The cerebellum is composed of two principal classes of neurons,
Purkinje cells and granule cells, which originate from the roof
plate of the metencephalon during early embryogenesis (Wang
and Zoghbi, 2001; Sillitoe and Joyner, 2007). During embryonic
day 11-13 of mouse development, Purkinje cells generated from
the ventricular zone of the fourth ventricle become postmitotic
and migrate radially within the developing cerebellar anlage.
After birth, Purkinje cells settle into a monolayer where they
differentiate and develop extensively arborized dendritic
processes. Granule cells, which arise from the rhombic lip,
migrate rostrally over the dorsal surface of the cerebellar anlage
to form the external granule cell layer (EGL). During the first 2
weeks after birth, cells in the EGL undergo extensive
proliferation to produce a granule cell progenitor (GCP) pool
that is required for generating a large number of granule cells.
Developing GCPs then exit the cell cycle, and migrate internally
past the Purkinje cells to form the inner granule cell layer (IGL).
Signaling between the granule cells and Purkinje cells is required
to orchestrate the proliferation and migration of the granule cells
to form the final, highly organized, structure of the mature
cerebellum (Jensen et al., 2002). Sonic hedgehog (Shh) secreted
from Purkinje cells serves as a potent mitogenic signal that
causes the expansion of the GCP population (Wallace, 1999;
Wechsler-Reya and Scott, 1999; Dahmane and Altaba, 1999).
Deletion of Shh in mouse Purkinje cells disrupts normal
cerebellar development, principally by blocking the proliferation
of GCPs in the EGL (Lewis et al., 2004). Thus, a tight regulation
of Shh expression by Purkinje cells is required for appropriate
GCP expansion during cerebellum development. However,
mechanisms involved in the control of Shh expression within the
Purkinje cells are poorly understood.
Skor2 was first identified by an in silico search for novel genes
homologous to Ski/Sno family of transcriptional co-repressors
(Arndt et al., 2005). It has also been referred to as FUSSEL18
(functional Smad suppressing element on chromosome 18) (Arndt
et al., 2005), or Corl2 (Minaki et al., 2008), owing to its high
degree of homology to co-repressor for LBX1 (Mizuhara et al.,
2005), of which the human counterpart is termed FUSSEL15
(Arndt et al., 2007). Although Corl1 has been shown to be a co-
repressor for LBX1 (Jagla et al., 1995), the role of Skor2 has not
been determined. Similar to Ski/Sno, Skor2 possesses two
structural domains in the N-terminal region, a DHD (Dachshund
homology domain) (Wilson et al., 2004), and an adjacent SAND
domain that is necessary for Ski/Sno to interact with Smad4 (Wu
et al., 2002). Ski/Sno has been shown to negatively regulate
transforming growth factor (TGF)/bone morphogenetic protein
(BMP) signaling pathways through binding to Smad proteins
(Deheuninck and Luo, 2009). Given the sequence similarity to
Ski/Sno, Skor2 may have similar repressive function on
TGF/BMP signaling pathways (Arndt et al., 2005).
Transposons are mobile genetic elements that can be used as
tools for the generation of insertional mutations, as exemplified by
the use of the P element in Drosophila genetics (Cooley et al.,
1988; Hummel and Klambt, 2008). Although endogenous mouse
transposons have not been identified, the Sleeping Beauty (Ivics et
al., 1997) and piggyBac transposons have been shown to be
functional in mice (Ding et al., 2005; Dupuy et al., 2001; Ivics et
al., 2009). In order to simplify the genetic monitoring of
transposition in vivo, we tagged the Sleeping Beauty transposon
with a tyrosinase minigene. Most albino strains of laboratory mice
have a mutation in their endogenous tyrosinase gene (Yokoyama et
al., 1990). Therefore, transgenic mice that carry the transposon with
Development 138, 4487-4497 (2011) doi:10.1242/dev.067264
© 2011. Published by The Company of Biologists Ltd
1Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA.
2Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA. 3Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX 77030, USA.
