Richard Wingate

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.

Consensus Paper: Cerebellar Development.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

Development of the cerebellum: simple steps to make a ‘little brain'

The cerebellum is a pre-eminent model for the study of neurogenesis and circuit assembly. Increasing interest in the cerebellum as a participant in higher cognitive processes and as a locus for a range of disorders and diseases make this simple yet elusive structure an important model in a number of fields. In recent years, our understanding of some of the more familiar aspects of cerebellar growth, such as its territorial allocation and the origin of its various cell types, has undergone major recalibration. Furthermore, owing to its stereotyped circuitry across a range of species, insights from a variety of species have contributed to an increasingly rich picture of how this system develops. Here, we review these recent advances and explore three distinct aspects of cerebellar development – allocation of the cerebellar anlage, the significance of transit amplification and the generation of neuronal diversity – each defined by distinct regulatory mechanisms and each with special significance for health and disease.

Neurogenetics of the cerebellar system.

The development of the cerebellum occurs in four basic steps. During the first epoch, genes that mark the cerebellar territory are expressed in a restricted pattern along the anterioposterior axis of the embryo. In the second, an embryonic region termed the rhombic lip generates precursors of the granule cell population of the cerebellar cortex, and the lateral pontine nucleus and olivary nucleus of the brain stem. In the third period, the program of neurogenesis of the granule neuron gives rise to the formation of the fundamental layers of the cerebellum and to the pattern of foliation. Concomitantly, programs of gene expression define the principal neuronal classes, the granule cell and Purkinje cell, that will establish the cerebellar circuitry in the postnatal period. Understanding the molecular mechanisms underlying these steps of development is likely to yield important insights into malformations such as Joubert syndrome. (J Child Neurol 1999;14:574-582).

Math-Map(ic)s

In this issue of Neuron, Wang et al. and Machold and Fishell present contrasting molecular fate maps of Math1, which redefine the derivatives of the embryonic rhombic lip and offer a conceptual overhaul of cerebellar and precerebellar development. These fate maps identify a common developmental thread linking diverse, functionally associated neurons and reveal an exquisite temporal organization in cell production within a precise spatially defined region of neuroepithelium.

Absence of an External Germinal Layer in Zebrafish and Shark Reveals a Distinct, Anamniote Ground Plan of Cerebellum Development

The granule cell layer of the cerebellum comprises the largest population of neurons in the vertebrate CNS. In amniotes, its precursors undergo a unique phase of transit amplification, regulated by Sonic hedgehog. They do so within a prominent but transient secondary proliferative epithelium, the external germinal layer, which is formed by tangential migration of precursor cells from the rhombic lip. This behavior is a hallmark of bird and mammal cerebellum development. Despite its significance for both development and disease, it is unclear whether an external germinal layer is a requirement for granule cell production or an expedient of transit amplification. Evidence for its existence in more basal vertebrates is contradictory. We therefore examined cerebellum development in the zebrafish, specifically in relation to the expression of the basic helix–loop–helix gene Atonal 1, which definitively characterizes granule cell precursors. The expression of Atoh1a–Atoh1c, in combination with patterns of proliferation and fate maps, define precursor pools at the rhombic lip and cerebellar midline but demonstrate that an external germinal layer is absent. Sonic hedgehog signaling is correspondingly absent in the zebrafish cerebellum. Sustained roof-plate-derived signals suggest that, in the absence of transit amplification, primary granule cell precursor pools are maintained throughout development. To determine whether this pattern is specific to zebrafish or reflects a more general anamniote organization, we examined the expression of similar genes in the dogfish, Scylliorhinus canicula. We show that these anamniotes share a common ground plan of granule cell production that does not include an external germinal layer.

Congenital hypoplasia of the cerebellum: developmental causes and behavioral consequences.

