Butts+Cerebellar+development+2014 PDF Review of Cerebellar Development

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 REVIEW Development of the cerebellum: simple steps to make a ‘little brain’ Thomas Butts1,2,*, Mary J. Green3, * and Richard J. T. Wingate1,‡ ABSTRACT...

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 REVIEW Development of the cerebellum: simple steps to make a ‘little brain’ Thomas Butts1,2,*, Mary J. Green3, * and Richard J. T. Wingate1,‡ ABSTRACT various morphogenic manifestations clearly representing variations The cerebellum is a pre-eminent model for the study of neurogenesis on a simple theme, the cerebellum provided the perfect template for and circuit assembly. Increasing interest in the cerebellum as a addressing adaptive developmental processes. However, the failure of participant in higher cognitive processes and as a locus for a range comparative anatomy to deliver on a mechanism resulted in insights of of disorders and diseases make this simple yet elusive structure the late 1960s languishing unattended in intervening years. an important model in a number of fields. In recent years, our Historically, studies of the cerebellum focussed on its description understanding of some of the more familiar aspects of cerebellar through fate mapping, its induction via FGF signalling or its role as growth, such as its territorial allocation and the origin of its various cell a locus for developmental cancer. However, in recent years, each of types, has undergone major recalibration. Furthermore, owing to its these perspectives has been subject to a more or less severe stereotyped circuitry across a range of species, insights from a variety reworking, and this revision has generated important insights into of species have contributed to an increasingly rich picture of how the organisation of neurogenesis, the cell lineages, temporal this system develops. Here, we review these recent advances and patterning and differentiation in the cerebellum. Collectively, this explore three distinct aspects of cerebellar development – allocation scrutiny has propelled the cerebellum into a pre-eminent model for of the cerebellar anlage, the significance of transit amplification and neural development, an understanding of which impacts on a range the generation of neuronal diversity – each defined by distinct regulatory mechanisms and each with special significance for health and disease. Box 1. Glossary Actinopterygian fish Ray-finned fish. One of the two branches of KEY WORDS: Granule cell, Atoh1, Autistic spectrum disorder, extant osteichthyeans (bony vertebrates) that comprises all of the extant Medulloblastoma, Ptf1a, Purkinje cell fish with the exception of the coelacanth and lungfish. The latter two, together with tetrapods, make up the other branch of bony vertebrates: the lobe-finned fish (sarcopterygians). Introduction Bergmann glia. A characteristic glial population of the cerebellum. In The cerebellum (‘little brain’) resides at the anterior end of the development, they function as scaffolds for the radial migration of hindbrain and is classically defined by its role in sensory-motor granule cell precursors from the EGL to the IGL. processing (Buckner, 2013). In amniotes, it represents one of the Cerebellar nuclei. Clusters of glutamatergic and GABAergic neurons most architecturally elaborate regions of the central nervous system located in the cerebellar white matter that are the synaptic targets of the majority of Purkinje cells. Projection neurons within nuclei account for the (CNS), and in humans it contains over half of the mature neurons in output of the cerebellum. Cerebellar nuclei are often termed ‘deep’ the adult brain (Butts et al., 2012). This morphological complexity although the designation is superfluous. belies histological simplicity: the cerebellar cortex is composed of a External germinal layer (EGL). A transient zone of granule cell very basic structure comprising a monolayer of inhibitory Purkinje precursors that is formed from cells that migrate tangentially from the cells (see Glossary, Box 1) sandwiched between a dense layer of rhombic lip to cover the pial surface of the developing cerebellum during excitatory granule cells (see Glossary, Box 1) and a sub-pial development. Subsequently, cells of the EGL migrate radially to their final position as mature granule cells within the internal granule layer. molecular layer of granule cell axons and Purkinje cell dendritic trees External ‘granule’ cell layer. A common, if less precise, substitution for (Fig. 1). Granule cells receive inputs from outside the cerebellum and external germinal layer. It adequately describes a transient superficial project to the Purkinje cells, the majority of which then project to a layer of an embryonic cerebellum that is either non-proliferative variety of cerebellar nuclei (see Glossary, Box 1) in the white matter. (amphibian) or proliferative (birds and mammals) but does not A less well-defined complement of locally interacting inhibitory discriminate between the two. interneuron cell types and glutamatergic unipolar brush cells (see Granule cells. Glutamatergic excitatory neurons in the internal granule Glossary, Box 1) complete the circuit, which famously promised layer that receive excitatory inputs from mossy fibres, the majority of which originate in the pons, medulla and spinal cord. They receive local to be the first of any vertebrate neural network to be fully inhibitory inputs from Golgi neurons. Granule cells extend T-shaped comprehended (Eccles et al., 1967). axons into the molecular layer where they synapse with Purkinje cell At around the same time as Eccles, Ito and Szentágothai were dendrites. publishing their famous treatise on the cerebellum as a neural machine Medulloblastoma. A developmental tumour that originates either from (Eccles et al., 1967), the variation in cerebellar structure across cells within the cerebellum or the dorsal hindbrain. DEVELOPMENT vertebrates emerged (Fig. 2), thus highlighting the cerebellum as an Purkinje cells. GABAergic inhibitory neurons in the cerebellar cortex that receive excitatory inputs from granule cell parallel fibres and important model for brain evolution (Nieuwenhuys, 1967). With its inhibitory input from climbing fibres of the inferior olive. The majority of Purkinje cell axons project to the deep cerebellar nuclei, while a subset 1 MRC Centre for Developmental Neurobiology, King’s College London, London directly innervates vestibular targets in the hindbrain. 