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.     

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.     

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.     

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.     

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.     

Segmental identity and cerebellar granule cell induction in rhombomere 1

Background  

Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment?     

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.     

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.     

Identification and expression of zebrafish Iroquois homeobox gene irx1

Iroquois homeoproteins are prepatterning factors that positively regulate proneural genes and control neurogenesis. We have identified a zebrafish Iroquois gene, irx1, which is highly homologous to Xenopus Xiro1, Gallus c-Irx1 and mouse Irx1. Expression of irx1 was initially detected at the bud stage. By 16 h post-fertilization (hpf), irx1 expression was exclusively limited to the prospective midbrain and hindbrain. By 24 hpf, irx1 expression was clearly detected in the acousticovestibual ganglia, tectum, tegmentum, cerebellum and rhombomere 1 but not in rhombomere 2 or mid-hindbrain boundary.     

Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression

Current evidence suggests that the anterior segment of the vertebrate hindbrain, rhombomere 1, gives rise to the entire cerebellum. It is situated where two distinct developmental patterning mechanisms converge: graded signalling from an organising centre (the isthmus) located at the midbrain/hindbrain boundary confronts segmentation of the hindbrain. The unique developmental fate of rhombomere 1 is reflected by it being the only hindbrain segment in which no Hox genes are expressed. In this study we show that ectopic FGF8 protein, a candidate for the isthmic organising activity, is able to induce and repress gene expression within the hindbrain in a manner appropriate to rhombomere 1. Using a heterotopic, heterospecific grafting strategy we demonstrate that rhombomere 1 is able to express Hox genes but that both isthmic tissue and FGF8 inhibit their expression. Inhibition of FGF8 function in vivo shows that it is responsible for defining the anterior limit of Hox gene expression within the developing brain and thereby specifies the extent of the rl territory. Previous studies have suggested that a retinoid morphogen gradient determines the axial limit of expression of individual Hox genes within the hindbrain. We propose a model whereby activation by retinoids is antagonised by inhibition by FGF8 in the anterior hindbrain to set aside the territory from which the cerebellum will develop.     

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.     

Morphogenetic and cellular movements that shape the mouse cerebellum; insights from genetic fate mapping

We used the cerebellum as a model to study the morphogenetic and cellular processes underlying the formation of elaborate brain structures from a simple neural tube, using an inducible genetic fate mapping approach in mouse. We demonstrate how a 90 degrees rotation between embryonic days 9 and 12 converts the rostral-caudal axis of dorsal rhombomere 1 into the medial-lateral axis of the wing-like bilateral cerebellar primordium. With the appropriate use of promoters, we marked specific medial-lateral domains of the cerebellar primordium and derived a positional fate map of the murine cerebellum. We show that the adult medial cerebellum is produced by expansion, rather than fusion, of the thin medial primordium. Furthermore, ventricular-derived cells maintain their original medial-lateral coordinates into the adult, whereas rhombic lip-derived granule cells undergo lateral to medial posterior transverse migrations during foliation. Thus, we show that progressive changes in the axes of the cerebellum underlie its genesis.     

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.     

Monofocal origin of telencephalic oligodendrocytes in the anterior entopeduncular area of the chick embryo

Oligodendrocytes are the myelin-forming cells in the central nervous system. In the brain, oligodendrocyte precursors arise in multiple restricted foci, distributed along the caudorostral axis of the ventricular neuroepithelium. In chick embryonic hind-, mid- and caudal forebrain, oligodendrocytes have a basoventral origin, while in the rostral fore-brain oligodendrocytes emerge from alar territories (Perez Villegas, E. M., Olivier, C., Spassky, N., Poncet, C., Cochard, P., Zalc, B., Thomas, J. L. and Martinez, S. (1999) Dev. Biol. 216, 98-113). To investigate the respective territories colonized by oligodendrocyte progenitor cells that originate from either the basoventral or alar foci, we have created a series of quail-chick chimeras. Homotopic chimeras demonstrate clearly that, during embryonic development, oligodendrocyte progenitors that emerge from the alar anterior entopeduncular area migrate tangentially to invade the entire telencephalon, whereas those from the basal rhombomeric foci show a restricted rostrocaudal distribution and colonize only their rhombomere of origin. Heterotopic chimeras indicate that differences in the migratory properties of oligodendroglial cells do not depend on their basoventral or alar ventricular origin. Irrespective of their origin (basal or alar), oligodendrocytes migrate only short distances in the hindbrain and long distances in the prosencephalon. Furthermore, we provide evidence that, in the developing chick brain, all telencephalic oligodendrocytes originate from the anterior entopeduncular area and that the prominent role of anterior entopeduncular area in telencephalic oligodendrogenesis is conserved between birds and mammals.     

