Received: 29 January 2019 Revised: 10 April 2019 Accepted: 16 May 2019

DOI: 10.1002/dvdy.64

REVIEW

The generation of granule cells during the development and

evolution of the cerebellum

Angelo Iulianella1 | Richard J. Wingate2 | Cecilia B. Moens3 | Emily Capaldo1

1
Department of Medical Neuroscience and
Brain Repair Centre, Life Science Research
Abstract
Institute, Dalhousie University, Halifax, The cerebellum coordinates vestibular input into the hindbrain to control balance and
Nova Scotia, Canada movement, and its anatomical complexity is increasingly viewed as a high-
2
MRC Centre of Neurodevelopmental
throughput processing center for sensory and cognitive functions. Cerebellum devel-
Disorders, King's College London,
London, UK opment however is relatively simple, and arises from a specialized structure in the
3
Division of Basic Sciences, Fred anterior hindbrain called the rhombic lip, which along with the ventricular zone of
Hutchinson Cancer Research Center, Seattle, the rostral-most dorsal hindbrain region, give rise to the distinct cell types that consti-
Washington
tute the cerebellum. Granule cells, being the most numerous cell types, arise from
Correspondence the rhombic lip and form a dense and distinct layer of the cerebellar cortex. In this
Angelo Iulianella, Department of Medical short review, we describe the various strategies used by amniotes and anamniotes to
Neuroscience and Brain Repair Centre, Life
Science Research Institute, Dalhousie generate and diversify granule cell types during cerebellar development.
University, 1348 Summer Street, Halifax,
NS, Canada B3H-4R2. KEYWORDS
Email: angelo.iulianella@dal.ca amniotes, anamniotes, Atoh1, cerebellum, Cux2, external granular layer, granule cells

Funding information
Canadian Institutes of Health Research,
Grant/Award Number: PJT-156364; Natural
Sciences and Engineering Council of
Canada, Grant/Award Number: RGPIN-
04475-2015

1 | INTRODUCTION granule cells, we are only beginning to appreciate the diverse

The cerebellum has been studied since the time of Santiago
Ramon y Cajal at the turn of the 20th century for its simple
three-layered structure, yet complex connectivity. Cajal once
described the intricate anatomy of the cerebellum, and particu-
larly the endlessly diverting beauty of a Purkinje cell (PC), the
main output/integrator neurons of the cerebellum, by comparison
to trees: “In our parks are there any trees more elegant and luxu-
rious than the PCs from the cerebellum?”1 While the cellular
anatomy and organization of the cerebellum have been studied
for more than a century, we have yet to have a comprehensive
understanding of how the different cell types arise and ultimately
organize into circuits. Even within a single-cell type, such as
1
Javier DeFelipe; Cajal's butterflies of the soul. https://blog.oup.com/2013/
11/cajal-butterflies-of-the-soul-cerebral-cortex/
molecular and developmental strategies used in different verte-
brates to diversify progenitor pools in the cerebellar primordium.
In this review, we focus on the morphogenesis of the cerebellum
and lineage diversification of its progenitors during develop-
ment, with particular focus on granule cell neurons (GCN).
2 | O V E R V I E W O F C E R E B E L L AR
ANATOMY
The cerebellum sits inferior to the cerebrum and posterior to
the brainstem, comprising the dorsal wall of the fourth ven-
tricle. It was once thought to only be involved in regulating
motor function, however recent research has suggested that
alterations in cerebellar anatomy and function can not only
lead to ataxias but can also contribute to autism spectrum

506 © 2019 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/dvdy Developmental Dynamics. 2019;248:506–513.

IULIANELLA ET AL.
disorder.1-5 Its study thus has important implications for our
understanding of neurodevelopmental disorders. Anatomi-
cally, the cerebellum is tightly organized as a dense tri-
laminar structure consisting of an internal granule cell layer
(IGL), Purkinje cell layer (PL), and molecular layer (ML),
comprised of interneurons, these layers combined pack a tre-
mendous degree of processing power. The anatomical orga-
nization and connectivity of the cerebellum allows it to
efficiently function as a byway for processing a myriad of
sensory modalities and cognitive tasks requiring rapid com-
munication between distinct brain centers. This includes ves-
tibular input to refine locomotion, sensory integration from
specific facial nuclei, principally including the trigeminal,
that processes information from the head and face and asso-
ciated glands, and input from autonomic motor pathways
emanating from hindbrain derivatives, particularly the pons.
The basic cerebellar circuit is highly compact (Figure 1).