2 SE1 1UL, UK. School of Biological and Chemical Sciences, Queen Mary, Unipolar brush cells. Glutamatergic interneurons that are found in the 3 University of London, London E1 4NS, UK. National Institute for Medical Research, internal granule layer in areas associated with the vestibular system. Mill Hill, London NW7 1AA, UK. They receive inputs from the vestibular system nuclei in the hindbrain and *These authors contributed equally to this work project locally to granule cells. ‡ Author for correspondence ([email protected]) 4031 REVIEW Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 A B Granule cell layer C Stellate/basket cells* Sagittal section Coronal section Vermis Purkinje cell (in PCL) Granule cell (in IGL) White matter Golgi/Lugaro cells* Pia Molecular layer White matter Cerebellar nucleus Key Dendritic arbour Granule cell Purkinje cell Interneuron Fig. 1. Structure of the cerebellum. (A) Viewed superficially, the cerebellar is divided into transverse folia. The mammalian cerebellum (green) is characterised by a medial expansion of the hemispheres into a vermis. (B) In sagittal section, each folia comprises distinct cellular layers with white matter beneath. Cerebellar nuclei lie within the white matter. Layering reflects the distribution of different cell types: Purkinje cell layer (blue), internal granule cell layer (red) and a molecular layer (not coloured) in which Purkinje cell dendrites and granule cell axons interact. Each layer also contains characteristic GABAergic interneuron subtypes (*). Of these, only the stellate neuron appears to be present in all vertebrates, whereas others have a variable distribution: Lugaro (mammals only), basket (birds and mammals), Golgi (birds, reptiles and mammals) (Lliná s and Hillman, 1969). Glutamatergic interneurons (unipolar brush cells) have also been found in the IGL in both birds and mammals (Takacs et al., 1999). (C) Schematic magnified views (sagittal and coronal sections) of the molecular layer of the cerebellum. Granule cell axons form parallel fibres arranged orthogonally to Purkinje cell dendritic arbours. of congenital and acquired disorders. The increasing recognition of An overview of cerebellar development the diversity of cerebellar-related syndromes reflects a growing Although it is easiest to consider how developmental phases fit understanding of the repertoire of brain regions influenced by together in the mammal, it is important to recognise that, beyond the cerebellar activity, as revealed by novel mapping techniques and stereotyped neuronal Purkinje-granule cell circuit, evolutionary implied from clinical studies. Most recently (Courchesne et al., variability in cerebellum form reflects variability in how these 1988; Brito et al., 2009; Schmahmann, 2010; Buckner, 2013), the phases are deployed in the embryo. Thus, the territory that will cerebellar circuit has achieved a new significance in the context of generate the cerebellum – its ‘anlage’ – is allocated during the early autistic spectrum disorder (ASD). Moreover, the recent explosion embryonic segmental phase of hindbrain development [in mouse, at of genetic developmental techniques means that the cerebellum can approximately embryonic day (E) 8.5] close to the boundary (the perhaps fulfil its potential as a model in comparative approaches in ‘isthmus’) between the hindbrain and the midbrain. However, as we biology. will describe, regulation of patterning in this earliest phase seems In this Review, we outline the advances made over this decade particularly important for the development of the uniquely in understanding cerebellum development and discuss their mammalian midline expanded region of the cerebellum known as significance for clinical science. Using insights gained from the ‘vermis’ (Fig. 1A). studies of sharks, paddlefish, zebrafish, frogs, chicks and mice, Lagging behind the establishment of rhombomere boundaries we focus on three distinct aspects of cerebellar development that (Simon et al., 1995), specific cell types are allocated along the represent autonomous phases of growth: the allocation of the dorsoventral axis. For glutamatergic cells of the cerebellum, this cerebellar anlage, the significance of transit amplification and is a remarkably prolonged and, importantly, a dynamic process the generation of neuronal diversity. that takes place at the most dorsal interface between neural and Lamprey** Spiny dogfish Paddlefish Trout Frog Pigeon Chimpanzee Adult Develpment DEVELOPMENT Internal GC Postmitotic No GC migration Proliferative EGL migration EGL Fig. 2. Variations in cerebellar morphology. Variation in the morphology and of the adult cerebellum (green) across vertebrates is reflected in developmental adaptations of the granule cell precursor pool (red). Cerebellar expansion in basal fish corresponds to linear extensions of the rhombic lip axially (spiny dogfish) or medially ( paddlefish). In other clades, granule cells ( pink shaded area) are distributed in an internal layer that is co-extensive with the overlying Purkinje cells (blue). To achieve this, granule cells migrate internal to (teleosts and tadpoles) or external to (metamorphic amphibians, birds and mammals) and then through the Purkinje cell layer. Only in birds and mammals do granule cell precursors themselves migrate in substantial numbers to form a transient external germinal layer. **A theoretical model of the as yet uncharacterised embryonic lamprey. 4032 REVIEW Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 non-neural ‘roof plate’ tissue: the rhombic lip (in mouse, at E10.5- E18.5). This phase generates the basic dichotomy between Box 2. Early cerebellar patterning defects and cognitive GABAergic and glutamatergic cell types that underlies the impairment conserved Purkinje-granule cell circuit, but, as we will see, it is The past 20 years have seen an increasing awareness of the role of the cerebellum in non-motor functions (Schmahmann, 2010). These functions also responsible for the diversity of cerebellum output connectivity are reflected in the higher cognitive function defects that accompany motor across species. dysfunction following cerebellar damage (Schmahmann and Sherman, Cell type allocation precedes a third, distinct temporal phase of 1998). Pre-term damage to the developing cerebellum also predicates development that extends into early prenatal life (postnatal day 21 in long-term cognitive deficits (Limperopoulos et al., 2007). Furthermore, mouse and up to 2 years in humans). Here, the principal derivative of congenital deficits, in particular vermal agenesis, lead to later the rhombic lip, the granule cell precursor, accumulates over the communicative and affective relational disorders (Tavano et