RESEARCH ARTICLE 4487
Development 138, 4487-4497 (2011) doi:10.1242/dev.067264
© 2011. Published by The Company of Biologists Ltd
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,*
SUMMARY
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
cerebellum development.
INTRODUCTION
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
1
Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA.
Department 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.
2
*Authors for correspondence ([email protected]; [email protected])
Accepted 10 August 2011
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 corepressor 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
KEY WORDS: Skor2, Sonic hedgehog, Ski, Cerebellum, Purkinje, Mouse
4488 RESEARCH ARTICLE
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
above background.
Histology and immunohistochemistry
Animals were perfused with 4% paraformaldehyde. Tissues were postfixed 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: anticalbindin D-28K (Chemicon), anti-phospho-Histone H3 (Chemicon), antiZic1 (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.
RESULTS
Sleeping Beauty-tyrosinase transposon-based
mutagenesis
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 sitesensitive 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
DEVELOPMENT
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 lossof-function alleles of Skor2 that exhibit severe defects in Shh
expression and cerebellar development.
Development 138 (20)
RESEARCH ARTICLE 4489
Fig. 1. Transposon mutagenesis and generation of transgenic mouse lines with deletion of Skor2. (A)Map of the transposon vector pT2tyro-gal4-SV40 used for microinjection. IR/DR, inverted repeat/direct repeat of the transposon; L3 and R3, primers for PCR detection of the
transposon concatemer. Sequences in the brackets are terminal transposon sequences (underlined) connected with the plasmid backbone
sequences (not underlined). (B)Example of stable gray color pigmentation in half of the offspring from a transgenic male of similar pigment without
transposase. (C)Example of offspring from a bigenic male (far left) with changes in coat color, representing distinct transposition events.
(D)Example of offspring from interbreeding of a hemizygous transgenic male and female showing the genotyping of wild-type (Yokoyama et al.,
1990), hemizygous (light brown) and homozygous (darker brown) pups by visual examination. (E)Cross of a homozygous mouse with an albino
partner yielded offspring all with lighter pigmentation, confirming homozygosity of the transposon inserted allele. (F)Schematic drawing of the
original integration site of founder 1799B, which had 15 concatemerized copies of the transposon integrated in chromosome 18B3. Arrow with
broken line indicates reintegration after mating to induce transposition. (G)Schematic drawing of the transposition events in Skor2 mutant lines
1799B CA3 and 1799B CA7. The chromosomal locations of the start and stop codons of Skor2, the integration junctions and the closest upstream
and downstream genes (Smad2, Ier3ip1 and Hdhd2) are indicated. Arrows indicate the primers (see Table S3 in the supplementary material) used
for genotyping shown in H. (H)PCR genotyping for the Skor2 mutant allele (CA7) and tyrosinase minigene (Tyro). (I)RT-PCR analysis shows lack of
the Skor2 transcript in the mutant. Expression of Gapdh was used as a positive control. (J)Relative expression of adjacent genes (Smad2, Ier3ip1
and Hdhd2) determined using real-time RT-PCR in the P0 cerebellum of control (ctr) and Skor2 mutant (ko) mice. Real-time PCR was performed as
described previously (Li et al., 2010). Primer sequences are shown in Table S3 in the supplementary material. Data are the mean ± s.e.m. of two
independent experiments with three samples per genotype. ***P<0.001 (Student’s t-test).
DEVELOPMENT
Role of Skor2 in cerebellum
4490 RESEARCH ARTICLE
Development 138 (20)
(CAGGS-SB10) (Dupuy et al., 2001). Pigmented male offspring
(transposon-positive) were screened for the presence of the
transposase transgene to identify males that carried both transgenes.
Excision and re-integration of the transposon is thus activated in
the germline of these males and new integration events can be
isolated from their offspring.
Males carrying either the Sleeping Beauty transposon only, or
both the transposase and the Sleeping Beauty transposon were
mated to albino FVB females, and the offspring were screened for
new coat colors. As expected, all offspring from crosses of the
single transgenic Sleeping Beauty transposon alone displayed the
identical coat color to the parental transgenic male (Fig. 1B). By
contrast, an average of 30% of the offspring from double transgenic
males showed a change in coat color (Fig. 1C). Overall, 64 mice
with coat color changes were recovered using the four initial
integration sites. These mice were bred to FVB partners to remove
the transposase and to establish new transgenic lines with stable
transposon integration, followed by interbreeding to generate
homozygous mice (Fig. 1D). Inverse PCRs [described previously
(Yang et al., 2004)] were carried out to obtain molecular
information about the transposition events for 52 of the families
(see Table S2 in the supplementary material). Sequencing results
were used for BLAST searches against the mouse genome database
to determine the precise location of each integration site. Putative
homozygous mice were mated to albino partners to test for fertility
and to confirm homozygosity (Fig. 1E).
