The migration of cerebellar rhombic lip derivatives

We have used cell labelling, co-culture and time-lapse confocal microscopy to investigate tangential neuronal migration from the rhombic lip. Cerebellar rhombic lip derivatives demonstrate a temporal organisation with respect to their morphology and response to migration cues. Early born cells, which migrate into ventral rhombomere 1, have a single long leading process that turns at the midline and becomes an axon. Later born granule cell precursors also migrate ventrally but halt at the lateral edge of the cerebellum, correlating with a loss of sensitivity to netrin 1 and expression of Robo2. The rhombic lip and ventral midline express Slit2 and both early and late migrants are repelled by sources of Slit2 in co-culture. These studies reveal an intimate relationship between birthdate, response to migration cues and neuronal fate in an identified population of migratory cells. The use of axons in navigating cell movement suggests that tangential migration is an elaboration of the normal process of axon extension.     

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.     

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.     

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.     

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.     

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.     

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.     

Lbx1 is required for muscle precursor migration along a lateral pathway into the limb

In mammalian embryos, myogenic precursor cells emigrate from the ventral lip of the dermomyotome and colonize the limbs, tongue and diaphragm where they differentiate and form skeletal muscle. Previous studies have shown that Pax3, together with the c-Met receptor tyrosine kinase and its ligand Scatter Factor (SF) are necessary for the migration of hypaxial muscle precursors in mice. Lbx1 and Pax3 are co-expressed in all migrating hypaxial muscle precursors, raising the possibility that Lbx1 regulates their migration. To examine the function of Lbx1 in muscle development, we inactivated the Lbx1 gene by homologous recombination. Mice lacking Lbx1 exhibit an extensive loss of limb muscles, although some forelimb and hindlimb muscles are still present. The pattern of muscle loss suggests that Lbx1 is not required for the specification of particular limb muscles, and the muscle defects that occur in Lbx1(-/-) mice can be solely attributed to changes in muscle precursor migration. c-Met is expressed in Lbx1 mutant mice and limb muscle precursors delaminate from the ventral dermomyotome but fail to migrate laterally into the limb. Muscle precursors still migrate ventrally and give rise to tongue, diaphragm and some limb muscles, demonstrating Lbx1 is necessary for the lateral, but not ventral, migration of hypaxial muscle precursors. These results suggest that Lbx1 regulates responsiveness to a lateral migration signal which emanates from the developing limb.     

Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors

We have utilized an in vivo-inducible genetic-fate-mapping strategy to permanently label cohorts of Math1-positive cells and their progeny that arise in the rhombic lip of the cerebellar primordium during embryogenesis. At stages prior to E12.5, with the exception of the deep cerebellar nuclei, we find that Math1 cells migrate out of the cerebellar primordium into the rostral hindbrain to populate specific nuclei that include cholinergic neurons of the mesopontine tegmental system. Moreover, analysis of Math1-null embryos shows that this gene is required for the formation of some of these nuclei. Around E12.5, granule cell precursors begin to be labeled: first, ones that give rise to granule cells that predominantly populate the anterior lobes of the adult cerebellum and later, those that populate progressing more caudally lobes until labeling of all granule cell precursors is complete by E17. Thus, we demonstrate that the cerebellar rhombic lip gives rise to multiple cell types within rhombomere 1.     

Widespread contribution of Gdf7 lineage to cerebellar cell types and implications for hedgehog-driven medulloblastoma formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling.     

A combination of chain and neurophilic migration involving the adhesion molecule TAG-1 in the caudal medulla.