al., 2007). surface of the cerebellum and undergoes further rounds of symmetric Accordingly, a study of structural brain abnormalities in mouse models of autistic spectrum disorder (ASD) revealed cerebellar-specific disruptions divisions in a process of transit amplification that exponentially (Steadman et al., 2013). Although this variety of sources suggests that expands its numbers. Growing evidence suggests that this most early patterning defects might generate significant cognitive impairment, investigated phase of cerebellum development is substantially reduced the most compelling evidence in support of this hypothesis is a recent or absent in aquatic vertebrates (Fig. 2). Because the final form of the analysis of a mouse model of human CHARGE syndrome. CHARGE mammalian cerebellum is so much a product of the first and third syndrome is reflected in a cluster of congenital abnormalities including phases of development, we will consider these first before looking at ASD-like behavioural problems in humans. In mice, mutation in the chromatin modifier Chd7 leads to a vermal hypoplasia that can be directly the less well-understood process of cell type allocation. linked to changes in Otx2 repression at the midbrain-rhombomere 1 boundary (Yu et al., 2013). This raises the possibility that other Defining the cerebellar anlage: molecular boundaries and unrecognised early patterning defects may underlie a range of human the role of Fgf8 cognitive deficit syndromes (Haldipur and Millen, 2013). A fundamental determinant of cerebellar morphology is the allocation of a territory in which its component cell types are specified. Despite the distinct structure and clear boundaries of the rostral boundary, Hoxa2 at the caudal boundary and Gbx2 cerebellum, this simple problem has proved more enduring than expressed in r1, abutting Otx2 and genes expressed at the MHB might have been anticipated. Similarly, the association of cerebellar organiser. Otx2 is required from an early stage to establish induction with the diffusible morphogen fibroblast growth factor 8 forebrain and midbrain territories, and its absence causes a rostral (Fgf8) has acquired a more nuanced perspective. It seems likely that expansion of the cerebellar Gbx2-positive territory at the expense FGF signalling has far-reaching evolutionary and developmental of midbrain tissue (Acampora et al., 1997). Conversely, ectopic significance for other aspects of brain development, such as the expression of Otx2 in the rostral hindbrain transforms this region allocation of isthmic territory and the origins of the mammalian into a Gbx2-negative midbrain identity with a caudal shift in the vermis, tying embryonic events at the early stages of axial position of the MHB (Broccoli et al., 1999; Katahira et al., 2000). specification to surprisingly profound clinical consequences for Most recently, it has been shown that the conditional deletion of higher cognitive function (Box 2). Otx2 throughout the dorsal midbrain, leaving the MHB intact, is sufficient to disrupt the differentiation of midbrain cell types and The cerebellar anlage sits between Hox and Otx domains induce a program of cerebellar development in the dorsalmost The anlage of the cerebellum is a product of the mechanisms of region of the midbrain (Di Giovannantonio et al., 2014). Thus, it is segmentation that establish iterated rhombomeric subdivisions within an absolute requirement that Otx2 is absent for cerebellar the early hindbrain just after neural tube closure. The establishment differentiation to begin. and maintenance of the boundaries defining the territory of the In a similar manner to Otx2, Hoxa2 expression is crucial for cerebellum has been a subject of several recent studies. These have determining the caudal limits of cerebellar differentiation. Loss of built our current understanding that all of the cells of the cerebellum Hoxa2 causes caudal expansion of the cerebellum (Gavalas et al., arise from dorsal rhombomere 1 (r1), a region that is definitively 1997), and ectopic Hoxa2 expression in r1 suppresses the characterised by an absence of the expression of Otx and Hox genes specification of cerebellar neurons (Eddison et al., 2004). By (Fig. 3). Early studies using quail-chick grafting to map boundaries of contrast, Gbx2 expression in r1 is required for the formation of the the neuromeres of the brain concluded that the majority of the cerebellum, which is replaced by an expanded midbrain in Gbx2 cerebellum arises from the metencephalic (rostral) hindbrain, but mutants (Wassarman et al., 1997). However, rather than playing a that as much as one-third of cerebellar granule cells originate from direct role in cerebellar differentiation, Gbx2 function appears to be the mesencephalon, which is rostral to the midbrain-hindbrain limited to the inhibition of Otx2. This is conclusively demonstrated in constriction (Hallonet et al., 1990). Later, this idea was overturned by zebrafish by the rescue of Gbx2-null mutations by reduction of Otx2 instead mapping the molecular boundary between the midbrain and expression (Foucher et al., 2006; Su et al., 2014). In experiments hindbrain as the caudal extent of Otx2 expression (Millet et al., 1996), where Gbx2 is overexpressed in the midbrain territory, cerebellar showing that all cerebellar cells are born from Otx2-negative tissue tissue can be induced (Millet et al., 1996; Katahira et al., 2000), but DEVELOPMENT and also demonstrating a surprising degree of anisotropic growth this is always coupled with downregulation of Otx2 expression in the proximal to the midbrain hindbrain-boundary (MHB). The caudal region. Therefore, it is likely that cerebellar differentiation occurs due boundary of cerebellar territory has also been mapped by chimeric to the repressive actions of Gbx2 on Otx2, rather than via a specific grafting to the r1/2 boundary, as marked by Hoxa2 expression inductive role of Gbx2. (Wingate and Hatten, 1999). Together, these studies demonstrate a key requirement for the The discovery that molecular, rather than morphological, absence of Otx2 and Hoxa2 expression in r1 to generate a boundaries are crucial in determining cerebellar territory was cerebellum. However, there is also a large body of work looking soon followed by studies looking at the function of these genes in at how the precise positions of these boundaries are set and determining the fate of their respective territories: Otx2 at the maintained as lineage restriction boundaries. For example, it has 4033 REVIEW Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 A expression is sufficient to rescue the loss of the cerebellum