In 15 of the 52 transposition events that were molecularly
characterized, the transposon was found to have integrated into
Gal4 sequences or tyrosinase sequences (see Table S2 in the
supplementary material), implying local hopping within the
original transgenic arrays. For the other 37 sites, 17 were located
within 15 different endogenous mouse genes. Fifteen of these sites
were intronic. Two integrations occurred in exons. For four of the
15 genes (Ppp3ca, Ace, Axin2 and Rgs9), mice with targeted
mutations have previously been described. However, 11 of the
integration sites were located in ‘new’ genes, i.e. genes for which
live mice with targeted alleles have not previously been described.
Nine of the integration sites produced homozygous mutant
phenotypes. Here, we describe our studies of one of the mutated
genes isolated from family 1799B. The other integration sites and
mutations will be described in later manuscripts.
Transposon integration sites for family 1799B
Founder 1799B had 15 tandem copies of the transposon
integrated in chromosome 18B3 (Fig. 1F). New genomic
transposition sites were characterized from founder 1799B and
are shown in Table 1. Two of the transposition sites (CA3 and
CA7) were located in the same predicted gene: Skor2 (NCBI
accession number XM_972803.2) (Fig. 1G). The CA3
integration site was located in exon 2. For CA7, the right
junction between transgenic and genomic sequences was located
in intron 8, right after the stop codon of Skor2; the left junction
was located 237 kb upstream from the start codon of Skor2.
Integration of the transgenic DNA was accompanied by a
deletion of 273 kb in mouse chromosome 18, including the entire
coding region of Skor2, but excluding any exons from the
predicted adjacent genes (Fig. 1G). Other investigators have
noted that concatemeric transposons can produce complicated
genomic alterations during transposition (Geurts et al., 2006).
Using primers located 5⬘ upstream of Skor2 gene, we showed
that the predicted 196 bp band was present in DNA from wildtype (+/+) or heterozygous (+/–) mice, but absent from DNA
taken from mice homozygous (–/–) for the transgene, indicating
that the transposition resulted in a deletion (Fig. 1H). Reverse
transcriptase-PCR (RT-PCR) analysis of RNA from P0
cerebellum using primers located in exon 2 of Skor2 showed a
complete absence of the Skor2 transcript in homozygous mutant
mice (Fig. 1I; see Fig. S1 in the supplementary material). These
mutants are thus referred to as Skor2 null or Skor2–/–. The same
analysis failed to amplify any transcripts containing the Gal4VP16 sequences (see Fig. S1A in the supplementary material),
indicating that Gal4-VP16 is not expressed in Skor2 mutants and
cannot contribute to the phenotypes present in the mutant
cerebellum. The expressions of known genes adjacent to Skor2,
including the upstream Smad2 and the downstream Ler3ip1 and
Hdhd2 were assessed by quantitative real-time PCR and the
levels were found to be similar between the Skor2-null mutants
and the littermate controls (Fig. 1J). Genomic PCR using primers
immediately upstream and downstream from the Skor2
integration sites also failed to detect any aberrant bands,
indicating that there were no unexpected genomic DNA
rearrangements in Skor2-null mutants (see Fig. S1B in the
supplementary material). These results, combined with the fact
that the same phenotypes were seen in two independent alleles
(see below) strongly support the notion that the phenotypes were
specifically caused by the disruption of the Skor2 gene.
Homozygous mice in both families CA3 and CA7 were found to
display very similar phenotypes. Most (90%) of the homozygotes
die within 48 hours of birth, probably owing to defective
neuromuscular junction formation and respiration failure (B.W. and
H.Z., unpublished). Homozygotes that survive are smaller than
their siblings and show an unstable gait throughout the life span.
The subsequent studies were performed with mice from strain
CA7.