Incoming climbing fibers (CFs) from the inferior olive synapse
primarily on PCs, while mossy fibers (MFs), which make up
the bulk of the cerebellar afferents, synapse predominantly on
granule cells, while both project collaterals to cerebellar
nuclei.6-10 Granule cells (GCs) and Golgi cells coinhabit
the granule cell layer, which in amniotes is the innermost
FIGURE 1 Schematic of the microarchitecture of the cerebellar
cortex circuitry depicting the three layers: molecular layer (ML),
Purkinje cell layer (PL), and granule cell layer (GL). The main
excitatory neuronal connections involve the mossy fibers (MFs),
climbing fibers (CFs), granule cell neurons (GCN), and parallel fibers
(PFs). The principal inhibitory connections involve Purkinje neurons
(PN), and a diversity of interneurons types including stellate cells
(SCs), basket cells (BCs), and Golgi cell (GC) interneurons. The local
circuit excitatory input pathways include CF and MF. MF synapse onto
GCN, which give rise to excitatory PF. Both PF and CF synapse
directly onto PN, which outputs signals from the cerebellar cortex
through synapses on the deep cerebellar nuclei (DCN)
507
layer of the cerebellar cortex. The MFs communicate with
GCs in glomeruli via ascending axons.11,12 PCs, along with
Bergmann glia, constitute the PL, which is the middle layer
of the cerebellar cortex. PCs receive input in the ML, for-
ming synapses with GCs, CFs, and interneurons.11,13-15
Finally, there are interneurons within the ML, exemplified
by the stellate and basket cells.16 Importantly, PCs form the
only output from the cerebellar cortex to extra-cerebellar
nuclei, as well as cerebellar nuclei, which in turn make up
the remaining output from the cerebellum.12,17-19
3 | DEVELOPMENT O F GRANULE
CELLS
The cerebellum arises from the most anterior hindbrain seg-
ment, rhombomere 1,20,21 and its formation is influenced by
the adjacent mid-hindbrain or isthmic region, which defines
the anterior boundary of the future cerebellum. Chimeric stud-
ies using the quail/chick marker system revealed that the cere-
bellum is derived from dorsal rhombomere 121-23 and that
GCs are specifically derived from the boundary between non-
neural fourth ventricle roof plate and neural epithelium21; a
region known as the rhombic lip (RL).24 The development of
the RL is partially dependent on the function of Zic125 and
completely dependent on the function of Atoh1/Math1,26-28
which is induced by bone morphogenetic protein (BMP) sig-
nals from the roof plate.29,30
Perhaps paradoxically, the first-born Atoh1-expressing
cells from the avian as well as mammalian cerebellar anlagen
are fated to an extra-cerebellar region.27,31 These Atoh1-
expressing cells are not RL derivatives but instead originate
from an FGF8-induced Atoh1 expression domain within the
isthmus.31 As development progresses, the retreat of fibroblast
growth factor signaling at the isthmus derepresses BMP sig-
nals from the roof plate, which promotes the formation of the
first migratory populations derived from Atoh1 expressing RL
progenitors in discrete temporal cohorts, as well as the precur-
sors of the deep cerebellar nuclei.8,31 Subsequently, RL pro-
genitors generate migratory GC precursors,27 which assemble
as a distinctive secondary proliferative layer on the surface of
the cerebellum. It is the formation of a secondary germinal
epithelium, the external granule layer (EGL) that allows GC
to become the most numerous neuronal cell type in the central
nervous system.32-34 The EGL subsequently gives rise to
inwardly, radially migrating postmitotic GCs that occupy the
IGL.13
Avian EGL precursors arise from the entire caudal border
of the cerebellum, from the midline to the caudolateral cor-
ner or RL.35 In rodents, there is more proliferation at the
caudal-lateral region of the cerebellar anlage, suggesting that
the RL gives rise to most of the cerebellar EGL.36 These pre-
cursors migrate rostrally, following a tangential migration
508
pattern up and over the developing fourth ventricle and cere-
bellar anlage, often crossing the midline from both medial
and lateral origins.22,37,38 Early born GC precursors accumu-
late furthest from the RL in the rostral cerebellar anlagen,
with later born cells filling in more caudally by migrating a
shorter distance.21,39 RL derivatives tend to be clonally
related and migrate as distinct streams that disperse along
the mediolateral axis as they emerge from the RL. As was
observed for the cerebellar anlage ventricular zone (VZ)
derivatives, clones can have either large or small migration
ranges.35 Interestingly, migrating cells within the EGL are
distinctly unipolar,21,35 suggesting a strong attractive and/or
repulsive drive, while cells found in the superficial, more
proliferative EGL tend to have more complex morphologies.