in Midbrain zebrafish fgf8 mutants (Foucher et al., 2006), demonstrating that Rhombomere 1 FGF signalling is not required to directly induce cerebellar differentiation and instead acts to maintain r1 as an Otx2-negative Hindbrain domain. However, FGF signalling does appear to have a role beyond B maintaining the caudal limit of Otx2 expression. Blockade of FGF signalling in r1, leaving MHB signalling intact, appears to affect elongation of the rhombic lip and r1, suggesting that FGF signalling mediates growth of the territory (Green et al., 2014). Given the anisotropic nature of growth at the isthmus (Millet et al., 1996) and the rostral origin of the cerebellar vermis in r1 (Sgaier et al., 2005), it Chick Mouse Fgf8-deficient is possible that the vermal dysplasia observed in Fgf8 hypomorphic mouse mice may be attributed to a reduction in Fgf8-mediated growth Key (Fig. 3), in addition to the loss of cerebellar territory through Otx2-derived Fgf8-dependent Fgf8-independent Hoxa2-derived expansion of the roof plate (Basson et al., 2008) and the Otx2 midbrain isthmic/vermal region cerebellum hindbrain domain (Sato and Joyner, 2009). Furthermore, in sprout 2 (Spry2) Fig. 3. FGF signalling regulates territorial allocation and anisotropic mutants, in which negative feedback of FGF signalling is reduced, cerebellar growth from rhombomere 1. (A) In early embryonic development, the cerebellar vermis is expanded (Yu et al., 2011), suggesting an the boundaries of rhombomere 1, from which the cerebellum derives, is increase in rostral r1 growth. defined by the exclusion of Otx (red) and Hox (blue) genes. FGF signalling Corresponding to this role as a proliferative node, the isthmic (yellow) is established at the anterior end of rhombomere 1. (B) Colour coding region of r1 is evolutionarily diverse. In actinopterygian fish (see indicates the contribution of territorial patterning mechanisms to regions of the adult cerebellum in birds and mammals. It seems likely that differences in their Glossary, Box 1), it is the origin of a sometimes hugely elaborate organisation reflect changes in the influence of isthmic FGF signalling on the and expanded valvulus (Chaplin et al., 2010; Kaslin et al., 2013). initial expansion of the anlage. The induction of both the mammalian vermis It also spawns a range of isthmic nuclei with sensory coordinating (a medial expansion that is absent in other vertebrates) and isthmic territory, roles across different vertebrates, which develop from a newly which lies just rostral to the cerebellum, is dependent on FGF (yellow). This identified FGF-dependent domain of isthmic Atoh1 expression suggests that the evolution of the mammalian vermis occurred at the expense (Green et al., 2014). The evolutionary emergence of the mammalian of a more-extensive isthmic territory. Cerebellar differentiation (green) is inhibited by isthmic signalling, suggesting that FGF expands the precursor vermis appears to have been at the expense of the development of a pools but is not directly involved in cerebellar specification. subset of isthmic nuclei (or a valvulus) (Fig. 3). This presents a paradox, given that the scale of both the isthmic structures and the vermis are dependent on FGF signalling. The resolution been shown that distinct pathways govern the morphological and of this contradiction lies in recent evidence that FGF, molecular features of the MHB downstream of the common perhaps paradoxically, inhibits cerebellar development: while transcription factor grainyhead-like 2 (Grhl2), with engrailed 2 FGF signalling increases the size of the cerebellar anlage, acting downstream of Grhl2 to promote cell survival and formation downregulation of FGF signalling is essential for the specification of the molecular boundary (Dworkin et al., 2012). In addition to the of cerebellar cell types (Suzuki-Hirano et al., 2010). Furthermore, co-repressive actions of Otx2 and Gbx2 at the boundary, Fgf8, Gbx2 overexpression of Fgf8 drives the specification of non-cerebellar, and Notch signalling (Sunmonu et al., 2011; Tossell et al., 2011) Lhx9-positive cell types in early r1 and at the isthmus in favour of promote cell sorting and, hence, lineage restriction at the boundary. later-born cerebellar cell types (Green et al., 2014). Hence, the The signalling molecule Fgf8 also has a key role in establishing and removal of FGF signalling after a period of establishing territory maintaining the cerebellar boundary. boundaries and promoting growth is essential for the onset of cerebellar development. FGF signalling: an inducer or repressor of cerebellar development? Fgf8 is the major signalling molecule in the MHB, and it is Transit amplification and the size and foliation of the expressed within the Gbx2-positive domain and abutting Otx2 cerebellum expression (Hidalgo-Sanchez et al., 1999). Previously, it was Although early events can significantly bias patterns of cerebellar considered that FGF signalling from this boundary induced growth, the final shape and size of the cerebellum of mammals and cerebellar development, due to the ability of ectopic Fgf8 in the birds ( possibly all reptiles) is the product of a remarkable example midbrain to induce a secondary cerebellum (Crossley et al., 1996; of a discrete phase of transit amplification that occurs much later in Liu et al., 1999; Martinez et al., 1999; Sato et al., 2001). Fgf8 is also development. This proliferative episode takes a small number of essential for the survival of the entire midbrain-hindbrain region and Atoh1-positive granule cell precursors and multiplies their numbers is required in a dose-dependent manner for the development of the by many fold through multiple symmetrical mitoses of single fated DEVELOPMENT vermis (Meyers et al., 1998; Chi et al., 2003; Basson et al., 2008). germinal cells. The transient appearance of this population of However, much like Gbx2, FGF signalling at the MHB appears to granule cell precursors over the surface of the cerebellum was act primarily by inhibiting Otx2 in the r1 territory. Where reduction quickly identified as a key feature of cerebellum development of Fgf8 causes loss of the vermis, an expansion of Otx2 expression is (Ramón y Cajal, 1894) and offered an intuitive explanation for the also seen in dorsal r1 (Sato et al., 2004; Sato and Joyner, 2009). massive foliation of the cerebellar surface in mammals. More Correspondingly, where ectopic Fgf8 induces cerebellar tissue in recently, the same logic has made the outermost layer of the the midbrain territory, a downregulation of Otx2 expression always cerebellum, the external germinal layer (EGL, see Glossary