Temporal identity transition in the avian cerebellar rhombic lip

The rhombic lip is a discrete strip of neuroepithelium bordering the roofplate of the fourth ventricle, which gives rise to a defined sequence of migratory neuronal derivatives. In rhombomere 1 of the chick, early born cells give rise to post-mitotic hindbrain nuclei, while later derivatives comprise of cerebellar granule cell precursors, a unique proliferative, migratory precursor population that forms the external granule cell layer. We have examined the temporal specification of these two populations using a heterochronic grafting strategy, in ovo. When transplanted into younger neural tube, rhombic lip cells maintain their characteristic molecular markers and migrate into the hindbrain. Granule cell precursor derivatives of late grafts are, in addition, able to exploit neural crest streams to populate the branchial arches. Within the neural tube, derivatives of early and late rhombic lip progenitors display patterns of migration and process extension, characterised by specific trajectories and targets, which are consistent with their temporal origin. However, the normal temporal progression of cell production is disrupted in grafted progenitors: transplanted early rhombic lip fails to subsequently produce granule cell precursors. This indicates that, while the behaviour of derivatives is intrinsically specified at the rhombic lip, the orderly temporal transition in cell type production is dependent on extrinsic cues present only in the later embryo.     

Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum

The rhombic lip (RL) is an embryonic proliferative neuroepithelium that generates several groups of hindbrain neurons. However, the precise boundaries and derivatives of the RL have never been genetically identified. We use beta-galactosidase expressed from the Math1 locus in Math1-heterozygous and Math1-null mice to track RL-derived cells and to evaluate their developmental requirements for Math1. We uncover a Math1-dependent rostral rhombic-lip migratory stream (RLS) that generates some neurons of the parabrachial, lateral lemniscal, and deep cerebellar nuclei, in addition to cerebellar granule neurons. A more caudal Math1-dependent cochlear extramural stream (CES) generates the ventral cochlear nucleus and cochlear granule neurons. Similarly, mossy-fiber precerebellar nuclei require Math1, whereas the inferior olive and locus coeruleus do not. We propose that Math1 expression delimits the extent of the rhombic lip and is required for the generation of the hindbrain superficial migratory streams, all of which contribute neurons to the proprioceptive/vestibular/auditory sensory network.     

The zebrafish cerebellar rhombic lip is spatially patterned in producing granule cell populations of different functional compartments

The upper rhombic lip, a prominent germinal zone of the cerebellum, was recently demonstrated to generate different neuronal cell types over time from spatial subdomains. We have characterized the differentiation of the upper rhombic lip derived granule cell population in stable GFP-transgenic zebrafish in the context of zebrafish cerebellar morphogenesis. Time-lapse analysis followed by individual granule cell tracing demonstrates that the zebrafish upper rhombic lip is spatially patterned along its mediolateral axis producing different granule cell populations simultaneously. Time-lapse recordings of parallel fiber projections and retrograde labeling reveal that spatial patterning within the rhombic lip corresponds to granule cells of two different functional compartments of the mature cerebellum: the eminentia granularis and the corpus cerebelli. These cerebellar compartments in teleosts correspond to the mammalian vestibulocerebellar and non-vestibulocerebellar system serving balance and locomotion control, respectively. Given the high conservation of cerebellar development in vertebrates, spatial partitioning of the mammalian granule cell population and their corresponding earlier-produced deep nuclei by patterning within the rhombic lip may also delineate distinct functional compartments of the cerebellum. Thus, our findings offer an explanation for how specific functional cerebellar circuitries are laid down by spatio-temporal patterning of cerebellar germinal zones during early brain development.     