Indeed, the migration of RL derivatives is driven by a com-
bination of slit-mediated chemorepulsion from the RL40 and
netrin-mediated attraction.40,41
Once precursors have migrated to fill in the EGL they
begin a variable number of rounds of transit amplifying,
symmetrical divisions,42 and are characterized by the expres-
sion of the neural progenitor marker Pax6.37 After their ter-
minal division, postmitotic GCs switch their migration
pattern from tangential (oriented in the anteroposterior axis
of the cerebellar analge) to radial (oriented ventrodorsally),
and translocate to the forming IGL (reviewed by Reference
43
). In birds, the migration of cells from the EGL to the IGL
occurs within “ribbons,” or raphes, which are molecularly
distinct stripes separating molecularly distinct cellular
domains first visible at E8.16,34,38 As GCs migrate radially
inwards to the IGL, they pass in between differentially
expressing EphA4 segments.34 These migrating cells express
the transcription factors Neurod1,44 Zic1,25,45 and Pax6.37
Neurod1 seems to be important for terminating the prolifera-
tion of EGL GC precursors by down regulating the expres-
sion of another helix-loop-helix transcription factor Atoh1,
thereby promoting migration to the IGL. When neurod1 is
aberrantly expressed in the early E4 chicken RL, EGL cell
proliferation is decreased, along with the loss of the prefer-
ential migration into the IGL directly.46
Shh is vitally important for GC precursor proliferation
and differentiation. When E11 or E12 chicken embryonic
cerebellar EGL explants are treated with Shh they express
higher levels of Gli1 and Zic1, two transcriptional targets of
Shh signaling in GCs.32,47 Shh from PCs acts directly on the
proliferation of GC in the outer EGL and indirectly influ-
ences their differentiation in the inner EGL by inducing the
differentiation of radial glia, which in turn stimulates GC
differentiation and migration to the IGL.32
It is well established that GC neurons arise from the RL
during development. However, work from Wojcinski et al48
has shown that there is a surprising secondary source of GCs
that are mobilized only under specific conditions. Namely,
IULIANELLA ET AL.
Nestin-positive glial cells act as a progenitor reservoir in the
adult cerebellum to supply newborn GCN upon cerebellar
damage, such as that caused by the massive cell death accom-
panying irradiation. Presumably, the embryonic origin of
these nestin-positive “reserve” GC precursor cells is the cere-
bellar VZ, which is distinct from the RL region. This finding
gives us hope for the regenerative properties of the cerebellum
after injury from oncogenesis and the side effects of using
chemotherapy and radiation. What is perhaps most stunning
about this discovery is that newborn GCs originating from the
damaged brain were able to migrate away from the interstices
of the cerebellum and reconstitute an EGL. Future work
should mine the intriguing mechanisms that guide the migra-
tion of these cells to maintain the overall anatomical architec-
ture and wiring of the adult cerebellum. In summary, there are
at least two independent mechanisms for generating GCs in
the cerebellum: an embryonic source derived from the RL,
and an adult reservoir residing in the radial glial cell niche.
This leads us to ask how diversity is generated within a
common pool of GC precursors, such as the Atoh1-positive
EGL pool, and whether different vertebrates have utilized
distinct mechanisms of achieving this diversity during the
course of evolution? This question is best addressed using a
comparative approach in the major classes of vertebrates.
4 | ATOH1 D I V E R S I F I C A T I O N
IN TELEOSTS
We know from studies in the mouse that Atoh1 plays a central
role in the diversification of RL progenitors and specification
of cerebellar GC. Atoh1 loss-of-function mutants display a
lack of the EGL, agenic RL, and cerebellar nuclei.26,28,49
Interestingly, in amniotes Atoh1 is expressed in the RL
throughout development, but its expression oscillates from
low to high levels along with the successive renewal of pro-
genitor populations.27,31 This suggests that a single Atoh1
gene is reused at several points during cerebellar development
to promote the formation of different cell types from RL pro-
genitors. The situation is more complex in teleosts, which due
to genome duplication deep in their phylogeny have more
paralogs of some genes than tetrapods do.50 Zebrafish pos-
sess three Atoh1 genes: atoh1a, atoh1b, and atoh1c. They
are all expressed in the anterior hindbrain during the forma-
tion of the cerebellar primordium, with important differences
in nonoverlapping domains.51-53 Atoh1a and atoh1b are
broadly expressed, encompassing the entire rhombic lip and
hindbrain, with the former being more strongly expressed. In
contrast, atoh1c is activated early in hindbrain development,
within the isthmic organizer at the mid-hindbrain junction,
and then highly restricted to the upper RL throughout cere-
bellar development, persisting there after the other paralogs
have been turned off. This suggests that the different
IULIANELLA ET AL.
zebrafish atoh1 paralogs have evolved to act within sub-
domains and at different times to specify distinct cell types,
contrasting with the reactivation of Atoh1 in the mammalian
RL to parse out the distinct cerebellar fates. To test this
hypothesis, the Moens lab set out to map out the derivatives
of atoh1c-expressing cells using a photoactivatable atoh1c
genetic lineage construct. They discovered that the early
atoh1c-expressing isthmic progenitors gave rise to anterior
hindbrain neurons outside the cerebellum, likely analogous
to the neurons derived from an early isthmic domain of
atoh1 expression described in the chick.31 Atoh1c RL pro-
genitors give rise to granule neurons as expected.53 Interest-
ingly the deletion of atoh1c eliminated most but not all
granule neurons in the cerebellar corpus, homologous to the
mammalian cerebellar vermis, however atoh1a; atoh1c dou-
ble mutants lacked all GC neurons in the corpus. Combined
with the finding that atoh1a and atoh1c mark non-
overlapping derivatives in the corpus, this data suggest that
the different atoh1 genes in the zebrafish genome may spec-
ify different GC neuron subpopulations. The teleost genome
duplication may thus have provided a mechanism for the
diversification of granule neuron types. Future single-cell
transcriptomic approaches in fish and mouse will reveal the
extent and conservation of this GC diversity.