Box 1), accompanies this switch of cell fate (Liu et al., 1999; Martinez et al., an obvious candidate for medulloblastoma (see Glossary Box 1), a 1999; Sato and Joyner, 2009). Furthermore, reduction of Otx2 devastating childhood cancer (Box 3). This has exemplified how 4034 REVIEW Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 differentiation and radial migration to their ultimate destination in Box 3. Medulloblastoma and the EGL the internal granule layer (Fig. 4). Medulloblastoma is a devastating paediatric cancer of the cerebellum. In The tempo of transit amplification within the EGL is driven by recent years, whole genome and transcriptome sequencing of clinical diffusible sonic hedgehog (Shh) secreted by underlying Purkinje cells samples has revealed a number of molecularly distinct subtypes of (Dahmane and Ruiz-i-Altaba, 1999; Wallace, 1999; Wechsler-Reya medulloblastoma (Jones et al., 2012; Pugh et al., 2012; Robinson et al., 2012) that frequently involve activation of the Shh and Wnt pathways. and Scott, 1999; Lewis et al., 2004), and the importance of this Disruption of transit amplification remains a compelling model for the Shh pathway in a subset of medulloblastomas has been established through subgroup of tumours, based on experimental disruption of Shh signalling a variety of experimental and genomic methodologies (Box 3). (Goodrich et al., 1997), and more recent developmental studies show Elegant studies manipulating the Shh signalling pathway appear to that commitment to the granule cell lineage is a prerequisite for tumour confirm the idea that foliation is a product of the surface expansion formation (Schuller et al., 2008; Yang et al., 2008; Li et al., 2013). generated by transit amplification (Corrales et al., 2004, 2006). Although Wnt signalling also affects cerebellar proliferation, its effects are restricted to non-granule cells (Pei et al., 2012; Selvadurai and Proliferation within the EGL has also been shown to be influenced Mason, 2012) and accordingly the Wnt-dependent subgroup of tumours, by a number of extracellular matrix (ECM) components, such as β1- along with some Shh subgroup tumours (Grammel et al., 2012), appears integrin, that are expressed both within the EGL (Blaess et al., 2004) to have a hindbrain origin (Gibson et al., 2010). Pathways that might and in cerebellar Bergmann glial cells (see Glossary, Box 1) (Frick supress transit amplification, such as BMP signalling (SMAD) (Aref et al., et al., 2012). Additionally, laminins and their α6 integrin receptor 2013), or promote differentiation (Barhl1) (Li et al., 2004) are thus subunits are confined to the outer EGL and promote granule associated with improved patient prognosis (Poschl et al., 2011), in contrast to those associated with regulating granule cell precursor progenitor proliferation in vitro, whereas vitronectin and receptor identity (Atoh1) (Schuller et al., 2008; Yang et al., 2008) or proliferation integrin subunit α5 are confined to the inner EGL (Pons et al., 2001) (Foxm1) (Schuller et al., 2007; Priller et al., 2011). Recent studies have (Fig. 4). Likewise, in contrast to its role in the cortical ventricular zone also shown that activation of the FGF (Emmenegger et al., 2013) and (Bizzoca et al., 2012), the lamina-specific expression of F3/contactin Wnt pathways (Anne et al., 2013) has tumour-supressing actions. This in the EGL suppresses Shh-dependent proliferation and is antagonised raises the possibility that other genes that antagonise granule cell by its binding partner Tag1 (Xenaki et al., 2011), the deletion of proliferation during development, such as Neurod1 (Butts et al., 2014a), which leads to ectopic subpial granule cell clusters in adult mice. may also provide a potential route to therapy. Correspondingly, premature misexpression of F3/contactin attenuates granule cell progenitor proliferation (Bizzoca et al., 2003). Taken together, these data highlight that the environment that granule insights from development both explain and offer therapeutic precursors face in the EGL is created by a balance of laminar-specific avenues for disease. ECM components, the interactions of which await detailed dissection. The factors governing how individual progenitors navigate this Balancing proliferation and differentiation in the external germinal environment and terminate transit amplification are less clear and layer yet equally important in development and disease. The lack of an The EGL (Fig. 4) is defined by its transience and proliferation, and internal cell division clock (Espinosa and Luo, 2008) has focussed by the expression of the bHLH transcription factor Atoh1 (Akazawa attention on cell non-autonomous factors such Wnt and bone et al., 1995; Ben-Arie et al., 1996, 1997), which is absolutely morphogenetic protein (BMP) pathway signals in the EGL. For required both for transit amplification (Flora et al., 2009) and for example, non-canonical Wnt signalling via Wnt3 has recently been supressing differentiation (Klisch et al., 2011). In mouse, the EGL shown to be capable of decreasing proliferation independently of persists until the third week of postnatal life, and the peak of BMP signalling (Anne et al., 2013). Conversely, multiple BMPs are proliferation occurs around birth (Espinosa and Luo, 2008). Ramón expressed in the cerebellum during EGL development and can y Cajal was able, in his first descriptions of the developing antagonise the Shh-dependent proliferation of granule progenitors cerebellum (Ramón y Cajal, 1894), to distinguish an outer EGL both in vitro and in slice cultures (Rios et al., 2004) through populated by proliferating progenitors and an inner EGL comprising regulation of Atoh1 (Zhao et al., 2008) and via miR22 (Berenguer cells that have exited the cell cycle and begun the process, of et al., 2013). Conditional deletions of intracellular mediators of TFs/receptors Fig. 4. Amniote granule cell progenitors expressed Atoh1 NeuroD1 proliferate in the outer EGL and begin α6-Integrin α5-Integrin differentiation in the inner EGL. As granule cell Cxcr4 L1/NrCAM precursors (red) mature, they make the transition from the outer to the inner EGL, which is coincident Proliferation Differentiation Mature Signals recieved Pia with exit from the cell cycle. Their presence in the Laminin outer EGL is dependent upon pia-derived SDF-1 Outer EGL Sdf1, Shh signalling via CXCR4. Although in the outer EGL, Vitronectin Inner EGL cells express the transcription factor Atoh1, which F3/contactin mediates proliferation in response to Shh secreted DEVELOPMENT PCL from Purkinje cells (blue). Subsequently, cells express Neurod1, downregulate