Mitotic granule cell precursors undergo highly dynamic morphological transitions throughout the external germinal layer of the chick cerebellum.

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). The EGL is comprised solely of granule cell precursors, whose progeny migrate inwardly to form the internal granule cell layer. While a range of cell morphologies in the EGL has long been known, how they reflect the cells' differentiation status has previously only been inferred. Observations have suggested a deterministic maturation from outer to inner EGL that we wished to test experimentally. To do this, we electroporated granule cell precursors in chick with plasmids encoding fluorescent proteins and probed labelled cells with markers of both proliferation (phosphohistone H3) and differentiation (Axonin1/TAG1 and NeuroD1). We show that granule cell precursors can display a range of complex forms throughout the EGL while mitotically active. Overexpression of full length NeuroD1 within granule cell precursors does not abolish proliferation, but biases granule cells towards precocious differentiation, alters their migration path and results in a smaller and less foliated cerebellum. Our results show that granule cells show a greater flexibility in differentiation than previously assumed. We speculate that this allows the EGL to regulate its proliferative activity in response to overall patterns of cerebellar growth.

Sox14 Is Required for a Specific Subset of Cerebello-Olivary Projections.

We identify Sox14 as an exclusive marker of inhibitory projection neurons in the lateral and interposed, but not the medial, cerebellar nuclei. Sox14+ neurons make up ∼80% of Gad1+ neurons in these nuclei and are indistinguishable by soma size from other inhibitory neurons. All Sox14+ neurons of the lateral and interposed cerebellar nuclei are generated at approximately E10/10.5 and extend long-range, predominantly contralateral projections to the inferior olive. A small Sox14+ population in the adjacent vestibular nucleus "Y" sends an ipsilateral projection to the oculomotor nucleus. Cerebellar Sox14+ and glutamatergic projection neurons assemble in non-overlapping populations at the nuclear transition zone, and their integration into a coherent nucleus depends on Sox14 function. Targeted ablation of Sox14+ cells by conditional viral expression of diphtheria toxin leads to significantly impaired motor learning. Contrary to expectations, associative learning is unaffected by unilateral Sox14+ neuron elimination in the interposed and lateral nuclei.SIGNIFICANCE STATEMENT The cerebellar nuclei are central to cerebellar function, yet how they modulate and process cerebellar inputs and outputs is still primarily unknown. Our study gives a direct insight into how nucleo-olivary projection neurons are generated, their projections, and their function in an intact behaving mouse. These neurons play a critical conceptual role in all models of cerebellar function, and this study represents the first specific analysis of their molecular identity and function and offers a powerful model for future investigation of cerebellar function in motor control and learning.

MCT8 deficiency in Purkinje cells disrupts embryonic chicken cerebellar development.

Inactivating mutations in the human SLC16A2 gene encoding the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in the Allan-Herndon-Dudley syndrome accompanied by severe locomotor deficits. The underlying mechanisms of the associated cerebellar maldevelopment were studied using the chicken as a model. Electroporation of an MCT8-RNAi vector into the cerebellar anlage of a 3-day-old embryo allowed knockdown of MCT8 in Purkinje cell precursors. This resulted in the downregulation of the thyroid hormone-responsive gene RORα and the Purkinje cell-specific differentiation marker LHX1/5 at day 6. MCT8 knockdown also results in a smaller and less complex dendritic tree at day 18 suggesting a pivotal role of MCT8 for cell-autonomous Purkinje cell maturation. Early administration of the thyroid hormone analogue 3,5,3'-triiodothyroacetic acid partially rescued early Purkinje cell differentiation. MCT8-deficient Purkinje cells also induced non-autonomous effects as they led to a reduced granule cell precursor proliferation, a thinner external germinal layer and a loss of PAX6 expression. By contrast, at day 18, the external germinal layer thickness was increased, with an increase in presence of Axonin-1-positive post-mitotic granule cells in the initial stage of radial migration. The concomitant accumulation of presumptive migrating granule cells in the molecular layer, suggests that inward radial migration to the internal granular layer is stalled. In conclusion, early MCT8 deficiency in Purkinje cells results in both cell-autonomous and non-autonomous effects on cerebellar development and indicates that MCT8 expression is essential from very early stages of development, providing a novel insight into the ontogenesis of the Allan-Herndon-Dudley syndrome.

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.

Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum.