Over the last 60 years, the spotlight of research has periodically returned to the cerebellum as new techniques and insights have emerged. Because of its simple homogeneous structure, limited diversity of cell types and characteristic behavioral pathologies, the cerebellum is a natural home for studies of cell specification, patterning, and neuronal migration. However, recent evidence has extended the traditional range of perceived cerebellar function to include modulation of cognitive processes and implicated cerebellar hypoplasia and Purkinje neuron hypo-cellularity with autistic spectrum disorder. In the light of this emerging frontier, we review the key stages and genetic mechanisms behind cerebellum development. In particular, we discuss the role of the midbrain hindbrain isthmic organizer in the development of the cerebellar vermis and the specification and differentiation of Purkinje cells and granule neurons. These developmental processes are then considered in relation to recent insights into selected human developmental cerebellar defects: Joubert syndrome, Dandy–Walker malformation, and pontocerebellar hypoplasia. Finally, we review current research that opens up the possibility of using the mouse as a genetic model to study the role of the cerebellum in cognitive function.

Segmental identity and cerebellar granule cell induction in rhombomere 1

BackgroundCerebellar 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?ResultsWe show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2.ConclusionsDifferent overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum.

Developmental origins of diversity in cerebellar output nuclei

BackgroundThe functional integration of the cerebellum into a number of different neural systems is governed by the connection of its output axons. In amniotes, the majority of this output is mediated by an evolutionarily diverse array of cerebellar nuclei that, in mice, are derived from the embryonic rhombic lip. To understand the origins of cerebellar nucleus diversity, we have explored how nucleus development is patterned in birds, which notably lack a dentate-like nucleus output to the dorsal thalamus.ResultsUsing targeted in ovo electoroporation of green fluorescent protein (GFP) and red fluorescent protein (RFP) in a variety of combinations and with different conditional enhancers, we show that cerebellar nuclei in chicks are produced, as in the mouse, at the rhombic lip. Furthermore, the comparison of fate-mapped neurons with molecular markers reveals a strict temporal sequence of cell fate allocation in establishing the avian lateral and medial cerebellar nuclei. In contrast to the mouse cerebellum, Lhx9 expression is confined to extracerebellar thalamic afferent nuclei corresponding to the absence, in chicks, of a dentate nucleus. Spatiotemporally targeted over-expression of Lhx9 in chick cerebellar nuclei (recapitulating in part the mammalian expression pattern) results in a loss of distinct nuclear boundaries and a change in axon initial trajectories consistent with a role for Lhx9 specifying targeting.ConclusionsOur results confirm the relationship between cell fate and a fine grain temporal patterning at the rhombic lip. This suggests that the lack of a cerebellar output to the dorsal thalamus of birds corresponds with a restricted expression of the LIM-homeodomain gene Lhx9 to earlier born rhombic lip cohorts when compared to mice. The evolution of cerebellar nucleus diversity in amniotes may hence reflect a heterochronic adaptation of gene expression with respect to the sequential production of rhombic lip derivatives resulting in altered axonal targeting.

Axonal Target Choice and Dendritic Development of Ferret Beta Retinal Ganglion Cells

We have investigated the relationship between axon targeting and dendritic morphology in beta retinal ganglion cells in the postnatal ferret. Axonal projections were assessed by making separate injections of different fluorescent retrograde tracers into either the superior colliculus or lateral geniculate nucleus in viva The dendritic morphology of retrogradely labelled cells was revealed by the in vitro intracellular injection of Lucifer yellow in fixed retina. In this way, 405 retinal ganglion cells were triple‐ or double‐labelled and characterized by their dendritic branching styles. Both the distinct dendritic morphology of beta cells and the characteristic restriction of their adult axonal terminals to the lateral geniculate nucleus emerge postnatally. Beta cell dendritic morphology is established between postnatal days 5 and 9. As in the cat (Ramoa et al, 1989), beta cells extend and then retract a projection to the superior colliculus as part of their normal development. Transient beta axonal collaterals to the superior colliculus persist beyond the period of cell death, but nearly all are withdrawn by postnatal day 15. No dendritically distinct beta cell projects to the superior colliculus alone, at any age.