from Sato et al. (2001) (a, b); Sato and Nakamura (2004) (c)).

and Irx2 expression extended rostrally up to the presumptive diencephalic region. Thus, the region where Gbx2 and Irx2 expression is induced may have changed its fate to differentiate into the cerebellum. En2 was so sensitive to Fgf8 that it was induced in the diencephalon by either Fgf8a or Fgf8b, which may explain the transformation of the fate of the presumptive diencephalon to the mesencephalon. Misexpression of Fgf8b in transgenic mice in which Fgf8b is misexpressed under Wnt1 regulation exerted similar effects on downstream gene expression (Liu et al., 1999). Although Fgf8a and Fgf8b exert different effects, quantitative experiments indicated that the type difference could be attributable to the difference in strength of the signal (Sato et al., 2001). Electroporation with 1/ 100 concentration of Fgf8b expression vector exerted

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Fgf8a-type effects. It was also shown in vitro that Fgf8b has stronger transformation activity (MacAuthur et al., 1995b). Structural analysis by surface plasmon resonance showed that Fgf8b binds more intimately to c isoforms of Fgf receptors 1–3 (FGFR1-3) according to the additional 11 amino acids in Fgf8b, phenylalanine 32 (F32) being the most important among them (Olsen et al., 2006). Mutation of F32 to alanine (F32A) reduced the affinity to FGFR, and the mutation functionally converted Fgf8b to Fgf8a; misexpression of this mutant Fgf8F32A exerted effects similar to those of Fgf8a (Olsen et al., 2007). These results together with that of Fgf8bbead implantation in the diencephalon, in which cerebellar structure differentiated in the center and the tectum in the periphery (Martinez et al., 1999), suggest that the region that is exposed to a strong Fgf8 signal may differentiate into the cerebellum and the region where the Fgf8 signal is weak may differentiate into the tectum. Indeed, in normal development, Fgf8 mRNA is expressed in the region of the presumptive cerebellum.

3.3.4 Other Fgfs and Fgf Receptors In the isthmic region, Fgf17 and Fgf18 are also expressed but in broader domains than Fgf8 including the midbrain and cerebellar territory (Maruoka et al., 1998; Ohuchi et al., 2000; Xu et al., 2000). The biochemical properties of Fgf8b, 17b, and 18 appear to be similar based on in vitro assays (Xu et al., 1999). Fgf8 regulates Fgf17 and Fgf18 as Fgf8b-soaked beads induce Fgf18 in explants (Liu et al., 2003), and expression of both genes is rapidly lost in Fgf8 mes/r1-specific conditional mutants (Chi et al., 2003). But, Fgf18 misexpression by electroporation does not affect Fgf8 expression (Chen et al., 2009; Picker et al., 1999). Misexpression of Fgf17/18 in chicks using electroporation induces expansion of the midbrain and alteration of marker genes similar to Fgf8a, not to Fgf8b (Liu et al., 2003), and disruption of Fgf17 in mice results in a decrease in the midbrain (Xu et al., 2000), in addition to a late reduction in proliferation of the medial cerebellum (vermis). Fgf18 mutant mice do not show an abnormal phenotype in the midbrain or cerebellum (Liu et al., 2002; Ohbayashi et al., 2002). Taking these data into consideration, Fgf8 may play most crucial role as the organizing molecule by collaborating with both Fgf17 and Fgf18. Only FgfRI is expressed near the isthmus (Liu et al., 2003; Walshe and Mason, 2000). Consistent with this, Fgf8b represses FgfR2/3, but not Fgf RI, in mouse brain explants (Liu et al., 2003), and FgfR3 is upregulated in zebrafish ace mutants (point mutation in Fgf8) (Sleptsova-Friedrich et al., 2001). Furthermore, conditional inactivation of Fgf RI in the mouse mes/r1 at E9 results in loss of the posterior midbrain and anterior cerebellum (Trokovic et al., 2003). In contrast, a similar loss

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of Fgf8 leads to death of the entire midbrain and r1 (Chi et al., 2003), suggesting redundancy of Fgf receptors.