The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2 weeks of embryonic development points to a potential role of this transporter in the uptake of T3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may serve as a buffering mechanism against high T3 levels during early embryonic development, a hypothesis supported by the pattern of expression of a fluorescent TH reporter in this lineage. Overall, this study builds a picture of the TH dependency in multiple cerebellar cell types starting from early embryonic development.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

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.

Transit amplification in the amniote cerebellum evolved via a heterochronic shift in NeuroD1 expression.

The cerebellum has evolved elaborate foliation in the amniote lineage as a consequence of extensive Atoh1-mediated transit amplification in an external germinal layer (EGL) comprising granule cell precursors. To explore the evolutionary origin of this layer, we have examined the molecular geography of cerebellar development throughout the life cycle of Xenopus laevis. At metamorphic stages Xenopus displays a superficial granule cell layer that is not proliferative and expresses both Atoh1 and NeuroD1, a marker of postmitotic cerebellar granule cells. Premature misexpression of NeuroD1 in chick partially recapitulates the amphibian condition by suppressing transit amplification. However, unlike in the amphibian, granule cells fail to enter the EGL. Furthermore, misexpression of NeuroD1 once the EGL is established both triggers radial migration and downregulates Atoh1. These results show that the evolution of transit amplification in the EGL required adaptation of NeuroD1, both in the timing of its expression and in its regulatory function, with respect to Atoh1.

The evolution of the vertebrate cerebellum: absence of a proliferative external granule layer in a non-teleost ray-finned fish.

The cerebellum represents one of the most morphologically variable structures in the vertebrate brain. To shed light on its evolutionary history, we have examined the molecular anatomy and proliferation of the developing cerebellum of the North American paddlefish, Polyodon spathula. Absence of an external proliferative cerebellar layer and the restriction of Atonal1 expression to the rhombic lip and valvular primordium demonstrate that transit amplification in a cerebellar external germinal layer, a prominent feature of amniote cerebellum development, is absent in paddlefish. Furthermore, expression of Sonic hedgehog, which drives secondary proliferation in the mouse cerebellum, is absent from the paddlefish cerebellum. These data are consistent with what has been observed in zebrafish and suggest that the transit amplification seen in the amniote cerebellum was either lost very early in the ray-finned fish lineage or evolved in the lobe-finned fish lineage. We also suggest that the Atoh1-positive proliferative valvular primordium may represent a synapomorphy (shared derived character) of ray-finned fishes. The topology of valvular primordium development in paddlefish differs significantly from that of zebrafish and correlates with the adult cerebellar form. The distribution of proliferative granule cell precursors in different vertebrate taxa is thus the likely determining factor in cerebellar morphological diversity.

Independently specified Atoh1 domains define novel developmental compartments in rhombomere 1.

The rhombic lip gives rise to neuronal populations that contribute to cerebellar, proprioceptive and interoceptive networks. Cell production depends on the expression of the basic helix-loop-helix (bHLH) transcription factor Atoh1. In rhombomere 1, Atoh1-positive cells give rise to both cerebellar neurons and extra-cerebellar nuclei in ventral hindbrain. The origin of this cellular diversity has previously been attributed to temporal signals rather than spatial patterning. Here, we show that in both chick and mouse the cerebellar Atoh1 precursor pool is partitioned into initially cryptic spatial domains that reflect the activity of two different organisers: an isthmic Atoh1 domain, which gives rise to isthmic nuclei, and the rhombic lip, which generates deep cerebellar nuclei and granule cells. We use a combination of in vitro explant culture, genetic fate mapping and gene overexpression and knockdown to explore the role of isthmic signalling in patterning these domains. We show that an FGF-dependent isthmic Atoh1 domain is the origin of distinct populations of Lhx9-positive neurons in the extra-cerebellar isthmic nuclei. In the cerebellum, ectopic FGF induces proliferation while blockade reduces the length of the cerebellar rhombic lip. FGF signalling is not required for the specification of cerebellar cell types from the rhombic lip and its upregulation inhibits their production. This suggests that although the isthmus regulates the size of the cerebellar anlage, the downregulation of isthmic FGF signals is required for induction of rhombic lip-derived cerebellar neurons.

Developmental origins of diversity in cerebellar output nuclei.

BACKGROUND: The 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. RESULTS: Using 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. CONCLUSIONS: Our 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.

The roof plate boundary is a bi-directional organiser of dorsal neural tube and choroid plexus development.