Neuronal populations destined to form several precerebellar nuclei are generated by the rhombic lip in the caudal hindbrain. These immature neurons gather into the olivary and the superficial migratory streams and migrate tangentially around the hindbrain to reach their final position. We focus on the cells of the superficial stream that migrate ventrally, cross the midline and form the lateral reticular (LRN) and external cuneate (ECN) nuclei. The cells of the superficial steam are preceded by long leading processes; in the dorsal neural tube, they migrate in close apposition to each other and form distinct chains, whereas they disperse and follow Tuj-1 immunoreactive axons on reaching the ventral hindbrain. This suggests that, in the superficial stream, neuronal migration combines both homotypic and heterotypic mechanisms. We also show that the adhesion molecule TAG-1 is expressed by the migrating cells. Blocking TAG-1 function results in alterations in the superficial migration, indicating that TAG-1 is involved in the superficial migration. Other members of the immunoglobulin superfamily and known ligands of TAG-1 are also expressed in the region of the migration but are not involved in the migration. These findings provide evidence that the TAG-1 protein is involved as a contact-dependent signal guiding not only axonal outgrowth but also cell migration.     

Multiple influences on the migration of precerebellar neurons in the caudal medulla.

Neurons destined to form several precerebellar nuclei are generated in the dorsal neuroepithelium (rhombic lip) of caudal hindbrain. They form two ventrally directed migratory streams, which behave differently. While neurons in the superficial migration migrate in a subpial position and cross the midline to settle into the contralateral hindbrain, neurons in the olivary migration travel deeper in the parenchyma and stop ipsilaterally against the floor plate. In the present study, we compared the behavior of the two neuronal populations in an organotypic culture system that preserves several aspects of their in vivo environment. Both migrations occurred in mouse hindbrain explants dissected at E11.5 even when the floor plate was ablated at the onset of the culture period, indicating that they could rely on dorsoventral cues already distributed in the neural tube. Nevertheless, the local constraints necessary for the superficial migration were more specific than for the olivary migration. Distinct chemoattractive and chemorespulsive signal were found to operate on the migrations. The floor plate exhibited a strong chemoattractive influence on both migrations, which deviated from their normal path in the direction of ectopic floor plate fragments. It was also found to produce a short-range stop signal and to induce inferior olive aggregation. The ventral neural tube was also found to inhibit or slow down the migration of olivary neurons. Interestingly, while ectopic sources of netrin were found to influence both migrations, this effect was locally modulated and affected differentially the successive phases of migration. Consistent with this observation, while neurons in the superficial migration expressed the Dcc-netrin receptor, the migrating olivary neurons did not express Dcc before they reached the midline. Our observations provide a clearer picture of the hierarchy of environmental cues that influence the morphogenesis of these precerebellar nuclei.     

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.     

Development of the cerebellum and cerebellar neural circuits

The cerebellum, a structure derived from the dorsal part of the most anterior hindbrain, is important for integrating sensory perception and motor control. While the structure and development of the cerebellum have been analyzed most extensively in mammals,recent studies have shown that the anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost) species, including zebrafish. In the mammalian and teleost cerebellum,Purkinje and granule cells serve, respectively, as the major GABAergic and glutamatergic neurons. Purkinje cells originate in the ventricular zone (VZ), and receive inputs from climbing fibers. Granule cells originate in the upper rhombic lip (URL) and receive inputs from mossy fibers. Thus, the teleost cerebellum shares many features with the cerebellum of other vertebrates, and isa good model system for studying cerebellar function and development. The teleost cerebellum also has features that are specific to teleosts or have not been elucidated in mammals, including eurydendroid cells and adult neurogenesis. Furthermore, the neural circuitry in part of the optic tectum and the dorsal hindbrain closely resembles the circuitry of the teleost cerebellum; hence,these are called cerebellum-like structures. Here we describe the anatomy and development of cerebellar neurons and their circuitry, and discuss the possible roles of the cerebellum and cerebellum-like structures in behavior and higher cognitive functions. We also consider the potential use of genetics and novel techniques for studying the cerebellum in zebrafish.     