5 | STRAT E GI E S FOR R E GU L A T I N G
G R A N U L E CE L L N U M B E R I N
VARIOUS VERTEBRATES
A key difference between amniotes and anamniotes is the
absence of an external EGL in the latter.54 Given the impor-
tance of the EGL in generating vast numbers of GC in birds
and mammals, how do fish, reptiles, and amphibians go
about diversifying and maximizing the number of GC? To
address this question requires a broader view of GC develop-
ment across a range of vertebrates. An interesting conclusion
from studies of cartilaginous and basal ray-finned fishes is
that GC number may be proportional to the rostral-caudal
extent of the Atoh1-positive RL of the cerebellum in ances-
tral vertebrates.51,54 In the absence of secondary prolifera-
tion, the number of adult GC is simply determined by the
length of the cerebellar RL and the duration of cell prolifera-
tion. In fish, the brain continues to grow throughout life.
An EGL first appears during the life cycle of the amphib-
ian. Remarkably, tadpoles have a fish-like embryonic cere-
bellum, but at metamorphosis and with the development of a
tetrapod body plan, GCs accumulate over the surface of the
cerebellum in a nonproliferative external layer. Work from
the Wingate lab has shown that in Xenopus a critical GC pre-
cursor determinant Neurod1 is expressed within this layer.46
By contrast, in amniotes Neurod1 expression is restricted to
inwardly migrating GC and is excluded from the more
509
superficially localized proliferative EGL.44 This finding sug-
gests that a shift in the timing of Neurod1 expression may
suppress a potentially transit-amplifying pool of GC precur-
sors in amphibians. Correspondingly, overexpressing
Neurod1 earlier than normal in the cerebellar primordium of
birds leads to a premature repression of Atoh1 expression and
promotes the radial migration of granule cell precursors.46
Why then do metamorphic amphibians have a non-
proliferative EGL? One possibility is that the appearance of
the EGL is related to the constraints on embryonic brain
growth in terrestrial vertebrates. A superficial GC layer
allows for a rapid and even distribution of GC across the cer-
ebellum within a limited embryonic period. The subsequent
emergence of proliferation in the EGL appears to have been
a secondary feature that then allowed the enormous expan-
sion of cerebellar size in birds and mammals, facilitated by
Shh expression in PCs. Intriguingly, in reptiles, where there
is a wide diversity of cerebellar form, it is unclear whether
Neurod1 plays a role in regulating EGL proliferation and
hence the size and shape of the cerebellum (Figure 2).
FIGURE 2 Vertebrate evolutionary relationships based on the
presence of proliferation in the EGL during cerebellar development. An
EGL that comprises transit-amplifying granule cell precursors first
emerged in the mammalian/reptilian lineage. While birds and mammals
both exhibit a transit-amplifying EGL (filled red lines), the status of the
EGL in non-avian reptiles is unknown (empty red lines). Tadpoles do
not exhibit an EGL, but at metamorphosis, a non-proliferative EGL
appears that is characterized by expression of Neurod1, which is
associated with neural differentiation (black lines). In birds and
mammals, where the EGL contains dividing cells, Neurod1 expression
is seen in the IGL only. There is a range of cerebellar anatomies in
reptiles, from the flat plate-like amphibian to the tri-foliated crocodilian
cerebellum. The presence of a Neurod1-positive EGL and IGL, and
Shh-expressing PCs are denoted by the filled-in boxes on the right.
(Phylogenetic tree adapted from Reference 55). EGL, external granule
layer; IGL, internal granule cell layer; PC, Purkinje cell
510 IULIANELLA ET AL.

FIGURE 3 Cux2 is a rhombic lip and external granule layer marker that identifies tangentially and radially migrating cerebellar precursor
cells. (A) 40 ,6-diamidino-2-phenylindole-stained section of an E12.5 mouse anterior hindbrain showing the formation of the cerebellum and RL in
the fourth ventricle region. (B) Section of the Cb showing tdTomato+ cells from an E16.5 Cux2CreER(T2);R26rTomato transgenic embryo pulsed
with tamoxifen at E12.5 to activate the tdTomato reporter gene. Cux2+ cells were found in the RL, EGL, and migratory precursor cells.
(C) Summary of Cux2+ cell movements in the fetal mouse brain. Tangentially migrating cells (red arrows) moved along the dorsal surface of the Cb
to invade the developing EGL (yellow arrows), and core of the cerebellar primordium, which migrate radially toward the EGL (black arrows).