3.3.5 Transduction of Fgf8 Signal Next, the question arises as to how the Fgf8 signal is transduced to organize cerebellar differentiation. Fgf signal is received by a receptor tyrosine kinase, whose signal is mainly transduced through the Ras–ERK signaling pathway (reviewed by Katz and McCormick, 1997; Rommel and Hafen, 1998). Indeed, ERK is activated around the isthmus where Fgf8 is expressed, which was shown immunohistochemically with an antidiphosphorylated ERK (dpERK) antibody (Sato and Nakamura, 2004). So, disrupting the Ras–ERK signaling pathway by misexpression of a dominant negative form of Ras (DN-Ras) was carried out. Misexpression of DN-Ras changed the property of the metencephalon to that of the mesencephalon (Figure 3.4(c)); Gbx2 expression in the metencephalon was repressed, and Otx2 expression was induced in the metencephalon. Finally, a tectum differentiated in place of the cerebellum (Sato and Nakamura, 2004). It was suggested that Irx2 is phosphorylated by ERK and be involved in cerebellar differentiation (Matsumoto et al., 2004). Thus, a strong Fgf8 signal activates the Ras–ERK signaling pathway to organize cerebellar differentiation.

3.3.6 Negative Regulators for Ras–ERK Signaling Pathway Fgf8 induces negative regulators of the Ras–ERK signaling pathway such as Sprouty2, Sef, and MKP3 in the isthmus (Chambers and Mason, 2000; Echevarria et al., 2005; Fu¨rthauer et al., 2002; Hacohen et al., 1998; Kawakami et al., 2003; Suzuki-Hirano et al., 2005; reviewed in Mason et al., 2006), by which activated ERK is down regulated in the presumptive cerebellar region. Expression of Sprouty2 is always overlapping to Fgf8 in the isthmus and in the anterior neural ridge (Chambers and Mason, 2000; Echevarria et al., 2005; Suzuki-Hirano et al., 2005). Misexpression of Fgf8 rapidly induced Sprouty2, but Sprouty2 downregulated ERK phosphorylation (Suzuki-Hirano et al., 2005, 2010). Misexpression of Sprouty2 exerted similar effects as that of DN-Ras. After Sprouty2 misexpression, Gbx2 expression was repressed and Otx2 expression was induced in the metencephalon; as a result, the tectum differentiated in place of the cerebellum (Suzuki-Hirano et al., 2005). On the other hand, misexpression of dominant negative form of Sprouty2 (DN-Sprouty2) kept ERK activated around the isthmus. After misexpression of DN-Sprouty2, posterior limit of the tectum shifted anteriorly (Suzuki-Hirano et al., 2005). The results indicate that Ras–ERK signaling should be regulated strictly for

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proper mesencephalic and metencephalic regionalization. If the signal flows too much, the mes/metencephalic boundary shifts anteriorly, and if the signal flows less, the mes/metencephalic boundary shifts posteriorly.

3.3.7 Differential Patterning of Midbrain, Isthmus, and Cerebellum by Fgf8 Signaling It is very interesting that distinct structures such as midbrain, isthmus, and cerebellum differentiate in response to a single organizing signal emanating from the isthmus. Analysis of Fgf8–Ras–ERK signaling and its negative regulators has been undertaken to answer the question. Fgf8 signaling activates the Ras–ERK pathway and at the same time, induces its negative regulators. For cerebellar differentiation, strong Fgf8 signaling activates the Ras–ERK pathway. But activated ERK would soon be downregulated (dephosphorylated) in the metencephalon in the course of cerebellar differentiation. In the mesencephalon, which had been misexpressed with Fgf8b and would differentiate to the cerebellum, ERK had been activated at first but was soon dephosphorylated. Continuous activation of ERK by misexpression of Fgf8b and DN-Sprouty2 in chick embryos was carried out under the assumption that activated ERK should be downregulated for cerebellar differentiation (Suzuki-Hirano et al., 2010). Mesencephalon did not transdifferentiate into the cerebellum but differentiated as the tectum. When Dn-Sprouty2 expression was turned off by tetracycline responding system after misexpression of Fgf8b and DN-Sprouty2, mesencephalon transdifferentiated into the cerebellum. These results indicate that Ras–ERK signaling should be activated once and that it should be soon downregulated by negative regulators for cerebellar differentiation. The effects exerted by DN-Mkp3 on ERK phosphorylation level in the isthmus are not as strong as those of DN-Sprouty2, which indicates that Sprouty2 is a stronger downregulator on the Ras–ERK pathway than Mkp3. After Fgf8b misexpression by electroporation in chick embryos, Sprouty2 was induced to overlap Fgf8, but Mkp3 was induced at the place where ERK activity is high. When Fgf8b and DN-Mkp3 were misexpressed, ERK phosphorylation level in the mesencephalon remained high, but dephosphorylation of ERK occurred in the posterior mesencephalon and the isthmus, perhaps due to Sprouty2, which was induced by Fgf8. After misexpression of Fgf8b and DN-Mkp3, the mesencephalon may have transdifferentiated into the isthmus because a new Fgf8 expression line appeared in the mesencephalon, and along the line, nerve fibers that resemble the trochlear nerve, that is, nerve fibers that go toward dorsal, appeared. The results indicate that after activation of the Ras–ERK pathway, ERK is differentially

