Supplemental information without figures - cell.com fileembryonic and adult brain sections were...
Transcript of Supplemental information without figures - cell.com fileembryonic and adult brain sections were...
Supplemental data
Figure S1 related to Figure 1. Sox6 expression is confined to SN dopamine
neurons. (A-‐D) Overview of Sox6 expression in dopamine neurons in the adult brain at
four distinct anteroposterior levels A most anterior and D most posterior. A’ and B’ are
higher magnification images of respectively A and B. C’ and D’ are higher magnification
images of the SN, while C’’ and D’’ are higher magnification images of the VTA shown in C
and D. (E-‐J) Immunohistochemical analysis of Sox6 expression in coronal midbrain
sections of E18.5 embryos. Sox6 expression in combination with Calbindin (Calb) in SN
(E) and VTA (F) neurons show that there is no overlap in expression. Sox6 co-‐localizes
with GlycoDat in SN neurons (G) and in a few neurons in the dorsolateral part of the VTA
(H). A subset of Sox6+ mDA neurons in the SN expresses Raldh1 (I), while all Raldh1+
neurons in the VTA are Sox6 negative (J). (K-‐N) Immunohistochemical analysis of Girk2
expression in dopamine neurons in the adult brain. L and M are higher magnification
images of the SN shown in K and N is a higher magnification image of the VTA. (O-‐R)
Expression analysis of Sox6 in combination with Girk2 showing overlap between Sox6
and Girk2 in SN neurons in the adult mouse brain. P and Q are higher magnification
images of the SN shown in O and R is a higher magnification image of the VTA. Scale
bars: 100 μm.
Figure S2 related to Figure 2. Detailed analysis of Sox6 expression in dopamine
progenitors and post-mitotic neurons during embryonic development.
(A-‐D) Analysis of Sox6 and Nolz1 expression in P0 Pitx3+/gfp heterozygous and Pitx3gfp/gfp
homozygous mutant midbrains. GFP marks Pitx3+ mDA neurons. Arrowheads in C and D
indicate Nolz1 positive GFP positive mDA neurons. (E-‐H) Sox6 fails to be expressed in
Pitx3 mutant mDA neurons in E12.5 (E, F) and E13.5 (G, H) embryos. Arrowheads in E
and G point to Sox6 expressing GFP positive mDA neurons. (I) Immunohistochemical
analysis of Sox6 and Corin in E11.5 ventral midbrain dopamine neural progenitors. (J)
Analysis of Sox6 expression in relation to Nurr1 in E11.5 and E12.5 embryonic ventral
midbrain by immunofluorescence. (K) Sox6 expression shown in midbrain by in situ
hybridization. Scale bars: 100 μm.
Figure S3 related to Figure 3. Otx2 is sufficient to repress Sox6 and analysis of
striatal innervation in E18.5 mutant embryos. (A-‐B) Otx2 is not necessary but
sufficient to repress Sox6 expression in post-‐mitotic mDA neurons. (A) Sox6 expression
is not ectopically induced in VTA neurons of DatCre;Otx2fl/fl mutant adult mice. (B)
Overexpression of Otx2 in post-‐mitotic mDA neurons represses Sox6 expression in SN
neurons. Coronal midbrain sections are taken from adult mice. (C-‐D) Reduced levels of
TH expressing fibres in the striatum of Sox6-/- mutant embryos. (C) Images represent
coronal sections through the striatum of E18.5 embryo stained with TH to visualize TH
expressing fibres. Area 1 marks the lateral part of the striatum while area 2 marks the
medial part. (D) Immunohistochemistry of TH innervation analysed by densitometry in
a lateral (area1) and medial (area 2) region of the striatum in wild-‐type and Sox6-/-
embryos, respectively. (n=4). Mean values +/-‐ standard deviation; **p<0.01. (E-‐F) No
defect in striatal innervation during embryonic development DatCre;Sox6fl/fl mutant
embryos. (E) Immunohistochemical analysis of TH expression in striatum of E18.5
DatCre;Sox6fl/fl mutant embryos. (F) Graph showing the relative signal intensity of TH
expression as measured by densitometry in the indicated striatal areas. Mean values +/-‐
standard deviation. Scale bars: 100 μm.
Figure S4 related to Figure 4. Sox6 is required for maintenance of SN neurons in
adults. (A) Immunostains showing TH expression in the caudal part of the striatum and
in the VTA of adult mice. (B) Immunofluorescence analysis of TH expression shows a
reduced number of fibers in the dorso-‐lateral part of the striatum in DatCre;Sox6fl/fl
mutant adult mice. Lower panel are higher magnification images of boxed areas in the
upper panel. (C) DA and DA metabolites (DOPAC and HVA) analysed by high
performance liquid chromatography from extracts of the ventral part of the striatum
(area 2) of 6 months old DatCre;Sox6fl/+ and DatCre;Sox6fl/fl mice. Values are shown as
percentages relative to the levels detected in extracts from DatCre;Sox6fl/+ mice (100%).