Table 1. Summary of family 1799B transposition events
1799B
1799B-CA9
1799B-CA18
1799B-CA6
1799B-CA3
1799B-CA7
1799B-CA8
1799B-CA1
Coat color
Chromosome
Coordinates
Gene
Medium brown
Light
Dark gray
18
18
18
18
18
18
18
18
76,860,053
31,804,712
73,902,476
76,830,644
77,097,862
77,133,014
77,206,994
84,875,090
None
Sap130
Elac1
None
Skor2
Skor2
Hdhd2
None
Dark brown
Medium brown
Light brown
Light brown
MGI ID
Site in gene
1919782
1890495
Intron 2
Intron 2
3645984
3645984
1924237
Exon 2
Intron 8
Intron 6
Homozygous
phenotype
Viable, fertile
Viable, fertile
Viable, fertile
Viable, fertile
Abnormal gait
Abnormal gait
E7 lethal
Viable, fertile
1799B represents the original transgenic integration line from which the transposition events (1799B-CA series) are derived. These are sorted by their genome coordinates
[from NCBI37/mm9 (July 2007)] along with the affected genes, the MGI ID, site of transposition within the gene and overt homozygous phenotypes. The lines used in this
study are in bold.
DEVELOPMENT
Line
Skor2 deficiency severely disrupts cerebellar
development
Given phenotypes indicative of cerebellar anomalies, we
performed morphological analysis of the Skor2-null cerebellum
at various developmental stages. Nissl staining of sagittal
sections from E15 wild-type and Skor2-null embryos revealed
equivalent cerebellar development (Fig. 2A, E15). However, in
P0 pups, the cerebellum from Skor2-null mice remained as a flat
structure, lacking the four principal fissures observed in the
control cerebellum. Additionally, the size of the cerebellum was
much smaller when compared with the control littermates (Fig.
2A, P0). This phenotype persisted during postnatal development
(Fig. 2A, P5) and to adulthood (Fig. 2B-D). Morphological
examination of the Skor2-null mice that survived to adulthood
revealed that, although the lateral cerebellar hemispheres were
relatively unaffected, the central vermis region was severely
reduced in size (Fig. 2B). In addition, analysis of serial sagittal
sections through the vermis revealed an abnormal foliation
pattern (Fig. 2C). Lobes 4-5 failed to develop entirely, whereas
lobes 6-8 did not divide normally into the sublobules seen in
wild-type littermates. Although the Skor2-null mutant
maintained a normal laminar structure, the molecular layer of the
cerebellar cortex was thinner compared with the wild-type
cerebellum (Fig. 2D). Examination of histological sections from
other brain regions did not reveal any gross morphological
abnormalities (see Fig. S2 in the supplementary material).
RESEARCH ARTICLE 4491
Skor2 mediates Purkinje cell differentiation and
granule cell proliferation
We analyzed Skor2 expression in both the developing and adult
CNS by in situ hybridization. At E15, strong expression was
detected in the central region of the cerebellum where primitive
Purkinje cells are located (Fig. 3A,B). At P0, we observed
predominant expression of Skor2 in developing Purkinje cell layers
(PCL). Interestingly, higher levels of Skor2 expression were
observed in the rostral lobes compared with caudal lobes (Fig. 3C).
In situ hybridization of whole adult mouse brain revealed that
Skor2 was exclusively expressed in the cerebellum (see Fig. S3 in
the supplementary material). Examination of higher magnification
images showed that Skor2 mRNA is restricted to Purkinje cell
bodies (Fig. 3E). The specificity of the probe was confirmed by the
absence of hybridization signal in Skor2-null brain slices (Fig. 5A).
Overall, our results demonstrate that Skor2 expression is specific
to developing and adult Purkinje neurons in the cerebellum.
The prominent expression of Skor2 in Purkinje cells prompted
us to ask whether phenotypes seen in Skor2-null mutants are due
to an intrinsic defect in Purkinje neurons. We performed
immunofluorescence labeling of brain slices with the Purkinje cell
marker calbindin D-28K at P0, P10 and 2 months of age (Fig. 3F).
At P0, Purkinje cells were present in multiple layers in both the
control and Skor2–/– mutant (Fig. 3F, P0), indicating that Skor2 is
not required for the specification or migration of Purkinje cells. At
P10, wild-type and Skor2-null Purkinje cells both settled into a
Fig. 2. Deletion of Skor2 severely disrupts cerebellar vermis development. (A)Nissl staining of sagittal sections of cerebella from wild-type
and Skor2–/– mice at E15.5, P0 and P5, showing reduced size and misformed cerebellar fissures in Skor2–/– mice relative to controls at P0 and P5.