downregulated by negative regulators, the isthmus differentiates in a region where ERK activity is high, and the cerebellum differentiates in a region where ERK activity is much more downregulated. Studies in mice have indicated that the duration of Fgf8 signaling and strength of Ras–ERK signaling may be responsible for differential patterning. Inhibition of Fgf8 signaling in mesencephalon to rhombomere 1 (r1) was carried out by conditional misexpression of Sprouty2 (Basson et al., 2008). Mice that misexpress Sprouty2 in mesencephalon-r1 showed increase in cell death in the anterior mesencephalon and cell fate change of the posterior mesencephalon to that of the anterior mesencephalon, that is, inferior colliculus was missing though superior colliculus differentiated in the posterior mesencephalon. In the r1, vermis was affected by conditional Sprouty2 misexpression (Basson et al., 2008). Temporal conditional ablation of Fgf8 was carried out, and it was shown that prolonged Fgf8 expression is required for the structures closer to the isthmus to form (Sato and Joyner, 2009). In mice of Fgf8-E8.5 CKO (treatment of tamoxifen at E8.5 and functional ablation of Fgf8 at E10.5), vermis and inferior colliculus were missing, whereas in mice of Fgf8-E9.5 CKO, anterior vermis and inferior colliculus were missing. In mice of Fgf8-E10.5 CKO, inferior colliculus was slightly smaller than the control, and only anterior most vermis was missing.

3.4 ISTHMIC ORGANIZER AND TECTUM POLARITY 3.4.1 Rostrocaudal Polarity of the Tectum Retinotectal projection is organized in a retinotopic manner and can be manipulated experimentally, so it has been a model system for the study of neuronal circuit formation. Pioneer work was carried out by Sperry (1963). He took advantage of the fact that severed optic nerve can regenerate in some fish and amphibian restoring vision. Sperry cut the optic nerve of the frog and rotated the eye 180 . After the nerve had regenerated, the frog behaved as if the vision had been reversed. When the bait was in the front, the frog jumped back and it turned right when the bait was at the left, which suggested that the growing axon projected to the same target that the original axon had projected. From eye rotation experiments in frogs, Sperry (1963) proposed the chemoaffinity theory, where the target cells and growing fibers carry some kind of individual chemical tags that distinguish them from other sets. Later, it was proposed that a gradient of guidance molecules along orthogonal axes of the retina and tectum exists. As examples of such molecules, an EphA3 gradient along the temporonasal and EphB2/B3 gradient along the ventrodorsal axes of

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the retina, and an ephrin A2/A5 gradient along the caudorostral and ephrin B1 gradient along the ventrodorsal axes of the tectum have been unraveled (Figure 3.3). Below the anteroposterior axis formation of the tectum in relation to the isthmic organizer is discussed. For the rostrocaudal polarity formation, sophisticated and successful series of works had been done in Bonhoeffer’s lab. A carpet of narrow stripes that were made by alternative arrangement of rostral and caudal tectal membrane fragments were made, and a strip of retina was explanted on the carpet (Walter et al., 1987a). Fibers from the explant were confronted with rostral and caudal tectal membrane fragments. Temporal retinal fibers extended only on the rostral tectal membranes, whereas nasal retinal fibers extended on both caudal and rostral tectal membranes (Walter et al., 1987a). Preference of the temporal retinal fibers for the rostral tectal membrane disappeared when the stripe had been prepared after heat treatment of the tectal membrane fragments. It was concluded that the preference of temporal retinal fibers for the rostral tectal membrane was due to repulsive signal from the caudal tectal membrane to the temporal retinal fibers (Walter et al., 1987a,b). In an extension of this line, a repulsive molecule that is expressed in the tectum in a gradient of caudal high and rostral low was cloned and named RAGS, repulsive axon guidance signal. RAGS is a GPI anchored protein and shows sequence homology to ligands for receptor tyrosine kinases of the Eph subfamily. On the other hand, Flanagan and his coworkers cloned ELF-1 in a search of the ligand for Eph family receptors, Mek4 and Sek. As ELF-1 was expressed in a gradient in the tectum, caudal high and rostral low, and Mek4 was expressed in the retina in a gradient of temporal high and nasal low, they speculated that the Mek4– ELF system may contribute to the retinotectal projection map. They showed that Mek4 expressing retinal fibers were repulsed by ELF-1 in vitro and in vivo. Later, Eph ligands and receptors were systematically renamed. Now RAGS is called as ephrin A5, and ELF-1 is called as ephrin A2. Mek4 is called as EphA3 (Figure 3.3). For the retinotectal projection along the rostrocaudal axis of the tectum, retinal fibers that express EphA3 are repulsed by ephrin A2 and A5 and project to the rostral tectum (Nakamoto et al., 1996). Those that express more EphA3 project to more rostral tectum (Figure 3.3(a)).

