Cerebellar

Development

Carolyn Brager, Tyler Kervella,

Monica Devaraju, and Sarie Haraguchi
 Cerebellum: Structure and Function
Latin for “Little Brain”
1. Massive capacity for signal processing
2. Critical for coordinated movement and
motor learning
3. Three layers of neurons
4. Folds of folia

Database Center for Life Science CC BY-SA 2.1

Anatomy Overview
Anatomical divisions: lobes and zones
Functional divisions
● Cerebrocerebellum
○ Formed by lateral hemispheres
○ Involved in planning movements
and motor learning
● Spinocerebellum
○ Vermis and intermediate zone
○ Error correction
● Vestibulocerebellum
○ Flocculonodular lobe
○ Balance and ocular reflexes

Superior View
Somatotopic Mappingotopic mapping

● cerebellar maps are of the

ipsilateral side of the body
● lesions of one side of the
cerebellum affect the
musculature of the same
side of the body
Neurons
● Extremely high density of neurons
○ Contains 4X more total neurons
than the entire cerebral cortex
● Three distinct cellular layers
overly an inner core of white
matter and deep cerebellar nuclei

1. Molecular layer

2. Purkinje cell layer

3. Granule cell layer

Circuitry
Layer Development
 Joyner, A. L. (1994)
Foliation Segal, R. A. (1997)

● Formation of gyri and sulci produce a characteristic set of folds

● Separated by fissures that run perpendicular to the anterior-posterior axis
● Genetically determined, primarily postnatal process
=WT
Less
Fissures are
pronounced
shallower in
fissures and
mutant
delayed
development
IGL density is
greater in WT
than in mutant

Lobes VIII
Lobules are and IX are
less distinct abnormally
connected
 Cerebellum Development Overview

1. Neurons arise from TWO germinal zones

2. Proliferation / Foliation

3. Shh signaling

4. Primary Cilium

Sillitoe et al. 2007

 Rhombic Lip (rl) and Ventricular Zone (VZ)
● RL is located at the outer aspect of the
cerebellar plate, adjacent to the roof
plate
○ Gives rise to granule cell (GC)
○ Defined by expression of Atoh1
○ GC migrate to form the external
granular layer (EGL) where they
are mitotically active
● VZ lines the fourth ventricle
○ Gives rise to purkinje cells (PC) Sillitoe et al. 2007
○ Defined by expression of Ptf1a
Proliferation of Granule Cells / Foliation

● 30-fold expansion of
cerebellar volume
● PC control the mitotic activity
of GC within the EGL
● Correct proliferation is
necessary for foliation

Goldowitz et. al 1998

Sonic Hedgehog (Shh)
 Sonic Hedgehog (Shh)

● Shh expressed first by the choroid plexus and later by purkinje cells
after E17.5
● Shh is a ligand for the receptor patched 1 (Ptch1)
○ In the absence of Shh, Ptch1 inhibits smoothened (Smo)
○ When Shh binds Ptch1, Smo is activated allowing downstream
activation of glioblastoma (Gli) proteins
● Shh targets include genes involved in cell growth and division, various
transcription factors and components of the Shh pathway (regulation)

What is the importance of Shh in cerebellar development?

What happens if we block Shh signaling?

Lewis et al. 2004

What about if we add too much Shh?

Corrales et al. 2004, 2006

 Shh Conclusions

● MAJOR MORPHOGEN IN CEREBELLUM FOLIATION

● Shh produced by PC control the proliferation of GC which in turn
controls the correct development of folia
● Shh signaling is highly regulated
○ Too little Shh leads to loss of conserved folia structures
○ Too much Shh leads to additional folia forming during
development
 Primary Cilium

● Microtubule-based protrusion on the

surface of nearly all nucleated cells
● Ptch1 and Smo localize at primary cilium
○ allows cells to be ready to respond
to Shh signaling
● Additionally, Shh/Ptch1 complex is
internalized and degraded allowing a
controlled number of proliferations of
the GC

De Luca et al. 2016

Joubert Syndrome
Joubert Syndrome (JS)
Causes: > 35 known genes

For diagnosis: Molar tooth sign (MTS)

on axial MRI

Inheritance pattern: autosomal

recessive, X-linked recessive

Prevalence: 1/100,000 live births

*prevalence disproportionately high

in some populations due to a high
carrier frequency
JS phenotypes
 Tissue and
Perturbed protein
Causal variants Cellular dysfunction developmental
function
defects

Mechanism of Joubert??
Ciliopathies
Ciliopathies are diseases caused by
defective ciliary function

JS is a ciliopathy
Primary cilia
Non-motile cilia

Cellular projections generated from a basal body, a cylinder comprised of triplet

microtubules

The primary cilia acts as an antenna to modulate downstream signaling processes

such as Sonic hedgehog (Shh)

Cilia are dynamic organelles, being resorbed prior to mitosis and extended after
cell- division
Primary cilia
The transition zone (TZ) controls the protein
composition of the ciliary membrane

- Acts like a gatekeeper

Several JS-associated proteins are components of the

transition zone
TZ disrupted in individuals with JS-specific TCTN2 mutation

Shi, Xiaoyu et al.