(B)Representative whole-brain micrograph of a wild-type littermate and Skor2–/– mutant at 2 months of age. The size of the cerebellum
(highlighted by broken blue lines), in particular the vermis, is dramatically reduced in the Skor2–/– mutant. (C)Nissl staining of mid-sagittal sections
of the cerebellum from a wild-type littermate and a Skor2–/– mutant. Cerebellar lobules are indicated by Roman numerals. (D)Magnified views of
Nissl stained sections from comparable lobules of control and Skor2-null cerebella. ML, molecular layer (with thickness marked by arrows); PCL,
Purkinje cell layer; IGL, internal granule cell layer. Scale bars: 200m in A; 400m in C; 50m in D.
DEVELOPMENT
Role of Skor2 in cerebellum
4492 RESEARCH ARTICLE
Development 138 (20)
Fig. 3. Impaired Purkinje cell dendritic arborization
in Skor2–/– mice. (A-E)In situ hybridization of sagittal
sections of whole E15 embryo (A,B), P0 (C) and 2month-old (D,E) wild-type cerebella using an antisense
probe against Skor2. Cerebellum regions outlined in A
and D are enlarged and shown in B and E, respectively.
(F)Calbindin immunostaining of sagittal cerebellar
sections of P0, P10 and 2-month-old wild-type and
Skor2–/– mice. A significant decrease in Purkinje cell
dendritic arborization in Skor2-null mice was observed.
M, molecular layer; P, Purkinje cell layer. Scale bars:
200m in B-D; 50m in E,F.
marker for differentiating and mature granule neurons, we
observed abundant granule cells expressing Zic1 in both the
EGL and IGL of the control and Skor2 mutant animals (Fig. 4C),
indicating normal granule cell localization and maturation in the
absence of Skor2. Likewise, the levels of apoptosis measured by
TUNEL staining were similar in the control and Skor2–/– mutant
(Fig. 4D,E). The data combined provide strong support for the
notion that reduced GCP proliferation resulting from defective
Purkinje cell signaling is the cause for the smaller cerebellum
observed in the Skor2-null mutant.
Skor2 positively regulates sonic hedgehog
expression
Shh is secreted from Purkinje cells and acts as a potent mitogenic
signal to expand the GCP population. We performed in situ
hybridization to determine the levels of Shh expression in the Skor2
mutant and control cerebellum. Consistent with earlier reports
(Lewis et al., 2004; Corrales et al., 2004), in wild-type animals Shh
is predominantly expressed in Purkinje cells with higher levels in
rostral lobes than in caudal regions (Fig. 5A). In sharp contrast, Shh
expression in Purkinje cells was greatly diminished in Skor2
mutant cerebellum (Fig. 5A), demonstrating that proper Shh
expression is dependent on Skor2. To further confirm that this is
associated with impaired Shh signaling, we performed real-time
RT-PCR analysis using P0 cerebellum from control and Skor2
mutant mice, and found significant reduction in the expression
DEVELOPMENT
monolayer and developed dendritic processes. However, the depth
of the molecular layer in which the dendrites of the Purkinje cells
project was significantly reduced when compared with the control
(Fig. 3F, P10), suggesting that the maturation and differentiation of
Purkinje cells is defective in the absence of Skor2. This phenotype
persisted into adulthood (Fig. 3F, 2 months). Quantification of the
molecular layer thickness showed that there was a 32.3% and
27.2% thinning in this layer in Skor2-null mice at P10 and 2
months, respectively.
Purkinje cells migrate along the Bergmann glia fibers and form
a single layer postnatally. The absence of ectopic Purkinje cells in
the Skor2–/– cerebellum indicates that Skor2 is not required for
normal positioning of Purkinje cells. Further examination of
Bergmann glia morphology by immunostaining for GFAP showed
ordered and linear morphology of glial fibers extending to the pial
surface in the Skor2 mutant, indistinguishable from the wild-type
control (see Fig. S4 in the supplementary material), suggesting that
Bergmann glia differentiation is not affected in the Skor2-null
mutants.