*Authors for correspondence (firstname.lastname@example.org; email@example.com)
Accepted 10 August 2011
Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How
Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty
transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family
transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe
reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain,
where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner
and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study
identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during
KEY WORDS: Skor2, Sonic hedgehog, Ski, Cerebellum, Purkinje, Mouse
Transposon mutagenesis with coat color genotyping
identifies an essential role for Skor2 in sonic hedgehog
signaling and cerebellum development
Baiping Wang1, Wilbur Harrison2, Paul A. Overbeek2,3,* and Hui Zheng1,2,3,*
a functional copy of tyrosinase will lead to dose-dependent and
integration site-sensitive pigmentation, thus allowing for coat-color
based genotyping. We have created a series of stable insertional
mutants in mouse using this strategy, and have recovered two loss-
of-function alleles of Skor2 that exhibit severe defects in Shh
expression and cerebellar development.
MATERIALS AND METHODS
RNA in situ hybridization
Sagittal serial sections of brains of wild-type control and Skor2–/–mice
were cut with a cryostat, and placed with adjacent sections on separate
slides. After paraformaldehyde fixation and acetylation, the slides were
assembled into flow-through hybridization chambers and placed in a Tecan
Genesis 200 liquid-handling robot (Mannedorf). Templates for synthesis of
digoxigenin-labeled riboprobes for Skor2 and Shh are N-terminal 810 bp
of Skor2 cDNA and full-length Shh cDNA, respectively. Antisense and
sense probes were transcribed with T7 and Sp6 polymerase, respectively,
from linearized vector. Hybridized probes were detected by catalyzed
reporter deposition using biotinylated tyramide; this was followed by
colorimetric detection of biotin with avidin coupled to alkaline
phosphatase. Hybridization with sense control probes did not yield signals
Histology and immunohistochemistry
Animals were perfused with 4% paraformaldehyde. Tissues were post-
fixed in 4% paraformaldehyde, dehydrated in ethanol, embedded in
paraffin, and sectioned at 10 m. Alternatively, fixed tissues were
incubated in 30% sucrose at 4°C overnight, frozen in OCT (TissueTek) and
sectioned at 30 m. For histological analysis, paraffin sections were stained
with Hematoxylin and Eosin (Sigma) or with thionin for Nissl staining.
Immunohistochemistry on frozen and paraffin sections was performed by
incubation overnight at 4°C using the following primary antibodies: anti-
calbindin D-28K (Chemicon), anti-phospho-Histone H3 (Chemicon), anti-
Zic1 (Rockland). Secondary antibodies used were: goat anti-mouse Alexa
Fluor 488 and goat anti-rabbit Alexa Fluor 555 (Invitrogen), which were
added for 2 hours at room temperature. Sections were counterstained with
Toto3 (Invitrogen) and mounted using Prolong Gold anti-fade reagent
(Invitrogen) and images were acquired with a Zeiss LSM 510 confocal
microscope. Apoptosis in the developing brain was assessed by the
terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling
(TUNEL) assay using DeadEnd Fluorometric TUNEL System (Promega)
according to the manufacturer’s protocol.
Plasmids, cell culture, reporter assays
Skor2 cDNA was amplified by RT-PCR from mouse cerebellum and cloned
into pcDNA 3.1 expression vector (Clontech). Deletion and mutation
constructs were generated by site-directed mutagenesis. Id1-Luc
(Korchynskyi and ten Dijke, 2002) was kindly provided by Peter ten Dijke
(LUMC, The Netherlands). p3TP-lux, pCMV5B-Flag-Smad3 and pCL-Neo
HA-hSki were purchased from Addgene (Cambridge, MA). Shh-Luc
reporter plasmids containing human Shh promoter (nucleotides 3347 to 1548
of a 1.9 kb human Shh promoter sequence) were kindly provided by
Vladimir A. Botchkarev (University of Bradford, UK) (Sharov et al., 2009).