SMAD4 is essential for generating subtypes of neurons during cerebellar development

Cerebellum development involves the coordinated production of multiple neuronal cell types. The cerebellar primordium contains two germinative zones, the rhombic lip (RL) and the ventricular zone (VZ), which generate different types of glutamatergic and GABAergic neurons, respectively. What regulates the specification and production of glutamatergic and GABAergic neurons as well as the subtypes for each of these two broad classes remains largely unknown. Here we demonstrate with conditional genetic approaches in mice that SMAD4, a major mediator of BMP and TGFβ signaling, is required early in cerebellar development for maintaining the RL and generating subsets of RL-derived glutamatergic neurons, namely neurons of the deep cerebellar nuclei, unipolar brush cells, and the late cohort of granule cell precursors (GCPs). The early cohort of GCPs, despite being deficient for SMAD4, is still generated. In addition, the numbers of GABAergic neurons are reduced in the mutant and the distribution of Purkinje cells becomes abnormal. These studies demonstrate a temporally and spatially restricted requirement for SMAD4 in generating subtypes of cerebellar neurons.     

Role of Pax6 in development of the cerebellar system.

Post-mitotic neurons generated at the rhombic lip undertake long distance migration to widely dispersed destinations, giving rise to cerebellar granule cells and the precerebellar nuclei. Here we show that Pax6, a key regulator in CNS and eye development, is strongly expressed in rhombic lip and in cells migrating away from it. Development of some structures derived from these cells is severely affected in Pax6-null Small eye (Pax6(Sey)/Pax6(Sey)) embryos. Cell proliferation and initial differentiation seem unaffected, but cell migration and neurite extension are disrupted in mutant embryos. Three of the five precerebellar nuclei fail to form correctly. In the cerebellum the pre-migratory granule cell sub-layer and fissures are absent. Some granule cells are found in ectopic positions in the inferior colliculus which may result from the complete absence of Unc5h3 expression in Pax6(Sey)/Pax6(Sey) granule cells. Our results suggest that Pax6 plays a strong role during hindbrain migration processes and at least part of its activity is mediated through regulation of the netrin receptor Unc5h3.     

Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system

Netrin 1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. Here we used tissue explants to study the action of netrin 1 in the migration of several cerebellar and precerebellar cell progenitors. We show that netrin 1 exerts a strong chemoattractive effect on migrating neurons from the embryonic lower rhombic lip at E12-E14, which give rise to precerebellar nuclei. Netrin 1 promotes the exit of postmitotic migrating neurons from the embryonic lower rhombic lip and upregulates the expression of TAG-1 in these neurons. In addition, in the presence of netrin 1, the migrating neurons are not isolated but are associated with thick fascicles of neurites, typical of the neurophilic way of migration. In contrast, the embryonic upper rhombic lip, which contains tangentially migrating granule cell progenitors, did not respond to netrin 1. Finally, in the postnatal cerebellum, netrin 1 repels both the parallel fibres and migrating granule cells growing out from explants taken from the external germinal layer. The developmental patterns of expression in vivo of netrin 1 and its receptors are consistent with the notion that netrin 1 secreted in the midline acts as chemoattractive cue for precerebellar neurons migrating circumferentially along the extramural stream. Similarly, the pattern of expression in the postnatal cerebellum suggests that netrin 1 could regulate the tangential migration of postmitotic premigratory granule cells. Thus, molecular mechanisms considered as primarily involved in axonal guidance appear also to steer neuronal cell migration.     

Direct imaging of in vivo neuronal migration in the developing cerebellum.

The upper rhombic lip (URL), a germinal zone in the dorsoanterior hindbrain, has long been known to be a source for neurons of the vertebrate cerebellum. It was thought to give rise to dorsally migrating granule cell precursors (Figure 1e); however, recent fate mapping studies have questioned the exclusive contributions of the URL to granule cells. By taking advantage of the clarity of the zebrafish embryo during the stages of brain morphogenesis, we have followed the fate of neuronal precursor cells generated within the upper rhombic lip directly. Combining a novel GFP labeling strategy with in vivo time-lapse imaging, we find, contrary to the former view, that most URL-descendants migrate anterior toward the midhindbrain boundary (MHB) and then course ventrally along the MHB (Figure 1f). As the migrating neuronal precursors reach the MHB, they form ventrally extending projections, likely axons, and continue ventral migration to settle outside of the cerebellum, in the region of the ventral brainstem. Thus, we define a new pathway for URL-derived neuronal precursor cells consistent with the recent fate maps. In addition, our results strongly suggest that the MHB plays a crucial role, not only in induction and patterning of the cerebellar anlage, but also in organizing its later morphogenesis by influencing cell migration.     