Given that Purkinje cell signaling is known to be required for
the proliferation and expansion of the GCPs, we performed
phospho-Histone H3 staining to visualize the mitotic, immature
granule cell precursors at P0 and P5. Indeed, compared with
wild-type controls, there was a significant reduction in the
number of proliferating cells in the EGL of Skor2–/– mice at both
stages (Fig. 4A,B). Using the Zic1 transcription factor as a
Role of Skor2 in cerebellum
RESEARCH ARTICLE 4493
levels of Shh and Shh target gene Gli1, but not of Gli2 or Gli3, in
Skor2–/– cerebellum, suggesting that Skor2 plays a role in
regulating specific Shh downstream targets (Fig. 5B).
We next performed in vitro luciferase assay using a Shh
promoter-luciferase reporter construct (Sharov et al., 2009).
Transfection of a Skor2 expression vector led to dose-dependent
increase in Shh luciferase activity (Fig. 5C), demonstrating that
Skor2 positively regulates Shh transcription. Together, these
findings suggest that decreased Shh signaling from Purkinje to
granule cells in the absence of Skor2 leads to cerebellar
hypoplasia.
Skor2 transcriptional activity is correlated with
nuclear localization and HDAC binding
Next, we investigated the biochemical and functional properties of
Skor2 by transfecting a deletion series of Flag- or Gal4-tagged
Skor2 expression vectors in cell culture (Fig. 6A,B), and
monitoring their subcellular localizations (Fig. 6C; see Fig. S5 in
the supplementary material) and transcriptional activities (Fig. 6D),
respectively. Expression of the deletion mutants was validated by
western blotting (Fig. 6B). Immunostaining revealed that fulllength Skor2 is localized to the nucleus (Fig. 6C, Skor2), as is
construct expressing the first 385 amino acids of the protein (Fig.
6C, 1-385). However, expressing the first 256 amino acids of Skor2
leads to its localization in both the nucleus and cytoplasm (Fig. 6C,
1-256), indicating that amino acids 256-385 of Skor2 contains a
nuclear localization signal (NLS). Analysis of the primary
sequences
within
this
region
using
PSORTII
(http://www.psort.org/) revealed that the KRPR residues at amino
acid 306-309 could potentially serve as a NLS. We, accordingly,
deleted the KRPR sequence from the full-length Skor2
(Skor2KRPR) and found that, indeed, this mutant resulted in a
predominantly cytoplasmic expression (Fig. 6C). We therefore
conclude that the KRPR motif spanning residues 306-309 of Skor2
directs its nuclear targeting.
The sequence similarity of Skor2 to the Ski/Sno family of corepressors prompted us to assess whether Skor2 has a repressive
function on transcription. We co-transfected Gal4-fused full-length
Skor2 or various deletion mutants (Fig. 6A, #1-#7) with a reporter
construct containing the GAL4 DNA-binding site and the Elb
promoter upstream of the luciferase sequence (Cao and Sudhof,
2001), and measured the luciferase activities of the transfected cells
(Fig. 6D). We found that full-length Skor2 potently repressed basal
transcription (Fig. 6A,D, #2), as was construct deleting the DHD
and HAND domains (Fig. 6A,D, #7), demonstrating that the
repression activity is conferred by the C-terminal sequences.
Transfection of the C-terminal deletion constructs (Fig. 6A,D, #3#6) showed that, interestingly, deletion mutant #4, which contains
the NLS but lacks other C-terminal domains, failed to suppress
luciferase activity (Fig. 6D, #4), suggesting that nuclear
localization is not sufficient for the repressor function of Skor2.
The full repression by expressing amino acids 1-592 of Skor2 (Fig.
6D, #5) indicates that sequences within amino acids 385-592 are
also crucial for the repression activity.
Because Ski and Sno have been shown to complex with nuclear
co-repressors (N-CoRs) and HDACs to repress transcription
(Akiyoshi et al., 1999), we hypothesized that Skor2 may exert
repression activity through association with HDACs. We cotransfected HEK293 cells with Flag-tagged Skor2 and HA-tagged
HDAC1 expression plasmids and performed immunoprecipitation
using anti-HA and anti-Flag antibodies. We found that Skor2 and
HDAC1 strongly interact (Fig. 6E,F). By performing interaction
assays using a panel of Skor2 deletion mutants, we narrowed down
the HDAC1 interaction domain to residues 385 and 592 of Skor2
DEVELOPMENT
Fig. 4. Reduced proliferation
of GCPs in Skor2–/– mice.
(A)Phospho-histone H3
immunofluorescence staining
of sagittal sections of P0 and
P5 cerebella showing decreases
in GCP proliferation in
Skor2–/– mice when compared
with wild-type littermates.