Origin and plasticity of the subdivisions of the inferior olivary complex

The precerebellar nuclei (PCN) originate from the rhombic lip, a germinal neuroepithelium adjacent to the roof plate of the fourth ventricle. We first report here that, in chicken, the Brn3a-expressing postmitotic medullary cells that produce the inferior olive (ION, the source of cerebellar climbing fibres) originate from a dorso-ventral domain roughly coinciding with the hindbrain vestibular column. Whereas Foxd3 expression labels the whole mature ION but is only detected in a subpopulation of ION neuroblasts initiating their migration, we report that Brn3a allows the visualization of the whole population of ION neurons from the very beginning of their migration. We show that Brn3a-positive neurons migrate tangentially ventralwards through a characteristic dorso-ventral double submarginal stream. Cath1 expressing progenitors lying just dorsal to the ION origin correlated dorso-ventral topography with the prospective cochlear column (caudal to it) and generate precerebellar nuclei emitting mossy-fiber cerebellar afferents. We used the chick-quail chimaera technique with homotopic grafts at HH10 to determine the precise fate map of ION precursors across the caudal cryptorhombomeric subdivisions of the medullary hindbrain (r8-r11). We demonstrate that each crypto-rhombomere contributes to two lamellae of the ION, while each ION sub-nucleus originates from at least two contiguous crypto-rhombomeres. We then questioned how rhombomere identity is related to the plasticity of cell type specification in the dorsal hindbrain. The potential plasticity of ectopically HH10 grafted ION progenitors to change their original fate in alternative rostrocaudal environments was examined. Heterotopic grafts from the presumptive ION territory to the pontine region (r4-r5) caused a change of fate, since the migrated derivatives adopted a pontine phenotype. The reverse experiment caused pontine progenitors to produce derivatives appropriately integrated into the ION complex. Grafts of ION progenitor domains to myelomeres (my) 2-3 also showed complete fate regulation, reproducing spinal cord-like structures, whereas the reverse experiment revealed the inability of my2-3 to generate ION cell types. This was not the case with more caudal, relatively less specified myelomeres (my5-6). Interestingly, when heterotopically grafted cells are integrated dorsally, they do not change their phenotype. Our results support the hypothesis that positional information present in the hindbrain and spinal cord at early neural tube stages controls the specific fates of ventrally migrating PCN precursors.     

The roof plate regulates cerebellar cell-type specification and proliferation

During embryogenesis, the isthmic organizer, a well-described signaling center at the junction of the mid-hindbrain, establishes the cerebellar territory along the anterior/posterior axis of the neural tube. Mechanisms specifying distinct populations within the early cerebellar anlage are less defined. Using a newly developed gene expression map of the early cerebellar anlage, we demonstrate that secreted signals from the rhombomere 1 roof plate are both necessary and sufficient for specification of the adjacent cerebellar rhombic lip and its derivative fates. Surprisingly, we show that the roof plate is not absolutely required for initial specification of more distal cerebellar cell fates, but rather regulates progenitor proliferation and cell position within the cerebellar anlage. Thus, in addition to the isthmus, the roof plate represents an important signaling center controlling multiple aspects of cerebellar patterning.     

Inhibitory effects of draxin on axonal outgrowth and migration of precerebellar neurons

The rhombic lip, a dorsal stripe of the neuroepithelium lining the edge of the fourth ventricle, is the site of origin of precerebellar neurons (PCN), which migrate tangentially towards the floor plate. After reaching the floor plate, they project their axons to the cerebellum. Although previous studies have shown that the guidance molecules Netrin/DCC and Slit/Robo have critical roles in PCN migration, the molecular mechanisms underlying this process remain poorly understood. Here, we report that draxin, a repulsive axon guidance protein, is involved in PCN development. We found that draxin is expressed in the rhombic lip and migratory stream of some PCN in the developing hindbrain of mice. In addition, draxin inhibited neurite outgrowth and nuclei migration from rhombic lip explants. These results suggest that draxin functions as a repulsive guidance cue for PCN migration. However, we observed no significant differences in PCN distribution between draxin^−/− and wild type embryos. Thus, draxin and other axon guidance cues may have redundant roles in PCN migration.