HepG2 and HaCat cells were grown in MEM containing 10% FBS. HEK293
and N2a cells were grown in DMEM containing 10% FBS. Cells were
transfected using Lipofectamine 2000 reagent (Invitrogen). Cells in 12-well
plates were co-transfected with expression plasmids for Skor2 and reporter
plasmids (p3TP-lux, Id1-luc or Shh-luc), together with Renilla luciferase
vector. Twenty-four hours after transfection, cells were treated for 12 hours
with or without 25 ng/ml BMP2 (R&D) and lysed with Passive Lysis Buffer
(Promega). The Dual-Luciferase Reporter Assay System (Promega) was
used to determine firefly and Renilla luciferase activities according to the
manufacturer’s instructions. Measurements were performed with a BD
luminometer (BD), and firefly luciferase values were normalized to Renilla
luciferase values. In all experiments, the internal control plasmid was used
to compensate variable transfection efficiencies. All assays were repeated
three times, data were pooled, mean ± s.e.m. was calculated, and statistical
analysis was performed using unpaired Student’s t-test.
Immunoprecipitation and western blotting
HEK293 cells were transfected with an appropriate combination of
expression plasmids, and solubilized in a buffer containing 50 mM Tris
HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40 supplemented with
Protease inhibitor cocktail (Roche). Lysates were cleared and incubated
with anti-FLAG and anti-HA antibody (Sigma), followed by incubation
with protein A/G-Sepharose beads (Amersham Pharmacia Biotech). The
beads were washed with solubilization buffer, and the immunoprecipitates
were eluted by boiling for 3 minutes in SDS sample buffer [100 mM Tris
HCl (pH 8.8), 0.01% Bromphenol Blue, 36% glycerol, 4% SDS]
containing 10 mM dithiothreitol and subjected to SDS-gel electrophoresis.
Sleeping Beauty-tyrosinase transposon-based
Transposable elements can be mobilized by the expression of a
transposase, which causes transposons to excise and reinsert
randomly in the genome. Thus, randomized mutagenesis can be
performed by the generation of a parental animal that contains the
transposon and transposase, and stable mutations screened in
offspring which lack expression of the transposase.
Most albino strains of laboratory mice have a mutation in their
endogenous tyrosinase gene. Expression of a tyrosinase reporter leads
to gene therapy for albinism, with pigment production in the fur, skin
and eyes (Overbeek et al., 1991). In order to simplify the genetic
monitoring of transposition in vivo, we have engineered the Sleeping
Beauty transposon, which expresses a tyrosinase minigene that
confers four particularly useful features (Lu et al., 2007). First, the
minigene is expressed at sufficient levels to give visible pigmentation
for most sites of integration in the genome. Second, the level of
tyrosinase expression is consistent for a given integration site,
resulting in hemizygous siblings that exhibit coat colors that are
nearly identical to each other and to their hemizygous parents. Third,
the level of minigene expression is usually rate limiting for melanin
synthesis, resulting in a gene dose effect for each integration site such
that homozygous transgenic mice, if viable, are more darkly
pigmented than their hemizygous littermates. Fourth, different
integration sites give different levels of minigene expression, and
yield mice with different coat colors for each different integration site.
Therefore, transgenic mice that carry the transposon with a functional
copy of tyrosinase will lead to dose-dependent and integration site-
sensitive pigmentation, thus allowing for coat color-based genotyping.
The 4.1 kb tyrosinase minigene isolated from plasmid Ty811C
(Yokoyama et al., 1990) was inserted into the pT2 version of the
Sleeping Beauty transposon (Cui et al., 2002). We also inserted a
Gal4-VP16-SV40 cassette in the opposite orientation to the
tyrosinase minigene, but detected no expression in the hemizygous
or homozygous mutants (see Fig. S1A in the supplementary
material). The tyrosinase-tagged transposon DNA (Fig. 1A) was
microinjected into one-cell stage inbred albino FVB/N embryos
and two pigmented transgenic founder mice were obtained. Upon
outbreeding to FVB males, both founder mice produced F1
offspring with two different coat colors, indicating two different
integration sites for each founder. The F1 mice were used to
establish four different transgenic families. Southern hybridization
and fluorescent in situ hybridization to metaphase chromosomes
were used to estimate the copy number for the transgenes in each
family and the initial chromosomal integration sites (see Table S1
in the supplementary material).
In order to mobilize the transposons from their initial integration
sites, we mated the transposon-carrying transgenic mice to
transgenic mice that express the transposase for Sleeping Beauty
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Role of Skor2 in cerebellum