Tub Ac = acetylated tubulin, a marker for the structural component of the primary cilium

Cep164= a basal body protein

TCTN2 = transition zone protein; when specifically mutated causes JS

Smoothened localizes to the TZ in wild-type cells
Shi, Xiaoyu et al.

Shi, Xiaoyu et al.

Tub Ac = acetylated tubulin, a marker for the structural component of the primary cilium

Cep164= a basal body protein

SMO= Smoothened, an essential mediator of the Shh signaling pathway

SAG= Smoothened AGonist; binds Smoothened & activates Shh

Take-aways
Diverse ciliary proteins accumulate at the transition zone in wild-type cells

Transition zone acts a selective gateway for proteins exiting and entering cilium

Disruption of TZ architecture in JS leads to a failure of SMO to accumulate in the

primary cilium, disrupting proper developmental signaling in JS (Shi et al.)
 Dandy Walker Malformation (DWM)
● Vermis of cerebellum either absent, very
small, or abnormally dispositioned
● Cavity between brainstem and
cerebellum, and posterior fossa
abnormally large
● Buildup of cerebrospinal fluid in fourth
ventricle
● Problems with coordination, intellect,
mood, polydactyly or syndactyly, and
abnormal facial features
● Suspected that DWM is
associated with deletion of
genes in the 6p25.3
chromosome
● Looked at large and small
deletions in 6p25.3
chromosome and compared
the anatomy
● Found very small vermis and
large posterior fossa in large
deletion and less severe
abnormalities in small
deletions
● all individuals showing
symptoms of DWM had a
deletion of FOXC1 gene
 FOXC1 Experiments in Mice

● FOXC1 stained with X-Gal in blue

● FOXC1 found in mesenchyme (a
primitive embryonic tissue) and not
expressed in cerebellum itself
● Disorganization between
rhombic lip (rl) and external
granule cells (EGL)
● Lack of distinction between
EGL and Purkinje cells (PC)
● Loss of FOXC1 decreases
expression of genes secreted from
hind mesenchyme (Tgfb1, Cxcl12,
Bmp2, Bmp4)
○ Molecules required for proper EGL
development
○ Bmp 2, Bmp 4, and Cxcl12 important for
Atoh1 expression
● No effect on genes expressed in
neural tube (Fgf15, Cxcr4)
● Atoh1 expression in green
● Atoh1 is a transcription factor
involved in rhombic lip
development
● In FOXC1 KO Atoh1 not
expressed in EGL and rhombic
lip fails to develop properly
 Dandy Walker Conclusion
● Loss of FOXC1 in 6p25.3 chromosome
→ loss of proper mesenchymal
signaling → cerebellar malformations
Questions?
References
Aldinger, K. A., Lehmann, O. J., Hudgins, L., Chizhikov, V. V., Bassuk, A. G., Ades, L. C., Krantz, I. D.,
Dobyns, W. B., … Millen, K. J. (2009). FOXC1 is required for normal cerebellar development and is a major
contributor to chromosome 6p25.3 Dandy-Walker malformation. Nature genetics, 41(9), 1037-42.

Corrales JD, Blaess S, Mahoney EM, Joyner AL. 2006. The level of sonic hedgehog signaling regulates the
complexity of cerebellar foliation. Development. 33:1811–21

De Luca, A., Cerrato, V., Fuca, E., Parmigiani, E., Buffo, A., and K. Leto. 2016. Sonic hedgehog patterning
during cerebellar development. Cellular and Molecular Life Sciences. 73:291-303

Goldowitz, D., and K. Hamre. 1998. The cells and molecules that make a cerebellum.Trends in Neuroscience.
21:375–38

Lewis PM, Gritli-Linde A, Smeyne R, Kottmann A, McMahon AP. 2004. Sonic hedgehog
signaling is required for expansion of granule neuron precursors and patterning of the
mouse cerebellum.Dev. Biol. 270:393–410
References
Millen, K. J., Wurst, W., Herrup, K., & Joyner, A. L. (1994). Abnormal embryonic cerebellar development
and patterning of postnatal foliation in two mouse engrailed-2 mutants. Development, 120(3), 695-706.

Romani, Marta, et al. “Joubert Syndrome: Congenital Cerebellar Ataxia with the Molar Tooth.” The Lancet
Neurology, vol. 12, no. 9, 2013, pp. 894–905., doi:10.1016/s1474-4422(13)70136-4.

Schwartz, P. M., Borghesani, P. R., Levy, R. L., Pomeroy, S. L., & Segal, R. A. (1997). Abnormal cerebellar
development and foliation in BDNF−/− mice reveals a role for neurotrophins in CNS patterning

Sillitoe, R., V., and A. L. Joyner. 2007. Morphology, Molecular Codes, and Circuitry Produce the
Three-Dimensional Complexity of the Cerebellum. Cellular and Developmental Biology. 23:549-577

Shi, Xiaoyu et al. “Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture
causes Joubert syndrome” Nature cell biology vol. 19,10 (2017): 1178-1188.
Question
Considering the Shh signaling pathway, what is the
significance of the transition zone in maintaining proper cell
division and growth?