(D) Still images of migrating Cux2+ cells form E14.5 Cux2CreER(T2);R26rtdTomato transgenic embryo pulsed with tamoxifen at E12.5 to label
the RL and EGL. Elapsed time (t) in slice cultures is indicated in minutes, showing the distinct unipolar morphology of migratory RL derivatives
that expressed Cux2. 4thV, fourth ventricle; Cb, cerebellar primordium; ChP, Choroid plexus; EGL/egl, external granular level; Mb, midbrain; NTZ,
nuclear transitory zone; PC plate, Purkinje cell plate; RL, rhombic lip; VZ, ventricular zone. Scale (A) 100 μm, (C) 50 μm

6 | D O E S TH E R H O M B I C L I P and unipolar brush cells (UBCs). Moreover, Atoh1 expression

POSSESS MULTIPOTENT OR
LINEAGE-RESTRICTED
PROGENITORS?
The next question we will address concerns the possible
molecular mechanisms that generate different cell fates that
arise from the RL. More specifically, does the RL possess
multipotent or a combination of lineage-restricted progeni-
tors? As described above, Atoh1 is a definitive marker of the
RL, and defines a lineage that includes cerebellar nuclei, GC,
throughout the time course of RL development is consistent
with it potentially defining a multipotent cerebellar progenitor.
However, recent work has identified the transcription factor
Cux2 as a fate-restricted marker of the RL (Figure 3B).56 Sim-
ilar to Atoh1, Cux2 is expressed early and widely in the RL
and the rostral hindbrain of both chicken and mouse embryos.
However, genetic lineage mapping of Cux2-expressing cells
during cerebellar development revealed a preference in the
labeling of proliferative GC precursors in the EGL, migratory
GC forming the IGL, and UBC. An inducible Cux2Cre driver
IULIANELLA ET AL.
was used in these studies, so it was surprising that irrespective
of when the driver was activated, the vast majority of cells
labeled were GC (Figure 3).56 Since GC and UBC arise at the
same time from the RL region,57 it is not surprising that there
was significant Cux2-lineage restriction to these cell types.
But, the highly restrictive fate mapping of Cux2-positive RL
contrasted with the wider potential of Atoh1-expressing pro-
genitors and argues for the presence of lineage-restricted
transit-amplifying progenitors coexisting with multipotent pro-
genitors. Interestingly, previous work demonstrated that Cux2
acts upstream of Neurod1 and downstream of Notch1 signal-
ing in spinal cord progenitors.58,59 Given that both Atoh1 and
Neurod1 function downstream of Notch1 signaling, it is possi-
ble that Cux2 acts within RL progenitors to reinforce
Atoh1-modulated gene regulatory networks to diversify RL
derivatives, including GCs. Alternatively, Cux2 may play a
role in the temporal segregation of RL fates by acting within
Atoh1-expressing cells to bias their development to transit-
amplifying populations. To test this idea, Cux2 was over-
expressed early in the chicken embryonic anterior hindbrain,
which gives rise to the RL. We observed a colocalization of
the Cux2-overexpressing cells with markers for transit ampli-
fying and migratory GC.56 However, Cux2 overexpression did
not appear to shift the proportion of progenitors developing
into GC over other cell fates. Moreover, Cux2 mouse mutants
did not show any major cerebellar defects (Capaldo and
Iulianella, unpublished), possibly due to a redundancy with the
related gene Cux1, which is also expressed in the developing
GCs.60 A third possibility is that Cux2 activity in the RL and
EGL delineate subsets of GC neurons, similar to what we
argue for Atoh1 gene diversification in teleosts. Interestingly,
fate mapping using a Cux2creER(T2);Rosa26rtdtomato
reporter mouse line showed a preference for the labeling of
GC in the anterior folia, irrespective of when Cre activity was
induced (Figure 3B).56 These data are consistent with previ-
ous findings suggesting the presence of distinct subtypes of
GCs along the anteroposterior (and mediolateral) axis of the
folia.44,61 It is unclear what the significance of the patterning
of GC subtypes is, but it is tempting to speculate that it under-
lies the microcircuitry responsible for the functional segrega-
tion of distinct behavior tasks within cerebellar lobules.62-64
Given its expression in the RL of amniotes, Cux2 may be
a defining characteristic of vertebrates that develop their cere-
bella through an EGL transit-amplifying stage. If this was the
case, then one would expect to see tangentially migrating
Cux2-positive RL cells contributing to EGL formation.
Indeed, time lapse imaging of Cux2-expressing cells in the
developing mouse RL revealed labeled cells with long
dynamic unipolar processes that invaded the developing EGL
(Figure 3B,D; AI, unpublished), in a manner highly reminis-
cent of the migratory behavior previously observed in the
avian RL.40 Based on these findings, one would predict that
511
Cux2 would be highly expressed in the RL of species that
possess a transit-amplifying GCL, and less so or not at all in
the species that lack it, such as amphibians. Future investiga-
tions across a range of vertebrates will determine whether a
regulatory network of Cux and proneural factors correlate
with the acquisition and expansion of an EGL.