The engrailed homeobox genes are required in multiple cell lineages to coordinate sequential formation of fissures and growth of the cerebellum

The layered cortex of the cerebellum is folded along the anterior-posterior axis into lobules separated by fissures, allowing the large number of cells needed for advanced cerebellar functions to be packed into a small volume. During development, the cerebellum begins as a smooth ovoid structure with two progenitor zones, the ventricular zone and upper rhombic lip, which give rise to distinct cell types in the mature cerebellum. Initially, the cerebellar primordium is divided into five cardinal lobes, which are subsequently further subdivided by fissures. The cellular processes and genes that regulate the formation of a normal pattern of fissures are poorly understood. The engrailed genes (En1 and En2) are expressed in all cerebellar cell types and are critical for regulating formation of specific fissures. However, the cerebellar cell types that En1 and En2 act in to control growth and/or patterning of fissures has not been determined. We conditionally eliminated En2 or En1 and En2 either in both progenitor zones and their descendents or in the two complementary sets of cells derived from each progenitor zone. En2 was found to be required only transiently in the progenitor zones and their immediate descendents to regulate formation of three fissures and for general growth of the cerebellum. In contrast, En1 and En2 have overlapping functions in the cells derived from each progenitor zone in regulating formation of additional fissures and for extensive cerebellar growth. Furthermore, En1/2 function in ventricular zone-derived cells plays a more significant role in determining the timing of initiation and positioning of fissures, whereas in upper rhombic lip-derived cells the genes are more important in regulating cerebellar growth. Our studies reveal the complex manner in which the En genes control cerebellar growth and foliation in distinct cell types.     

Hindbrain rhombic lip is comprised of discrete progenitor cell populations allocated by Pax6

The lower rhombic lip (LRL) is a germinal zone in the dorsal hindbrain productive of tangentially migrating neurons, streaming extramurally (mossy fiber neurons) or intramurally (climbing fiber neurons). Here we show that LRL territory, operationally defined by Wnt1 expression, is parceled into molecular subdomains predictive of cell fate. Progressing dorsoventrally, Lmx1a and Gdf7 expression identifies the primordium for hindbrain choroid plexus epithelial cells; Math1, for mossy fiber neurons; and immediately ventral to Math1 yet within Wnt1(+) territory, a climbing fiber primordium dominated by Ngn1-expressing cells. Elimination of Pax6 results in expansion of this Ngn1(+) progenitor pool and reduction in the Math1(+) pool, with accompanying later enlargement of the climbing fiber nucleus and reductions in mossy fiber nuclei. Pax6 loss also disrupts Msx expression cell-nonautonomously, suggesting Pax6 may influence LRL progenitor identity indirectly through potentiating BMP signaling. These studies suggest that underlying the diversity and proportions of fates produced by the LRL is a precise suborganization regulated by Pax6.     

Cre-mediated recombination in rhombic lip derivatives

To study the development of the cerebellum, we generated a transgenic mouse line Tg(malpha6-cre)B1LFR that expresses CRE recombinase under the GABA(A) receptor alpha6 subunit promoter. In this line, recombination of an R26R reporter allele occurred postnatally in granule cells of the cerebellum and dorsal cochlear nucleus, as well as in a subset of precerebellar nuclei in the brainstem. All neurons in which recombination occurred originated during embryogenesis from the rhombic lip. This might be explained by a very early specification event at the rhombic lip that primes cells derived from this structure to express the transgene during neuronal maturation. As no recombination occurred in the inferior olive, it may be derived from a distinct subset of precursors at the rhombic lip. No recombination occurred in any of the interneurons in the cerebellum (stellate cells, basket cells, and Golgi cells), consistent with the hypothesis that they are not derived from the same embryonic tissue as the granule cells.     