The roof plate is a signalling centre positioned at the dorsal midline of the central nervous system and generates dorsalising morphogenic signals along the length of the neuraxis. Within cranial ventricles, the roof plate gives rise to choroid plexus, which regulates the internal environment of the developing and adult brain and spinal cord via the secretion of cerebrospinal fluid. Using the fourth ventricle as our model, we show that the organiser properties of the roof plate are determined by its boundaries with the adjacent neuroepithelium. Through a combination of in ovo transplantation, co-culture and electroporation techniques in chick embryos between embryonic days 3 and 6, we demonstrate that organiser properties are maintained by interactions between the non-neural roof plate and the neural rhombic lip. At the molecular level, this interaction is mediated by Delta-Notch signalling and upregulation of the chick homologue of Hes1: chairy2. Gain- and loss-of-function approaches reveal that cdelta1 is both necessary and sufficient for organiser function. Our results also demonstrate that while chairy2 is specifically required for the maintenance of the organiser, its ectopic expression is not sufficient to recapitulate organiser properties. Expression of atonal1 in the rhombic lip adjacent at the roof plate boundary is acutely dependent on both boundary cell interactions and Delta-Notch signalling. Correspondingly, the roof plate boundary organiser also signals to the roof plate itself to specify the expression of early choroid plexus markers. Thus, the roof plate boundary organiser signals bi-directionally to acutely coordinate the development of adjacent neural and non-neural tissues.

Can clues from evolution unlock the molecular development of the cerebellum?

The cerebellum sits at the rostral end of the vertebrate hindbrain and is responsible for sensory and motor integration. Owing to its relatively simple architecture, it is one of the most powerful model systems for studying brain evolution and development. Over the last decade, the combination of molecular fate mapping techniques in the mouse and experimental studies, both in vitro and in vivo, in mouse and chick have significantly advanced our understanding of cerebellar neurogenesis in space and time. In amniotes, the most numerous cell type in the cerebellum, and indeed the brain, is the cerebellar granule neurons, and these are born from a transient secondary proliferative zone, the external granule layer (EGL), where proliferation is driven by sonic hedgehog signalling and causes cerebellar foliation. Recent studies in zebrafish and sharks have shown that while the molecular mechanisms of neurogenesis appear conserved across vertebrates, the EGL as a site of shh-driven transit amplification is not, and is therefore implicated as a key amniote innovation that facilitated the evolution of the elaborate foliated cerebella found in birds and mammals. Ellucidating the molecular mechanisms underlying the origin of the EGL in evolution could have significant impacts on our understanding of the molecular details of cerebellar development.

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.

Retinoic acid is a potential dorsalising signal in the late embryonic chick hindbrain.

BACKGROUND: Human retinoic acid teratogenesis results in malformations of dorsally derived hindbrain structures such as the cerebellum, noradrenergic hindbrain neurons and the precerebellar system. These structures originate from the rhombic lip and adjacent dorsal precursor pools that border the fourth ventricle roofplate. While retinoic acid synthesis is known to occur in the meninges that blanket the hindbrain, the particular sensitivity of only dorsal structures to disruptions in retinoid signalling is puzzling. We therefore looked for evidence within the neural tube for more spatiotemporally specific signalling pathways using an in situ hybridisation screen of known retinoic acid pathway transcripts. RESULTS: We find that there are highly restricted domains of retinoic acid synthesis and breakdown within specific hindbrain nuclei as well as the ventricular layer and roofplate. Intriguingly, transcripts of cellular retinoic acid binding protein 1 are always found at the interface between dividing and post-mitotic cells. By contrast to earlier stages of development, domains of synthesis and breakdown in post-mitotic neurons are co-localised. At the rhombic lip, expression of the mRNA for retinoic acid synthesising and catabolising enzymes is spatially highly organised with respect to the Cath1-positive precursors of migratory precerebellar neurons. CONCLUSION: The late developing hindbrain shows patterns of retinoic acid synthesis and use that are distinct from the well characterised phase of rostrocaudal patterning. Selected post-mitotic populations, such as the locus coeruleus, appear to both make and break down retinoic acid suggesting that a requirement for an autocrine, or at least a highly localised paracrine signalling network, might explain its acute sensitivity to retinoic acid disruption. At the rhombic lip, retinoic acid is likely to act as a dorsalising factor in parallel with other roofplate signalling pathways. While its precise role is unclear, retinoic acid is potentially well placed to regulate temporally determined cell fate decisions within the rhombic lip precursor pool.

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.

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? RESULTS: We 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. CONCLUSIONS: Different 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.

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.