7 | CONCLUSION
Cerebellar GCs are the most numerous cells in the cerebel-
lum. This allows for the high neuronal density and ability of
this ancient part of the vertebrate brain to process multiple
sensory modalities from the brain stem, and regulate fine
motor control. Emerging evidence from a number of labs
suggests that multiple strategies have been used during the
course of evolution to diversify and increase the numbers of
cerebellar GC produced from the RL. This leads us to specu-
late that the formation of a dense and computationally pow-
erful cerebellar GC layer is under strong selection pressure
in vertebrates. The expansion of the GC layer can be
achieved in any number of ways, such as diversifying the
function of Atoh1 paralogs, by expanding the Atoh1 within
the RL, or by altering the timing of Neurod1 expression to
promote the production of transit-amplifying granule precur-
sor cells. Also important is the role of other regulatory fac-
tors such as Neurod1, Cux2, and Zic1 that act within the RL
and EGL to expand and diversify cerebellar cell types during
development.
ACKNOWLEDGMENTS
The AI lab is supported by funds from the Natural Sciences
and Engineering Council of Canada (RGPIN-04475-2015)
and the Canadian Institutes of Health Research (PJT-156364).
We thank Samantha Moore (AI lab) for making Figure 1 and
comments to the manuscript. This review originated from the
participation of AI, RJW, and CBM in a mini-symposium
entitled as “New Perspectives in the Development and Evolu-
tion of the Cerebellum,” held at the annual meeting of the
American Association of Anatomists/Experimental Biology
on April 21st to 24th 2018 at San Diego.
OR CI D
Angelo Iulianella https://orcid.org/0000-0003-0712-4295
REF ER ENC ES
1. Ito M. Control of mental activities by internal models in the cere-
bellum. Nat Rev Neurosci. 2008;9(4):304-313.
512
2. Schmahmann JD. Disorders of the cerebellum: ataxia, dysmetria of
thought, and the cerebellar cognitive affective syndrome. J Neuro-
psychiatry Clin Neurosci. 2004;16(3):367-378.
3. Schmahmann JD, Caplan D. Cognition, emotion and the cerebel-
lum. Brain. 2006;129(Pt2):290-292.
4. Strick PL, Dum RP, Fiez JA. Cerebellum and nonmotor function.
Annu Rev Neurosci. 2009;32:413-434.
5. Timmann D, Drepper J, Frings M, et al. The human cerebellum
contributes to motor, emotional and cognitive associative learning.
A review. Cortex. 2010;46(7):845-857.
6. Eccles JC, Llinas R, Sasaki K. The excitatory synaptic action of
climbing fibres on the Purkinje cells of the cerebellum. J Physiol.
1966;182(2):268-296.
7. Fink AJ, Englund C, Daza RA, et al. Development of the deep cer-
ebellar nuclei: transcription factors and cell migration from the
rhombic lip. J Neurosci. 2006;26(11):3066-3076.
8. Green MJ, Wingate RJ. Developmental origins of diversity in cere-
bellar output nuclei. Neural Dev. 2014;9(1):1.
9. Shinoda Y, Sugihara I, Wu HS, Sugiuchi Y. The entire trajectory
of single climbing and mossy fibers in the cerebellar nuclei and
cortex. Prog Brain Res. 2000;124:173-186.
10. Wu HS, Sugihara I, Shinoda Y. Projection patterns of single
mossy fibers originating from the lateral reticular nucleus in the rat
cerebellar cortex and nuclei. J Comp Neurol. 1999;411(1):97-118.
11. Gray EG. The granule cells, mossy synapses and Purkinje spine
synapses of the cerbellum: light and electron microscope observa-
tions. J Anat. 1961;95(Pt3):345-356.
12. Voogd J, Glickstein M. The anatomy of the cerebellum. Trends
Cogn Sci. 1998;2(9):307-313.
13. Cajal SRY. Les Nouvelles Idées sur la Structure du Système
Nerveux chez l'Homme et chez les Vertébrés. 1st ed. Paris:
C. Reinwald & Cie; 1894.
14. Lordkipanidze T, Dunaevsky A. Purkinje cell dendrites grow in
alignment with Bergmann glia. Glia. 2005;51(3):229-234.
15. White JJ, Sillitoe RV. Development of the cerebellum: from gene
expression patterns to circuit maps. Wiley Interdiscip Rev Dev
Biol. 2013;2(1):149-164.
16. Consalez GG, Hawkes R. The compartmental restriction of cere-
bellar interneurons. Front Neural Circuits. 2012;6:123.
17. Arndt K, Nakagawa S, Takeichi M, Redies C. Cadherin-defined
segments and parasagittal cell ribbons in the developing chicken
cerebellum. Mol Cell Neurosci. 1998;10(5/6):211-228.
18. Ito M, Yoshida M. The cerebellar-evoked monosynaptic inhibition
of Deiters' neurones. Experientia. 1964;20(9):515-516.
19. Ito M, Yoshida M, Obata K. Monosynaptic inhibition of the
intracerebellar nuclei induced from the cerebellar cortex. Experientia.