Atoh1, and exit the cell cycle, no longer responding to Shh ligand, but IGL instead interacting with ECM components, including vitronectin and F3/contactin, that are specific to the inner EGL. Corresponding ligands and receptors in the EGL are shown in the same colour. Key Atoh1 granule Atoh1 proliferating NeuroD1 differentiating Mature granule Purkinje cell precursor granule cell precursor granule cell cell cell 4035 REVIEW Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 BMP signalling result in cerebellar defects (Fernandes et al., 2012; Tong and Kwan, 2013), although the interpretation of such Box 4. ASD and cerebellar cell types experiments is confounded by earlier roles for BMP signalling The heterogeneous nature of autistic spectrum disorder (ASD) is during dorsal neural tube patterning (Alder et al., 1999; Lee and reflected in the range of its different, potential developmental causes. Perhaps surprisingly, the most consistent pathological correlates of ASD Jessell, 1999; Broom et al., 2012). are found in the cerebellum (Courchesne, 1997). Furthermore, a recent The extrinsic events that terminate proliferation may also meta-analysis suggests that a signature constellation of anatomical include mechanisms that remove granule cell precursors from the deficits makes cerebellar damage in ASD distinct from that in either sub-pial surface of the cerebellum adjacent to basal membranes. A ADHD or developmental dyslexia (Stoodley, 2014). These include basal lamina attachment is exhibited by all granule precursors localised folia hypoplasia (Courchesne et al., 1988) or the specific loss or (Hausmann and Sievers, 1985), raising the possibility that, alteration of Purkinje cells (Ritvo et al., 1986; Fatemi et al., 2002). Specific disruption to white matter in the superior cerebellar peduncle analogous to cortical intermediate precursors in the might be associated with a loss of cerebellar output to the thalamus (Brito subventricular zone (Fietz and Huttner, 2011; Molnar, 2011; et al., 2009). The dentate nucleus, which supplies this projection, is a Borrell and Gotz, 2014; Florio and Huttner, 2014), contact with crucial link in the cortico-cerebellar close loop circuits that potentially the outer lamina is the factor that defines transit amplifying modulate higher cognitive functions in primates (Kelly and Strick, 2003; precursors. In support of this idea, the onset of radial migration is Strick et al., 2009) and humans (Kipping et al., 2013). The highly complex mediated by loss of responsiveness to the chemokine Sdf1, which and enlarged dentate nucleus in humans shows a pronounced left-right is secreted by the meninges that surround the neural tube (Lu et al., asymmetry (Baizer, 2014) and, correspondingly, consistent unilateral reduction in dentate projections is inferred from a study of individuals with 2001; Zhu et al., 2002, 2004; Vilz et al., 2005). Additional recent Asperger’s (Catani et al., 2008). Finally, a recent transgenic study in genomic analysis of mouse cerebellum following a nervous which mutation of the tuberous sclerosis gene associated with human system-specific knockout of the Sdf1 receptor Cxcr4 suggests a ASD was targeted specifically to Purkinje cells resulted in an ASD-like link between changes in responsiveness to Sdf1 and the interaction mouse phenotype (Tsai et al., 2012). Collectively, these observations with the ECM (Huang et al., 2014). This potentially suggest that, by virtue of cortico-cerebellar connectivity, selective mechanistically links the mode of migration to the interactions cerebellar cell loss can mimic the effects of what are more readily perceived as ‘cortical’ syndromes (Schmahmann and Pandya, 2008). with the ECM discussed above. However, although a large number of additional pathways have been implicated in the different tangential and radial phases of granule cell migration (Chedotal, 2010), the mechanisms that mediate the decision of individual amplification emerges as an opportunistic expedient to generate a granule cells to switch their mode of migration and exit the cell greater number of granule cells within this transient organisation. cycle remain poorly understood. Nevertheless, the presence of separable regulatory mechanisms Differentiation of progenitor zones and the generation of governing the cessation of proliferation and onset of inward radial cellular diversity migration is exemplified in the evolution of the EGL. In the cerebellum Although the territorial allocation of the cerebellum and the of the amphibian, which is the simplest tetrapod cerebellum, a sub-pial expansion of granule cell numbers that shapes cerebellar granule layer forms transiently at metamorphosis over the cerebellum morphogenesis have received a wealth of experimental scrutiny, but fails to proliferate (Uray et al., 1987). Here, Atoh1-positive cells the factors that generate cell diversity in the cerebellum have express Neurod1, which in amniotes is required for (Miyata et al., received relatively little attention. This is despite a literature that 1999) and is sufficient to trigger granule cell differentiation (Butts hints at important evolutionary changes in the diversity of neuronal et al., 2014a), and yet are held at the cerebellar surface. As for the subtypes (Llinás and Hillman, 1969; Nieuwenhuys et al., 1998) and intermediate precursors in the EGL of birds and mammals, this layer is points to a changing functional role for the cerebellum as new a transient feature of the developing cerebellum. Inward migration of networks of connections emerged in amniotes. Most recently, the amphibian post-mitotic granule cells into the internal granule layer importance of cerebellar connectivity as a potential locus of ASD (IGL) is triggered by thyroid hormone and correlated with the end of (Box 4) emphasises the need for a clear understanding of cellular metamorphosis (Gona, 1972; Hauser et al., 1986). specification mechanisms within cerebellar precursor pools. The status of the frog external granule – as opposed to germinal (proliferative) – cell layer (see Glossary, Box 1) raises the issue of Blurred lines: GABAergic and glutamatergic progenitor domains are whether the original evolutionary requirement of an EGL was for not lineage-restriction compartments proliferation alone or reflects different developmental demands. The In the same way as the definition of the territorial boundaries of the limited number of aquatic anamniotes and pre-metamorphic cerebellum was transformed by genetic insights, our understanding amphibians so far examined lack this transient structure (Rodriguez- of the origins of different neuronal