The sections were
counterstained with Toto3
(blue). (B)Quantification of
GCP proliferation. Data are
mean + s.e.m.; *P<0.05.
(C)Immunofluorescence
staining of sagittal sections of
P5 cerebellum with an anti
Zic1 antibody. Toto3 staining
of nuclei is shown in blue.
(D)TUNEL analysis of sagittal
sections of P5 control and
Skor2–/– cerebella.
(E)Quantification of TUNEL
analysis. Data are mean +
s.e.m. Scale bars: 100m in A;
50m in C,D.
4494 RESEARCH ARTICLE
Development 138 (20)
Fig. 5. Skor2 regulates the expression of Shh. (A)RNA in situ
hybridization on adjacent sagittal sections of wild-type and Skor2-null
cerebellum at P0 with antisense riboprobes specific for Skor2 and Shh.
Shh expression is significantly reduced in the PCL of Skor2–/– mice
compared with wild-type control. (B)Relative expression of Shh, Gli1,
Gli2 and Gli3 determined using real-time RT-PCR in the cerebellum of
P0 control (ctr) and Skor2 mutant (ko) mice. Data are the mean + s.e.m.
of two independent experiments with three samples per genotype;
**P<0.01 (Student’s t-test). (C)Co-transfection of Skor2 with a Shh
promoter-luciferase reporter in HepG2 cells led to a dose-dependent
increase in luciferase activity. Data representing three independent
experiments are shown as mean + s.e.m.
(Fig. 6G). As this region is also important for transcription
repression (Fig. 6D, #4 versus #5), the results combined suggest
that interaction with HDAC1 may be required for the repressive
activity of Skor2.
Skor2 represses BMP-mediated transactivation
Having established the repressor function of Skor2 in a
heterologous reporter system, we next sought to test whether it
mediates TGF or BMP signaling similar to Ski. Previous studies
have shown that the N terminus of Skor2 binds Smad2/3 and
inhibits TGF signaling (Arndt et al., 2005). In order to ascertain
the effect of full-length Skor2 on TGF signaling, we cotransfected Skor2 with a TGF-responsive promoter fused to
luciferase (p3TP-Lux) in Hep3B cells (Wrana et al., 1992) (Fig.
7A, –Smad3). A subset of the cells was also co-transfected with
Smad3 to activate the TGF promoter (Fig. 7A, +Smad3).
Expression of Ski was used as a positive control (Ski), whereas
transfection with the empty vector was used as the negative control
(Ctrl). Both the basal and Smad3-induced TGF reporter activities
were suppressed by co-expressing the TGF co-repressor Ski (Fig.
7A, Ski). However, Skor2 failed to suppress either the basal or
Smad3-activated TGF signaling pathway (Fig. 7A, Skor2),
suggesting that Skor2 cannot repress TGF-induced transcription.
DISCUSSION
Using Sleeping Beauty transposon tagged with a tyrosinase
reporter, we demonstrate here that transposition events can be
identified by simple visual inspection of the offspring from bigenic
males, providing a valuable tool to uncover genes that play crucial,
but heretofore uncharacterized, roles in normal vertebrate
development. Transposition and generation of new insertional
mutations requires only a two generation breeding strategy. There
is no need for micromanipulation of mouse embryos to generate
new integration sites. Identification of each transposition event is
accomplished by visual inspection of the mice, making screening
for insertional mutations straightforward and reliable. Additionally,
owing to the dose-dependent expression of the tyrosinase minigene
reporter, homozygous mice can be visually distinguished from
hemizygous animals. Even though transposition usually results in
reintegration near the original integration site (Keng et al., 2005),
a significant fraction of the integration sites can result in new
insertional mutations. Here, we describe one of the insertion
mutants generated, the Skor2 loss-of-function animal that displays
defects in cerebellar development. Loss of Skor2 leads to defective
Purkinje cell development, a severe reduction of granule cell
proliferation and a malformed cerebellum.
Skor2 contains DHD and SAND homology domains
characteristic of the Ski/Sno family of transcription co-repressors.
Although Skor2 shares structural similarities, it is unique in several
aspects. First, in contrast to the widespread expression of Ski/Sno
in multiple tissues including the CNS (Reed et al., 2001), Skor2 is
specifically expressed in cerebellar Purkinje cells in the brain.