1964;20(10):575-576.
20. Millet S, Bloch GE, Simeone A, Alvarado MR. The caudal limit
of Otx2 gene expression as a marker of the midbrain/hindbrain
boundary: a study using in situ hybridisation and chick/quail
homotopic grafts. Development. 1996;122(12):3785-3797.
21. Wingate RJ, Hatten ME. The role of the rhombic lip in avian cere-
bellum development. Development. 1999;126(20):4395-4404.
22. Hallonet ME, Teillet MA, Le Douarin NM. A new approach to the
development of the cerebellum provided by the quail-chick marker
system. Development. 1990;108(1):19-31.
23. Hidalgo-Sánchez M, Millet S, Bloch-Gallego E, Alvardo-
Mallart RM. Specification of the meso-isthmo-cerebellar region:
the Otx2/Gbx2 boundary. Brain Res Rev. 2005;49(2):134-149.
IULIANELLA ET AL.
24. His W. Die entwickelung des menschlichen rautenhirns vom ende
des ersten bis zum beginn des dritten monats. I Verlängertes Mark
Abh Kön Sächs. Ges d Wiss Mat Phys Kl. 1890;29:1-74.
25. Aruga J, Minowa O, Yaginuma H, et al. Mouse Zic1 is involved
in cerebellar development. J Neurosci. 1998;18(1):284-293.
26. Ben-Arie N, Bellen HJ, Armstrong DL, et al. Math1 is essential
for genesis of cerebellar granule neurons. Nature. 1997;390(6656):
169-172.
27. Machold R, Fishell G. Math1 is expressed in temporally discrete
pools of cerebellar rhombic-lip neural progenitors. Neuron. 2005;
48(1):17-24.
28. Wang VY, Rose MF, Zoghbi HY. Math1 expression redefines the
rhombic lip derivatives and reveals novel lineages within the
brainstem and cerebellum. Neuron. 2005;48(1):31-43.
29. Alder J, Lee KJ, Jessell TM, Hatten ME. Generation of cerebellar
granule neurons in vivo by transplantation of BMP-treated neural
progenitor cells. Nat Neurosci. 1999;2(6):535-540.
30. Broom ER, Gilthorpe JD, Butts T, Campo-Paysaa F, Wingate RJ.
The roof plate boundary is a bi-directional organiser of dorsal neu-
ral tube and choriod plexus development. Development. 2012;139
(22):4261-4270.
31. Green MJ, Myat AM, Emmenegger BA, Wechsler-Reya RJ,
Wilson LJ, Wingate RJ. Independently specified Atoh1 domains
define novel developmental compartments in rhombomere 1.
Development. 2014;141(2):389-398.
32. Dahmane N, Ruiz i, Altaba A. Sonic hedgehog regulates the
growth and patterning of the cerebellum. Development. 1999;126
(14):3089-3100.
33. Hatten ME, Heintz N. Mechanisms of neural patterning and speci-
fication in the developing cerebellum. Annu Rev Neurosci. 1995;
18:385-408.
34. Karam SD, Burrows RC, Logan C, Koblar S, Pasquale EB,
Bothwell M. Eph receptors and ephrins in the developing chick
cerebellum: relationship to sagittal patterning and granule cell
migration. J Neurosci. 2000;20(17):6488-6500.
35. Ryder EF, Cepko CL. Migration patterns of clonally related gran-
ule cells and their progenitors in the developing chick cerebellum.
Neuron. 1994;12(5):1011-1028.
36. Altman J, Bayer SA. Prenatal development of the cerebellar sys-
tem in the rat. I. Cytogenesis and histogenesis of the deep nuclei
and the cortex of the cerebellum. J Comp Neurol. 1978;179(1):
23-48.
37. Lin JC, Cai L, Cepko CL. The external granule layer of the
developing chick cerebellum generates granule cells and cells of
the isthmus and rostral hindbrain. J Neurosci. 2001;21(1):
159-168.
38. Lin JC, Cepko CL. Granule cell raphes and parasagittal domains
of Purkinje cells: complementary patterns in the developing chick
cerebellum. J Neurosci. 1998;18(22):9342-9353.
39. Jensen P, Smeyne R, Goldowitz D. Analysis of cerebellar develop-
ment in math1 null embryos and chimeras. J Neurosci. 2004;24
(9):2202-2211.
40. Gilthorpe JD, Papantoniou EK, Chedotal A, Lumsden A,
Wingate RJ. The migration of cerebellar rhombic lip derivatives.
Development. 2002;129(20):4719-4728.
41. Alcántara S, Ruiz M, De Castro F, Soriano E, Sotelo C. Netrin
1 acts as an attractive or as a repulsive cue for distinct migrating
neurons during the development of the cerebellar system. Develop-
ment. 2000;127(7):1359-1372.
IULIANELLA ET AL.
42. Espinosa JS, Luo L. Timing neurogenesis and differentiation:
insights from quantitative clonal analyses of cerebellar granule
cells. J Neurosci. 2008;28(10):2301-2312.