The role of the rhombic lip in avian cerebellum development.

We have used a combination of quail-chick fate-mapping techniques and dye labelling to investigate the development of the avian cerebellum. Using Hoxa2 as a guide for the microsurgical construction of quail-chick chimaeras, we show that the caudal boundary of the presumptive cerebellum at E6 maps to the caudal boundary of rhombomere 1. By fate mapping the dorsoventral axis of rhombomere 1, we demonstrate that granule cell precursors are generated at the rhombic lip together with neurons of the lateral pontine nucleus. DiI-labelling of cerebellum explants reveals that external germinal layer precursors have a characteristic unipolar morphology and undergo an orientated, active migration away from the rhombic lip, which is apparently independent of either glial or axon guidance or 'chain' formation.     

Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.     

The rhombic lip and early cerebellar development

Recent studies have transformed our understanding of the embryonic rhombic lip by revealing the inductive cues, regional origins and guidance molecules that pattern the development of this important structure and its derivatives. In the cerebellum, a precise combination of anteroposterior and dorsalising cues induces a stream of migratory progenitors that give rise to the external granule cell layer, while more caudally, Netrin orchestrates the migration of hindbrain rhombic lip derivatives to form the precerebellar nuclei. The rhombic lip is thus emerging as a spatiotemporally distinct epithelium whose late appearance in both development and evolution is instrumental in generating a complex, functionally related but spatially distributed neural system.     

Generation of cerebellar neuron precursors from embryonic stem cells

Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.     

A novel role for the choroid plexus in BMP-mediated inhibition of differentiation of cerebellar neural progenitors

Cerebellar granule cells, the most abundant neurons in the mammalian brain, arise in the rhombic lip located at the roof of the brain's fourth ventricle. Bordering the rhombic lip is the choroid plexus, a non-neuronal structure, composed of blood vessels enveloped by epithelial cells. Here, we show a striking decrease in neural differentiation of rhombic lip-derived cells, which failed to extend neuritic processes and attenuate Math1 promoter activity, when co-cultured with choroid plexus cells. Moreover, a blocking antibody against BMP7, a morphogenetic protein expressed in the choroid plexus, blocked the inhibitory effect of the choroid plexus, whereas purified BMP7 mimicked this effect, demonstrating causal involvement of BMP. On the other hand, the BMP antagonist NBL1 promoted neurogenesis in rhombic lip cultures from Math1 null mice displaying arrested differentiation. Our data indicate that besides its secretory and barrier functions, the choroid plexus has a novel role in attenuating the differentiation of adjacent neural progenitors.     

Assembly of the brainstem cochlear nuclear complex is revealed by intersectional and subtractive genetic fate maps

The cochlear nuclear complex (CN) is the entry point for central auditory processing. Although constituent neurons have been studied physiologically, their embryological origins and molecular profiles remain obscure. Applying intersectional and subtractive genetic fate mapping approaches, we show that this complex develops modularly from genetically separable progenitor populations arrayed as rostrocaudal microdomains within and outside the hindbrain (lower) rhombic lip (LRL). The dorsal CN subdivision, structurally and topographically similar to the cerebellum, arises from microdomains unexpectedly caudal and noncontiguous to cerebellar primordium; ventral CN subdivisions arise from more rostral LRL. Magnocellular regions receive contributions from LRL and coaxial non-lip progenitors; contrastingly, ensheathing granule cells derive principally from LRL. Also LRL-derived and molecularly similar to CN granule cells are precerebellar mossy fiber neurons; surprisingly, these ostensibly intertwined populations have separable origins and adjacent but segregated migratory streams. Together, these findings provide new platforms for investigating the development and evolution of auditory and cerebellar systems.     

Aberrant Otx2 expression enhances migration and induces ectopic proliferation of hindbrain neuronal progenitor cells

Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation.