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Cerebellar Rhombic Lip Derivatives 4397 Development 126, 4395-4404 (1999) 4395 Printed in Great Britain © The Company of Biologists Limited 1999 DEV2426 The role of the rhombic lip in avian cerebellum development Richard J. T. Wingate* and Mary E. Hatten Laboratory of Developmental Neurobiology, Rockefeller University, 1230 York Avenue, New York, NY 10021-10034, USA *Author for correspondence (e-mail: richard. wingate@kcl. ac. uk) Accepted 3 August; published on WWW 27 September 1999 SUMMARY We have used a combination of quail-chick fate-mapping neurons of the lateral pontine nucleus. DiI-labelling of techniques and dye labelling to investigate the development cerebellum explants reveals that external germinal layer of the avian cerebellum. Using Hoxa2 as a guide for the precursors have a characteristic unipolar morphology and microsurgical construction of quail-chick chimaeras, we undergo an orientated, active migration away from the show that the caudal boundary of the presumptive rhombic lip, which is apparently independent of either glial cerebellum at E6 maps to the caudal boundary of or axon guidance or ‘chain’ formation. rhombomere 1. By fate mapping the dorsoventral axis of rhombomere 1, we demonstrate that granule cell Key words: Granule cell, Lateral pontine nucleus, Quail-chick precursors are generated at the rhombic lip together with chimaera, Cell migration, Hoxa2 INTRODUCTION constriction, or isthmus, that separates these two embryonic vesicles is an important signalling centre and a number of The vertebrate neuraxis is patterned by dorsoventral and studies have demonstrated that genes induced at the isthmus anteroposterior molecular cues into regionally distinct regulate both cerebellum and midbrain development (Lumsden subdivisions each characterised by a different repertoire of and Krumlauf, 1996). However, it is only recently that the neuronal types. In the early neural tube, these complementary boundaries of the anlage have been investigated. By accurately patterning systems can be interpreted in terms of the constructing a fate map according to molecular anatomy, Millet orthogonal axes of a Cartesian co-ordinate system (Lumsden et al. (1996) showed that the posterior boundary of Otx2 and Krumlauf, 1996). However, for CNS structures generated expression indelibly marks the caudal limit of the presumptive later in development, these basic motifs are often obscured by midbrain and hence, by inference, the rostral boundary of the gross morphogenic changes. We have re-examined the cerebellar anlage. Accordingly, targeted mutation of this gene development of one such structure, the cerebellum, in the light leads to a rostral expansion of cerebellum (Acampora et al., of recent molecular evidence indicating that its development 1997). Conversely, a targeted mutation of Hoxa2, the most may be far simpler than previously presumed. In particular, we anterior Hox gene in the hindbrain, results in a caudal have investigated the role of the rhombic lip, which lies at the enlargement of the cerebellum (Gavalas et al., 1997). Does interface between the cerebellum and roofplate, in the Hoxa2 define the caudal limit of presumptive cerebellum? At induction of cerebellar granule neurons. Over half the adult early embryonic stages, there are no structural landmarks brain consists of cerebellar granule cells, which receive mossy delimiting the region that lies between the limits of Hoxa2 and fibre input chiefly from the pons and participate in the relays Otx2 expression (Prince and Lumsden, 1994; Millet et al., that govern Purkinje cell activity and hence cerebellar output. 1996). However, in the later embryo, this domain resolves to a Granule cell development involves two distinct phases of single defined neuromere designated as rhombomere (r) 1. proliferation and migration (Cajal, 1911). Of these, the second The role of dorsoventral patterning in cerebellar phase of cell division in the external granule cell layer (EGL) development, and in particularly the induction of granule cells, and subsequent, radial, inward migration to form the internal is currently unclear: recent molecular data indicate that granule granule cell layer has been extensively investigated (Hatten and cells are a dorsally derived cell population, but this conclusion Heintz, 1995). By comparison, the first phase of precursor is contradicted by fate-map data and classical neuroanatomy. induction leading to the formation of the EGL is poorly Key to understanding this apparent anomaly is determining the understood. The precise anteroposterior and dorsoventral derivatives and dorsoventral origins of the rhombic lip. Various origins of EGL precursors and their mode of migration over authors have argued that granule cells are born exclusively at the pial surface of the cerebellum remain key questions in the rhombic lip and subsequently migrate over the surface of developmental neurobiology. the cerebellum to form the EGL (Schaper,1897, adapted by The cerebellum arises in a region that initially encompasses Harkmark, 1954a; Miale and Sidman, 1961; Altman and Bayer, the boundary between the mesencephalon and the 1978, 1987, 1997; Hynes et al., 1986; Ryder and Cepko, 1994; metencephalon (Hallonet et al., 1990; Millet et al., 1996). The Hatten and Heintz, 1995). This is supported by recent studies 4396 R. J. T. Wingate and M. E. Hatten Fig. 1. (A) Dorsal view of the mesencephalon/metencephalon (mes/met) region of an E2 (stage 10) embryo triple-labelled by in situ hybridisation for Otx2 (blue), Hoxa2 (blue) and Gbx2 (red). Dark-grey shading in the accompanying schematic diagram shows the area from which r1 grafts were derived. (B) Four types of unilateral graft, of varying dorsoventral extent, are represented schematically as transverse sections of r1. showing that a number of granule neuron markers are initially also expressed at the rhombic lip (Hatten et al., 1997). A targeted mutation of one of these genes, the pro-neural atonal homologue, Math-1, leads to a complete loss of granule cells (Ben-Arie et al., 1997). Similarly, dissociation studies show that all rhombic lip derivatives are apparently positive for a granule cell lineage-specific marker, RU49 (Alder et al., 1996). Both Math-1 and RU49 can be induced in presumptive cerebellum by the dorsalising factor, BMP (Alder et al.,1999), Fig. 2. The relationship between r1, r2 and the E6 cerebellum. (A) Ventricular view of an E3 chimaeric hindbrain showing the indicating a role for dorsoventral patterning in granule cell distribution of cells derived from an r2 graft (green) with respect to precursor determination. However, nearly all anatomical rhombomere boundaries (enhanced by Nomarski optics). Scale bars models suggest that the rhombic lip is a caudal structure and in A,C, 200 µm. (B) Lateral view a whole stage 19 chimaera hence under the influence of a very different set of showing r1 derivatives (brown) in the CNS and cranial nerves. Quail anteroposterior patterning cues (Altman and Bayer, 1978, neural crest cells from r1 can be seen in the ophthalmic lobe of the 1985; Avarez Otero et al., 1993; Ryder and Cepko, 1994). trigeminal ganglion (Vo) and ensheathing the oculomotor (III) and Moreover, a precise fate map of the dorsoventral axis of the trochlear (IV) nerves. (C) Ventricular view of an E6 chimaeric metencephalon at E2 shows that granule cells are derived from hindbrain showing the distribution of cells derived from an r2 graft all but the most ventral neural tube (Hallonet and Le Douarin, relative to Hoxa2 expression. (D) Schematic diagrams explaining the 1993). Therefore, whether the rhombic lip is the sole source of dissection and orientation of the flat-mounted cerebellum (cb) and hindbrain (hb): rhombic lip is shaded blue. In all subsequent figures, granule cells remains unclear and, significantly, the proposal anterior is to the top of the page. (E) At E6, the caudal limit of an r1 that rhombic lip is a dorsally derived structure has yet to be graft maps to the caudal limit of the cerebellum. Quail cells can be confirmed. seen in the trigeminal (V) but not the facial/vestibuloacoustic We have used a combination of techniques to assess the (VII/VIII) ganglia. (F) A ventricular view shows negligible caudal precise caudal boundary of the cerebellar anlage at E6 and the spread from r1. Ventral midline, (vml) is left, while the rhombic lip dorsoventral origins of the rhombic lip. These show that the (rl), fringe (f) and roofplate (rp) are to the right. (G) At E7, Hoxa2 is rhombic lip is a dorsally derived structure and the source of excluded from the cerebellum. (H) In the ventricular layer at E6, the EGL precursors. The caudal limit of presumptive cerebellar anterior limit Hoxa2 matches the r1/2 boundary identified by µ territory corresponds to the boundaries between r1 and r2, grafting. Scale bar for F, H, 300 m. suggesting that, at E6, the cerebellar anlage is derived from r1. EGL precursors have a distinctive morphology and mode of MATERIALS AND METHODS early migration. Perhaps surprisingly, granule cell precursors are generated alongside a population of ventrally migrating Quail-chick chimaeras neurons that give rise to the lateral pontine nucleus. This The metencephalon of E2 (Hamburger and Hamilton stages 10 to 11: suggests a possible lineage relationship between two Hamburger and Hamilton, 1951) Japanese quail embryos (Treslow functionally connected, but topographically and Farms, Chester Town, Maryland) was isolated in vitro and treated with phenotypically distinct neuronal populations. dispase (1 mg/ml in L15 for 15 minutes). Mesoderm and ectoderm Cerebellar rhombic lip derivatives 4397 Immunoresearch Laboratories, West Grove, Pennsylvania). Some were vibratome-sectioned at 100 µm. Brains fixed in Dent’s fixative were double-labelled with Q¢PN and the quail-specific neuronal antibody, QN (Tanaka et al., 1990), and FITC-conjugated and Cy3- conjugated secondary antibodies, respectively. BrdU-labelled cells were identified by a monoclonal antibody (Bio-Science Products AG, Emmenbrücke, CH) and a Cy3-conjugated secondary antibody. Quail explants were labelled with the endothelial and quail cell-specific monoclonal antibody, QH1 (Hybridoma Bank) and a FITC- conjugated secondary antibody. PF fixed tissue was cleared in PBS/glycerol (1:10) with 2.
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