Cadherin-2 Controls Directional Chain Migration of Cerebellar Granule Neurons

Long distance migration of differentiating granule cells from the cerebellar upper rhombic lip has been reported in many vertebrates. However, the knowledge about the subcellular dynamics and molecular mechanisms regulating directional neuronal migration in vivo is just beginning to emerge. Here we show by time-lapse imaging in live zebrafish (Danio rerio) embryos that cerebellar granule cells migrate in chain-like structures in a homotypic glia-independent manner. Temporal rescue of zebrafish Cadherin-2 mutants reveals a direct role for this adhesion molecule in mediating chain formation and coherent migratory behavior of granule cells. In addition, Cadherin-2 maintains the orientation of cell polarization in direction of migration, whereas in Cadherin-2 mutant granule cells the site of leading edge formation and centrosome positioning is randomized. Thus, the lack of adhesion leads to impaired directional migration with a mispositioning of Cadherin-2 deficient granule cells as a consequence. Furthermore, these cells fail to differentiate properly into mature granule neurons. In vivo imaging of Cadherin-2 localization revealed the dynamics of this adhesion molecule during cell locomotion. Cadherin-2 concentrates transiently at the front of granule cells during the initiation of individual migratory steps by intramembraneous transport. The presence of Cadherin-2 in the leading edge corresponds to the observed centrosome orientation in direction of migration. Our results indicate that Cadherin-2 plays a key role during zebrafish granule cell migration by continuously coordinating cell-cell contacts and cell polarity through the remodeling of adherens junctions. As Cadherin-containing adherens junctions have been shown to be connected via microtubule fibers with the centrosome, our results offer an explanation for the mechanism of leading edge and centrosome positioning during nucleokinetic migration of many vertebrate neuronal populations.     

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.     

BMP and non-canonical Wnt signaling are required for inhibition of secondary tail formation in zebrafish

The role of bone morphogenetic protein (BMP) signaling in specifying cell fate in the zebrafish tailbud has been well established. In addition to a loss of ventral tissues, such as ventral tailfin and cloaca, some embryos with compromised BMP signaling produce an additional phenotype: a ventrally located secondary tail containing both somitic muscle and notochord. This phenotype has been proposed to reflect a fate-patterning defect due to a change in a hypothesized BMP activity gradient. Here, we show that a defect in morphogenetic movements, not fate patterning, underlies the formation of secondary tails in BMP-inhibited embryos. Our data indicate that BMP signaling is activated in the ventroposterior tailbud to promote cell migration during tailbud protrusion, and that defective migration of these cells in BMP mutants ultimately leads to bifurcation of the caudal notochord. Additionally, we show that non-canonical Wnt signaling is also required for proper tail morphogenesis, possibly by maintaining cohesion of notochord progenitors by regulation of cadherin localization. We propose a model in which BMP and the non-canonical Wnt pathway regulate tail morphogenesis by controlling cell migration and cell adhesion within the tailbud.     

Presenilin 1 in migration and morphogenesis in the central nervous system

Morphogenesis of the central nervous system relies in large part upon the correct migration of neuronal cells from birthplace to final position. Two general modes of migration govern CNS morphogenesis: radial, which is mostly glia-guided and topologically relatively simple; and tangential, which often involves complex movement of neurons in more than one direction. We describe the consequences of loss of function of presenilin 1 on these fundamental processes. Previous studies of the central nervous system in presenilin 1 homozygote mutant embryos identified a premature neuronal differentiation that is transient and localized, with cortical dysplasia at later stages. We document widespread effects on CNS morphogenesis that appear strongly linked to defective neuronal migration. Loss of presenilin 1 function perturbs both radial and tangential migration in cerebral cortex, and several tangential migratory pathways in the brainstem. The inability of cells to execute their migratory trajectories affects cortical lamination, formation of the facial branchiomotor nucleus, the spread of cerebellar granule cell precursors to form the external granule layer and development of the pontine nuclei. Finally, overall morphogenesis of the mid-hindbrain region is abnormal, resulting in incomplete midline fusion of the cerebellum and overgrowth of the caudal midbrain. These observations indicate that in the absence of presenilin 1 function, the ability of a cell to move can be severely impaired regardless of its mode of migration, and, at a grosser level, brain morphogenesis is perturbed. Our results demonstrate that presenilin 1 plays a much more important role in brain development than has been assumed, consistent with a pleiotropic involvement of this molecule in cellular signaling.