Consequently, Skor2 deletion results in a cerebellum-specific
phenotype, whereas the forebrain structure is preserved. Second,
different from Ski/Sno, which are co-repressors of both TGF and
BMP pathways, Skor2 represses only BMP but not TGF
signaling, even though it binds to Smads associated with both BMP
and TGF pathways. Third, Skor2 has weak binding to Smad4.
Ski/Sno have been shown to bind to Smad4 through its SAND
domain, which is an evolutionarily conserved sequence motif
involved in chromatin-dependent transcriptional regulation
(Bottomley et al., 2001). Crystal structure of the Ski SAND domain
in complex with the MH4 domain of Smad4 established that the
highly conserved structure motif (I loop) from Ski/Sno proteins is
essential for binding to Smad4 (Wu et al., 2002). Comparison of
the Skor2 SAND domain with that of Ski revealed that the essential
hydrogen bonds in the I loop, which provides the specificity for the
recognition of Smad4 by Ski, are divergent in Skor2 (see Fig. S6
in the supplementary material). The zinc-binding motif (CCHH)
DEVELOPMENT
We then tested the ability of Skor2 to repress BMP-dependent
transcriptional activity using Id1-Lux, a BMP responsive reporter
(Korchynskyi and ten Dijke, 2002), in the absence or presence (+
BMP2) of exogenous BMP2 expression (Fig. 7B) (Rios et al.,
2004). Similar to that of Ski, expression of Skor2 in Hep3B cells
potently suppressed BMP2 signaling under both basal and BMP2activated conditions (Fig. 7B). These data suggest that Skor2
selectively inhibits BMP- but not TGF-dependent transcriptional
activation.
As BMP signaling activates the Smad family of proteins, we
tested whether Skor2 interacts with Smads 1, 2, 3 and 4 by
performing immunoprecipitations on lysates from co-transfected
cells. We detected strong interactions between Skor2 and Smad 1
and 3, and weaker interactions with Smad 2 and 4 (Fig. 7C-F),
suggesting that Skor2 may mediate transcriptional co-repression
through physical association with Smad1 and Smad3.
Role of Skor2 in cerebellum
RESEARCH ARTICLE 4495
within the SAND domain of Ski, which contributes to its structure
stability (Wu et al., 2002), is preserved in Skor2, indicating that
Skor2 shares the conserved feature of zinc binding. It is worth
pointing out that the biochemical assays performed here are based
on in vitro overexpression of tagged proteins. The physiological
formation of the Skor2 complexes in vivo remains to be confirmed.
Shh produced from Purkinje cells functions as a potent
mitogenic molecule to promote cerebellar granule progenitor
proliferation. Differential signaling by Shh across the cerebellar
cortex, resulting from its uneven distribution along the rostral to
caudal areas of the Purkinje cell layer, leads to the differential
growth of the EGL and the complexity of the cerebellar foliation
(Corrales et al., 2004; Corrales et al., 2006; Lewis et al., 2004).
Our findings that Skor2 and Shh are co-expressed in Purkinje
cell layers in almost identical patterns, that Shh expression is
diminished in Skor2-deficient cerebellum, and that Skor2
potently activates the Shh promoter demonstrate that Skor2 is a
crucial upstream regulator of Shh expression in Purkinje cells.
The differential expression of Skor2 in rostral and caudal regions
suggests that Skor2 might be one of the genetic cues responsible
DEVELOPMENT
Fig. 6. In vitro analysis of Skor2 expression and function. (A)Schematic representation of the Skor2 constructs. Numbers on top indicate the
amino acid position in the Skor2 protein. Each construct was tagged with Flag (for biochemical and localization studies) or with Gal4 (for reporter
assays), as represented by ovals: (1) control (Flag or Gal4 alone); (2) Flag- or Gal4-tagged Skor2 full-length protein; (3-8) a series of Flag- or Gal4tagged Skor2 deletion mutants. (B)Western blot analysis of Flag-tagged wild-type or Skor2 deletion mutants illustrated in A using an anti-Flag
antibody. Anti-tubulin blots are shown as loading controls. (C)Subcellular localization of Skor2 proteins. HEK 293 cells were transfected with
plasmids encoding full-length Flag-tagged Skor2 (Skor2), Skor2 N-terminal 256 amino acids (1-256), N-terminal 385 amino acids (1-385) and Skor2
with deletion of amino acids KRPR, and stained with the anti-Flag antibody. Locations of nuclei of the same cells were visualized by DAPI staining.