3.4.2 Positional Specification of the Tectum Positional specification of the tectum is the prerequisite for proper retinotectal projection. At an early stage of the tectum development, En2 is expressed in a caudorostral gradient (Patel et al., 1989). After reversion of the rostrocaudal axis of the tectum primordium before the

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20-somite stage in chick embryos, En2 expression pattern, histogenesis, and retinal projection along the rostrocaudal axis were all regulated as the host pattern (Alvarado-Mallart et al., 1990; Ichijo et al., 1990; Itasaki and Nakamura, 1992; Itasaki et al., 1991; Matsuno et al., 1991). Thus, En2 had been regarded as the molecule that was responsible for the rostrocaudal specification. Involvement of En2 in tectum rostrocaudal polarity formation was further confirmed by heterotopic transplantation of the mesencephalic alar plate to the diencephalon and by misexpression of En. When the mesencephalic alar plate of a chick embryo was transplanted to the diencephalon at stage 10, the transplant kept its original fate and differentiated as the tectum in the ectopic place (Alvarado-Mallart and Sotelo, 1984; Nakamura, 1990). But En2 expression pattern became a mirror pattern to the host, and retinal projection to the ectopic tectum also became mirror image to that to the tectum proper (Itasaki and Nakamura, 1992; Itasaki et al., 1991). On the other hand, in the transplantation of E2 tectal primordium to the E3 diencephalon, the En expression pattern was kept as the original pattern, caudal high and rostral low, and the caudal part of the transplant received nasal retinal fibers (Itasaki and Nakamura, 1992; Itasaki et al., 1991). The results indicated that En conferred posterior characteristics to the tectum. This possibility had been challenged by misexpression of En by retrovirus vector in the rostral part of the tectum (Friedman and O’Leary, 1996; Itasaki and Nakamura, 1996; Logan et al., 1996). En was misexpressed by the retrovirus vector, and retinotectal projection was examined. Nasal retinal fibers arborized ectopically at the En-misexpressing sites. When so many cells were infected with the virus, temporal retinal fibers could not enter the tectum. En1 and En2 exerted identical effects. Since it was shown that ectopic En induced ephrin A2 and ephrin A5 (Logan et al., 1996; Shigetani et al., 1997), it was indicated that rostrocaudal polarity of the tectum may be first established by En2, and the ephrinA2/A5 gradient may be established according to the En2 expression pattern.

3.4.3 Isthmic Organizer and the Rostrocaudal Polarity of the Tectum The tectum primordium has plasticity in its rostrocaudal axis before 20-somite stage, and a rostrocaudal axis of the transplant that is reversed of the axis is regulated as the host pattern (Ichijo et al., 1990; Itasaki et al., 1991; Matsuno et al., 1991). Since En2 and Fgf8 were shown to be in a positive feedback loop for their expression (Funahashi et al., 1999), Fgf8 may play a role in regulation of the rostrocaudal polarity of the reversed tectum by regulating En2 expression. When mesencephalic alar plate was ectopically transplanted to the diencephalon,