subtypes within the cerebellar Moldes et al., 2008; Kaslin et al., 2009, 2013; Chaplin et al., 2010; anlage has been transformed in recent years. A key clarifying Butts et al., 2014a,b). However, many anamniotes develop an concept was identification of the origins of granule cell precursors elaborate and sizable cerebellum. This suggests that either an EGL is at the rhombic lip, a thin strip of neuroepithelium that borders the present in such species or that the indefinite developmental period non-neuronal roof plate of the fourth ventricle (Alder et al., 1996; DEVELOPMENT afforded in aquatic vertebrates may alone be sufficient to generate Wingate, 2001). Although it spans the entire rhombencephalon, large numbers of cerebellar neurons. If so, an external granule layer contributing to a variety of distinct auditory, proprioceptive and would therefore seem to be a requirement of an adaptation to land interoceptive hindbrain circuits (Rodriguez and Dymecki, 2000; colonisation and definitive embryogenesis (Chaplin et al., 2010). One Landsberg et al., 2005; Maricich et al., 2009; Rose et al., 2009), the possibility is that this facilitates homogeneous distribution of granule rhombic lip of the cerebellar anlage (rhombomere 1) is the cells within an established laminar circuitry. This implies that, within exclusive source of granule cell precursors that then migrate the frog, the accumulation of granule cells at the surface of the tangentially to form the EGL (Wingate and Hatten, 1999). The cells cerebellum is a means for distributing cells evenly across the anlage in the rhombic lip that contribute to the EGL already express Atoh1, prior to integration into the cerebellar cortex. In such a model, transit which is induced by TGFβ signals secreted from the neighbouring 4036 REVIEW Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 roof plate (Alder et al., 1999; Fernandes et al., 2012; Tong and A Lateral Dorsal Sagittal Kwan, 2013). Although this might suggest that the rhombic lip is a dorsally allocated progenitor pool, it is perhaps more appropriate to mb cb consider it as a zone of dynamic induction at the edge of the rp cb ventricular zone. The production of Atoh1-positive cells depends both on local Delta-Notch signalling and direct contact with the rp roof plate (Broom et al., 2012). Furthermore, once Atoh1 is switched on, cells rapidly migrate away from the rhombic lip (Machold and Fishell, 2005). Consistent with this dynamic definition of the rhombic lip as an B Wild type C Ptf1a–/– inductive interface, the lineage boundaries between the ventricular mb zone, rhombic lip and roof plate are somewhat blurred (Fig. 5). The egl ventricular zone of the cerebellum is characterised by Ptf1a cbn expression (Fig. 5A,B) and gives rise to GABAergic interneurons rl rp (Hoshino et al., 2005). By contrast, the roof plate comprises non- cb neural Lmx1a- (Mishima et al., 2009) and Gdf7-positive lineages vz (Currle et al., 2005) and gives rise to the choroid plexus (Fig. 5A,B). Atoh1-positive cells at the interface between these two zones are largely glutamatergic (Machold and Fishell, 2005; Wang et al., 2005; Rose et al., 2009). However, blurring is seen when genetic D Atoh1–/– E Lmx1a–/– labelling with Lmx1a- and Gdf7-driven Cre lines, which might be expected to be confined to the roof plate and its choroid plexus derivative, is also found unexpectedly in both glutamatergic and GABAergic descendants (Chizhikov et al., 2010; Cheng et al., 2012). This is significant in that it could suggest that lineages are not restricted. Furthermore, this blurring is increased on deletion of Lmx1a (Chizhikov et al., 2010), whereas genetic deletion of either Ptf1a or Atoh1 leads to increased mixing of lineages (Fig. 5C-E) (Wang et al., 2005; Pascual et al., 2007; Millen et al., 2014) due to the mutually repressive functions of these genes (Yamada et al., 2014). Loss of Ptf1a also leads to mixing between dorsal and ventral Key (non-cerebellar) ventricular derivatives (Millen et al., 2014). These Dark shading - progenitor domains Purkinje cell results suggest a dynamic segregation of lineages that is dependent Light shading - migration path Inhibitory interneuron Atoh1 Granule cell precursor on their genetically determined post-mitotic identity. Lmx1a Cerebellar nucleus projection neuron As in the EGL, the proliferation of both roof-plate and ventricular- Ptf1a/Ascl1 Ventral Lmx1b-positive neuron derived cells is, at late stages, sensitive to Shh signalling. In the roof Ascl1 (Ptf1a negative) Roof plate cell plate, endogenous Shh production stimulates secondary proliferation Fig. 5. Blurring the boundaries of lineages by genetic deletion of from non-neural precursors adjacent to the rhombic lip (Huang et al., transcriptional regulators. (A) The relationship between the cerebellum (cb), 2009; Nielsen and Dymecki, 2010). Shh secreted into the midbrain (mb) and the roof plate of the IVth ventricle (rp) in a vertebrate embryo cerebrospinal fluid (Huang et al., 2010) acts to drive early in lateral, dorsal and sagittal view. (B) A schematic sagittal section through the ventricular zone proliferation whereas, later, Shh secreted from midbrain, cerebellum and roof plate (green line in A) showing the distinct Purkinje cells also acts on a population of secondary precursors of progenitor zones at the ventricular surface (green, extra-cerebellar; blue, cerebellar; red, rhombic lip; yellow, roof plate) that contribute to different cell inhibitory neurons and glia that reside in the prospective white matter populations following distinct migratory paths (colour-coded shaded regions). (Leto et al., 2009; Fleming et al., 2013). The contributions of these progenitors zones to different cell populations are perturbed following knockdown of: (C) ventricular zone Ptf1a (Pascual et al., Timing and diversity: how cells become specified 2007; Millen et al., 2014); (D) rhombic lip Atoh1 (Machold and Fishell, 2005; Although Shh-dependent late-born populations represent the last Wang et al., 2005); or (E) roof plate Lmx1a (Chizhikov et al., 2010). egl, stages of cell production in the cerebellum, a clear temporal order of external granule later; vz, cerebellar ventricular zone; rl, rhombic lip; cbn, cell production precedes this stage. This temporal pattern is cerebellar nuclei. superimposed onto dynamically maintained progenitor zones. Thus, in the rhombic lip, the production of granule cell precursors As cerebellar nuclei represent the output connection of the proceeds alongside that of a population of small unipolar brush cells cerebellum, this diversity is functionally significant. For example, (Kita