43. Chedotal A. Should I stay or should I go? Becoming a granule
cell. Trends Neurosci. 2010;33(4):163-172.
44. Miyata T, Maeda T, Lee JE. NeuroD is required for differentiation
of the granule cells in the cerebellum and hippocampus. Genes
Dev. 1999;13(13):1647-1652.
45. Mecha M, Pena-Melian AL, Blanco MJ. Microarchitectural
changes during development of the cerebellar cortex. Int J Dev
Biol. 2010;54(4):691-698.
46. Butts T, Hanzel M, Wingate RJ. Transit amplification in the amni-
ote cerebellum evolved via a heterochronic shift in NeuroD1
expression. Development. 2014a;141(14):2791-2795.
47. Wallace VA. Purkinje-cell-derived sonic hedgehog regulates gran-
ule neuron precursor cell proliferation in the developing mouse
cerebellum. Curr Biol. 1999;9(8):445-448.
48. Wojcinski A, Lawton AK, Sumru Bayin N, Lao Z, Stephen DN,
Joyner AL. Cerebellar granule cell replenishment postinjury by
adaptive reprogramming of Nestin+ progenitors. Nat Neurosci.
2017;20(10):1361-1370.
49. Yeung J, Ha TJ, Swanson DJ, Choi K, Tong Y, Goldowitz D. Wls
provides a new compartmental view of the rhombic lip in mouse
cerebellar development. J Neurosci. 2014;34(37):12527-12537.
50. Amores A, Force A, Yan YL, et al. Zebrafish hox clusters and ver-
tebrate genome evolution. Science. 1998;282(5394):1711-1714.
51. Chaplin N, Tendeng C, Wingate RJ. Absence of an external germi-
nal layer in zebrafish and shark reveals a distinct, anamniote gro-
und plan of cerebellum development. J Neurosci. 2010;30(8):
3048-3057.
52. Kani S, Bae YK, Shimizu T, et al. Proneural gene-linked neuro-
genesis in zebrafish cerebellum. Dev Biol. 2010;343(1–2):1-17.
53. Kidwell CU, Su CY, Hibi M, Moens CB. Multiple zebrafish atoh1
genes specify a diversity of neuronal types in the zebrafish cerebel-
lum. Dev Bio. 2018;438(1):44-56.
54. Butts T, Modrell MS, Baker CV, Wingate RJ. The evolution of the
vertebrate cerebellum: absence of a proliferative external granule
layer in a non-teleost ray-finned fish. Evol Dev. 2014b;16(2):92-100.
55. Field DJ, Gauthier JA, King BL, Pisani D, Lyson TR, Peterson KJ.
Toward consilience in reptile phylogeny: miRNAs support an
513
archosaur, not lepidosaur, affinity for turtles. Evol Dev. 2014;16
(4):189-196.
56. Capaldo E, Iulianella A. Cux2 serves as a novel lineage marker of
granule cell layer neurons from the rhombic lip in mouse and chick
embryos. Dev Dyn. 2016;245(8):881-896.
57. Englund C, Kowalczyk T, Daza RA, et al. Unipolar brush cells of
the cerebellum are produced in the rhombic lip and migrate through
developing white matter. J Neurosci. 2006;26(36):9184-9195.
58. Iulianella A, Sharma M, Durnin M, Vanden Heuvel GB,
Trainor PA. Cux2 (Cutl2) integrates neural progenitor develop-
ment with the cell cycle progression during spinal cord neuro-
genesis. Development. 2008;135(4):729-741.
59. Iulianella A, Sharma M, Vanden Heuvel GB, Trainor PA. Cux2
functions downstream of notch signaling to regulate dorsal inter-
neuron formation in the spinal cord. Development. 2009;136(14):
2329-2334.
60. Topka S, Glassmann A, Weisheit G, Schuller U, Schilling K. The
transcription factor Cux1 in cerebellar granule cell development
and medulloblastoma pathogenesis. Cerebellum. 2014;13(6):
698-712.
61. Hawkes R, Faulkner-Jones B, Tam P, Tan SS. Pattern formation in
the cerebellum of murine embryonic stem cell chimeras. Eur J
Neurosci. 1998;10(2):790-793.
62. Guell X, Schmahmann JD, Gabrieli J, Ghosh SS. Functional gradi-
ents of the cerebellum. Elife. 2018;7:e36652.
63. Jörntell H. Cerebellar physiology: links between microcircuitry
properties and sensorimotor functions. J Physiol. 2017;595(1):
11-27.
64. Witter L, De Zeeuw CI. Regional functionality of the cerebellum.
Curr Opin Neurobiol. 2015;33:150-155.
How to cite this article: Iulianella A, Wingate RJ,
Moens CB, Capaldo E. The generation of granule
cells during the development and evolution of the
cerebellum. Developmental Dynamics. 2019;248:
506–513. https://doi.org/10.1002/dvdy.64