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FIGURE 3.5 Schematic drawing to show regionalization in response to Fgf8 signal A: at late neurula stages, Otx2 is expressed in the presumptive prosencephalon, mesencephalon, and anterior rhombencephalon, and Gbx2 is expressed in the rhombencephalon (a). Their expression overlaps at the isthmic region. Expression of Fgf8 is induced in r0 and r1 at the interface of Otx2 and Gbx2 expression overlapping to Gbx2 expression (a). The region where Fgf8 is expressed receives a strong Fgf8 signal, and the Ras–ERK signaling pathway is activated to organize cerebellar differentiation. (b) By repressive interaction between Otx2 and Gbx2, their expression domains clearly abut at the isthmus. ERK activity is downregulated by negative regulators. In r1, ERK activity is downregulated to differentiate into cerebellum. In the isthmic region (r0), ERK activity is slightly higher than the r1 region and the region differentiates into the isthmus. In the mesencephalon, ERK is in a gradient, posterior high and anterior low, which may contribute to the tectum polarity formation.

rostrocaudal polarity of the transplant was reversed (Itasaki and Nakamura, 1992; Itasaki et al., 1991). This may be caused by the Fgf8 signal emanating from the anterior neural ridge (Figure 3.1). In normal development, the Fgf8 signal may at first determine the fate of the mesencephalon and metencephalon and then regulate the rostrocaudal polarity of the tectum for proper retinotectal projection.

3.5 CONCLUSION The midbrain and hindbrain differentiate in response to the isthmus organizing signal (Figure 3.5). From the very early stages of development, Otx2 is expressed in

the presumptive forebrain and midbrain region, and Gbx2 is expressed in the hindbrain. Otx2 and Gbx2 expression prepattern the midbrain and hindbrain region, and Fgf8 expression is induced at the boundary of Otx2–Gbx2 expression, overlapping Gbx2 expression. Consequently, Fgf8 is expressed in the r1, which receives a strong Fgf8 signal and its alar plate differentiates into the cerebellum. The posterior mesencephalon would also receive a rather strong Fgf8 signal, but owing to Otx2, the alar plate of the posterior mesencephalon differentiates into the tectum. Fgf8 activates Ras–ERK signaling and induces its negative regulators. Negative regulators set differential ERK activation level, and the tectum, isthmus, and cerebellum may differentiate according to the ERK activation level. In the next step, the Fgf8 signal may regulate rostrocaudal polarity of the tectum by maintaining an En2 gradient, which may in turn translate into an ephrin A2 or ephrin A5 gradient. This chapter is related to Chapters 11, 23, ephrin/Eph117, ephrins, and Eph receptors.

References Acampora, D., Mazan, S., Lallemand, Y., et al., 1995. Forebrain and midbrain regions are deleted in Otx2 / mutants due to a defective anterior neuroectoderm specification during gastrulation. Development 121, 3279–3290. Adams, K.A., Maida, J.M., Golden, J.A., Riddle, R.D., 2000. The transcriptional factor Lmx1b maintains Wnt1 expression within the isthmic organizer. Development 127, 1857–1867. Agoston, Z., Schulte, D., 2009. Meis2 competes with the Groucho corepressor Tle4 for binding to Otx2 and specifies tectal fate without induction of a secondary midbrain–hindbrain boundary organizer. Development 136, 3311–3322. Alvarado-Mallart, R.M., Martinez, S., Lance-Jones, C., 1990. Pluripotentiality of the 2-day-old avian germinative neuroepithelium. Developmental Biology 139, 75–88. Alvarado-Mallart, R.-M., Sotelo, C., 1984. Homotopic and heterotopic transplantations of quail tectal primordia in chick embryos: Organization of the retinotectal projections in the chimeric embryos. Developmental Biology 103, 378–398. Ang, S.-L., Ou, J., Rhinn, M., et al., 1996. A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 122, 243–252. Araki, I., Nakamura, H., 1999. Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate. Development 126, 5127–5135. Aroca, P., Puelles, L., 2005. Postulated boundaries and differential fate in the developing rostral hindbrain. Brain Research. Brain Research Reviews 49, 179–190. Asano, M., Gruss, P., 1992. Pax-5 is expressed at the midbrain– hindbrain boundary during mouse development. Mechanisms of Development 39, 29–39. Bally-Cuif, L., Alvarado-Mallart, R.M., Darnell, D.K., Wassef, M., 1992. Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon. Development 115, 999–1009. Bally-Cuif, L., Cholley, B., Wassef, M., 1995a. Involvement of Wnt-1 in the formation of the mes/metencephalic boundary. Mechanisms of Development 53, 23–34.

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3.5 CONCLUSION

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