et al., 2013) that also express the T-box gene Tbr2 (Englund birds lack the most lateral of the mammalian nuclei, the Lhx9- DEVELOPMENT et al., 2006). This represents the final phase in a sequence of cell positive dentate nucleus, which in mammals targets the thalamus specification. Granule cell precursor production is preceded by the (Arends and Zeigler, 1991; Green and Wingate, 2014). This generation of glutamatergic cerebellar nuclei, which briefly express connection allows the cerebellum to participate in regulating Atoh1 but do not undergo transit amplification. The number of cortical functions and its absence in birds marks a major cerebellar nuclei varies between major amniote orders, with two in difference in brain organisation. reptiles and between three and five divisions in mammals Cerebellar nucleus neurons are the first cerebellar cells to be (Nieuwenhuys et al., 1998). These accumulate in a sequence with generated, but are not the earliest Atoh1 cells to be generated in the most lateral being born first (Hagan and Zervas, 2012; Green rhombomere 1. At pre-cerebellar stages, the rhombic lip is patterned by and Wingate, 2014). FGF signalling from the isthmus and generates Lhx9-positive neurons 4037 REVIEW Development (2014) 141, 4031-4041 doi:10.1242/dev.106559 that migrate into ventral and isthmic r1 (Machold and Fishell, 2005; development and evolution of cell diversity. The identity of this Wang et al., 2005; Green et al., 2014), contributing cells to multiple coordinating signal remains an important issue for future research. nuclei that form part of a wider hindbrain network of nuclei controlling In an analogous manner that again points to homologies between proprioception, interoception and arousal (Rose et al., 2009). seemingly diverse cell types, it has become clear that the secondary The switch from the production of cerebellar neurons to granule proliferative zones that emerge from precursor pools (the EGL, white cells at E12.5 in mouse (Machold and Fishell, 2005; Wang et al., matter stem cells and roof plate) all respond to a common signal: Shh. 2005) is paralleled by a switch in the production of GABAergic Thus, the elaboration of cerebellar structure brought about by the EGL neurons in the ventricular zone from Purkinje cells to other types of is, in mammals, elegantly coupled to the number and perhaps diversity interneurons and glia (Sudarov et al., 2011). This correlates with the of interneurons. At the centre of this relationship, the early embryonic changing patterns of Olig2 and Gsx1 expression between E12.5 and interactions that link choroid plexus and rhombic lip development E14.5. The expression of Gsx1, which marks interneuron (Broom et al., 2012) may graduate during development into an intra- progenitors, gradually expands dorsally and into the Olig2 lineage ventricular signalling mechanism that coordinates different aspects of that, before E12.5, gives rise to only cerebellar nucleus and Purkinje cerebellar development (Johansson et al., 2013). Whether the cells (Seto et al., 2014). In contrast to the rhombic lip, where the evolutionary emergence of the EGL was accompanied by the outcome of a single inductive interaction changes over time, emergence of other secondary, transit-amplifying epithelia would temporal patterning in the rest of the ventricular zone may reflect provide an interesting perspective on this argument. dynamic reorganisation of variously identified dorsoventral regions Perhaps the most subtle, though important, revision of conventional (Chizhikov et al., 2006; Zordan et al., 2008; Grimaldi et al., 2009; wisdom on cerebellar development is the demonstration that the Mizuhara et al., 2010; Florio et al., 2012). cerebellar anlage is superseded by an isthmic anlage of Lhx9-positive The correlation in the timing of fate switches and the observation extra-cerebellar nuclei (Green et al., 2014). Rhombomere 1 does not that this occurs even when Atoh1 or Ptf1a are misexpressed in the equate to the cerebellum, nor does FGF induce its development. ventricular zone or rhombic lip, respectively (Yamada et al., 2014), Rather, the temporal dynamics of FGF signalling appear to affect a suggest that a common, non-autonomous factor regulates the overall balance of cell production and are capable of generating a diversity of temporal development of the cerebellum and support the idea that isthmic specialisations. Crucially, the human vermis, the genesis of progenitor populations share common features. Transplantation which seems vital for a range of cognitive and affective behaviours, is studies of both GABAergic (Leto et al., 2006, 2009) and a product of this process (Yu et al., 2013). glutamatergic rhombic lip progenitors (Wilson and Wingate, 2006) Finally, cerebellar functions beyond the traditional role of support the concept of an extrinsic cue for developmental timing. The sensorimotor integration appear to rely on cerebellar communication choroid plexus, which is generated from the roof plate lineage and with the cortex via the thalamus that depends explicitly on the dentate whose development is at least partially regulated by the rhombic lip nucleus. Understanding the genetics underlying the production of (Broom et al., 2012), is an attractive candidate for orchestrating distinct nuclei and their distinct projection patterns is an important coordinated changes in cell fate through the secretion of a range of future challenge. In this regard, the cerebellum is likely to remain at the factors, including Shh (Huang et al., 2010), Igf2 (Lehtinen et al., forefront of developmental neuroscience. 2011), retinoic acid (Yamamoto et al., 1996; Wilson et al., 2007) and thyroid hormone (Koibuchi, 2008). The choroid plexus may thus Competing interests The authors declare no competing financial interests. prove to be a factor in early cerebellar dysgenesis and offer a locus for understanding the coordinated diversification of glutamatergic and Funding GABAergic neuronal subtypes during cerebellar evolution. The authors’ research was supported by grants (R.J.T.W.) from the Biotechnology and Biological Sciences Research Council, Wellcome Trust and a Medical Research Conclusions Council studentship (M.J.G.). As with the study of many parts of the developing brain, specialised References interests in specific phases of growth of subtypes of cells sometimes Acampora, D., Avantaggiato, V., Tuorto, F. and Simeone, A. (1997). Genetic obscure a larger picture of coordinated programmes of control of brain morphogenesis through Otx gene dosage requirement. development. 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