Scale bar: 20m. (D)N2a cells were transfected with Gal4-fused Skor2 as illustrated in A, together with the Gal4 responsive luciferase reporter
plasmid pG5E1B-luc and pRL-TK (the latter is used for normalization of Renilla luciferase activity). The transcriptional repression of the luciferase
reporter is represented as fold repression relative to the Gal4-only construct. Data from one representative experiment are shown as mean + s.e.m.,
and the same experiment was repeated at least twice with similar results. (E,F)Skor2/HDAC1 interaction upon co-transfection of Flag-tagged Skor2
and HA-tagged HDAC1 in HEK293 cells, and detected by immunoprecipitation (IP) followed by western blotting (WB) using the antibodies
indicated. Input: blotting of total cell lysates as expression control. IgG: IP with IgG as the negative control. (G)IP and WB using Skor2 truncation
mutants identified amino acid residues 385-592 in Skor2 as the HDAC1 interaction site.
4496 RESEARCH ARTICLE
Development 138 (20)
Shh expression in Purkinje cells with Skor2 as an upstream
regulator. The suppression of BMP signaling by Skor2 during
normal cerebellum development ensures sufficient Shh expression
and granule cell proliferation. This model is supported by the
finding that, even though BMP ligands such as BMP2, BMP4 and
BMP7 are abundantly expressed in all three layers of the
developing cerebellar cortex (Rios et al., 2004), BMP signaling in
Purkinje cells detected by phospho-Smad immunostaining is very
low under physiological conditions (Qin et al., 2006), suggesting
the presence of negative regulators. We performed phosphoSmad1/5/8 staining and found no detectable difference between
Skor2-null and control cerebellum (see Fig. S7 in the
supplementary material). Combining the in vivo result with our in
vitro Skor2 localization studies, we propose that Skor2 modulates
BMP signaling in the nucleus, downstream of Smad
phosphorylation, and by actions involving binding of both Smads
and HDAC. Although the exact mechanism is not clear, our data
indicate that Skor2 act as a negative regulator to suppress BMP
signaling, which in turn allows Shh expression in Purkinje cells.
Nevertheless, it remains possible that Skor2 directly activates the
Shh promoter independently of BMP signaling.
In conclusion, using a novel Sleeping Beauty transposon
mutagenesis strategy coupled with coat color-based genotyping, we
created mutant alleles with a disrupted Skor2 locus. We reveal here
an essential physiological function for Skor2, a new member of the
Ski/Sno family of transcriptional co-repressors, in cerebellar
development. We demonstrate that Skor2, probably via a HDACand Smad-dependent co-repressor function on BMP signaling, is a
regulator of Shh expression in Purkinje cells and thereby exerts a
crucial influence over the proliferation of granule cells during
cerebellum development.
for establishing the Shh signaling gradient to determine the final
size and shape of the cerebellum. The severely reduced size and
compromised foliation of Skor2-null cerebellum support this
conclusion.
In addition to Skor2, previous studies on staggerer mice have
implicated retinoid-related orphan receptor  (ROR) as a positive
regulator of Shh expression (Gold et al., 2003). Whether Skor2
regulated Shh transcription involves the ROR pathway is not
clear. Our studies suggest that the transcriptional activation of Shh
by Skor2 is mediated through a repression of BMP signaling
involving Smad and HDAC complexes. The negative regulation of
Shh expression by BMP has been previously suggested in a number
of developmental and neoplastic contexts (Bastida et al., 2009;
Zhang et al., 2000; Piedra and Ros, 2002; Sharov et al., 2009). We
propose the existence of a similar antagonistic effect of BMP on
Acknowledgements
We thank C. Thaller and A. Liang and R. Atkinson for assistance in in situ
hybridization and confocal imaging, respectively, and the Eunice Kennedy
Shriver Intellectual and Developmental Disabilities Research Center (IDDRC
HD024064) at BCM for supporting the core facilities. We are grateful to D.
Largaespada for the CAGGS-SB10 line, to P. ten Dijke for Id1-luc, to V.
Botchkarev for Shh-luc, to A. Geurts for tyrosinase minigene constructs and to
N. Justice for critical reading of the manuscript.
Funding
This work was supported by grants from NIH (AG020670 and AG032051).
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.067264/-/DC1
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DEVELOPMENT
Role of Skor2 in cerebellum