Mean values +/-‐ standard deviation. (D) The openfield test measured the total
locomotor activity of the control (n=25) and DatCre;Sox6fl/fl (n=26) mutant mice. The
graphs presents mean values +/-‐ SEM; *p<0.05. (E) Sox6 and TH expression in
neuromelanin positive neurons of the human SN. Staining for Sox6 and TH was
performed on consecutive sections and images were taken from similar regions of brain.
Distribution of Sox6 (dark blue) and TH (brown) is localized to neuromelanin-‐positive
(dark brown) neurons in the SN. Scale bars: 100 μm.
Supplemental experimental procedures
Mouse strains Sox6fl/+ animals (Dumitriu et al., 2006) were crossed with DatCre/+;Sox6fl/+
animals (Ekstrand et al., 2007) to obtain DatCre/+;Sox6fl/fl mutant offspring. No differences
were observed between the control animals carrying different combinations of alleles. To
generate constitutive Sox6-/- mutant embryos Sox6fl/fl mice were crossed with CmvCre/+ mice to
generate CmvCre/+; Sox6fl/+ offspring. CmvCre/+; Sox6fl/+ mice were crossed to C57bl6 wild-
type animals to obtain Sox6+/- heterozygous mutant animals. Sox6-/- mutant embryos were
obtained from crossings between Sox6+/- heterozygous mice. Pitx3eGFP/+ heterozygous mutant
animals were interbred to obtain Pitx3eGFP/eGFP mutant embryos (Zhao et al., 2004). Otx2
mutant mouse strains were kept and bred as described in Giovannantonio et al., 2013 and
DiSalvio et al., 2010.
Immunohistochemistry Embryos were fixed for 1-3 hrs in 4% PFA and cryosectioned at a
thickness of 12 µm and further processed as described before (Briscoe et al., 2000). Adult
mice were perfused with 4% PFA as described by Kadkhodaei et al., 2009 and dissected
brains were post-fixed for 4 hrs in 4%PFA. Sections were cut on a freezing microtome at a
thickness of 30 µm. Immunofluoresence labelled cells were analyzed using the LSM5 Exciter
Zeiss confocal microscope. Pictures were taken at 20x magnification. Otx2 mutant embryos
and adult mouse brains were processed and analysed as described before (Giovannantonio et
al., 2013 and DiSalvio et al., 2010). To analyse TH+ and DAT+ fibre density in the striatum
embryonic and adult brain sections were quenched with 3% H2O2 (Merck), incubated with
primary antibody and biotinylated secondary antibody (Jackson Immunoresearch), followed
with avidin–biotin-peroxidase complex (ABC Elite; Vector Laboratories), and visualized
using 3,3-diaminobenzidine as a chromogen. Sections were analyzed and photographed using
Eclipse E1000K (NIKON) microscope equipped with a digital camera (Spot2, Diagnostic
Instruments Inc.) using 10x magnification.
Fluorogold retrograde tracing Animals (4-5 months old; n=4) were anesthetised with
isofluorane. Animals received an unilateral stereotaxic injection in the striatum (Bregma
coordinates AP: +0.8mm, DL: +2.0mm, DV coordinate from Dura: -‐3mm) using a 10µl
Hamilton microsyringe (33 gauge cannula) with 0.75µl Fluorogold
(hydroxystilbamidine, 4%, Biotium) injected over 2 minutes. The needle was left in situ
for further 4 minutes before slowly retracted. Animals were perfused 24hrs after the
injection and isolated brains were fixed for an additional 4 hrs in 4% PFA. Sections were
cut on a freezing microtome at a thickness of 30 µm and analysed by immunofluorescence.
Cell counting and optical densitometry analysis To count the number of Calbindin,
GlycoDat and Raldh1 expressing TH positive neurons in the substantia nigra a minimum of 3
embryos were analysed. Of each embryos every 4th section was counted manually. Statistical
significance was calculated by one-tailed unpaired students t-test and data is presented as
mean±sd. Striatal fibre density was measured by densitometry using ImageJ software. The
measured values were corrected for non-specific background staining by subtracting values
obtained from the cortex. Of each embryo every 4th section was analysed and of the adult
brain every 6th section. At least 3 different embryos and adult brains of each time point were
analysed. Data expressed as a percentage of the control value and the students t-test was used
to determine significance.
HPLC Brains of 6 months old animals (n=10) were dissected and snap frozen in dry ice
cooled isopentane. The frozen brain was sectioned at a thickness of 1mm and regions of
interest were taken using a 1 mm and 1.2 mm thick neural punch. Tissue was pooled from
both hemispheres, weighted and stored at -80 °C. Dopamine and the metabolites DOPAC and
HVA were determined by HPLC with electrochemical detection following minor
modifications as described elsewhere (Kehr, 1999; Kehr and Yoshitake, 2006). Significance
was determined using the unpaired student’s t-test.
Behavioural analysis Open field: The test was performed a 55 x 35 x 30cm arena with lines
(squares of 7 x 35cm) painted on its floor. The animals (control n=25; DatCre;Sox6fl/fl n=26)
aged between 5 and 7 months were positioned in the center of the box habituated for 10
minutes and filmed for another 15 minutes. The number of lines crossed during the
monitoring period was measured. Crossing a line was only considered and counted when the
mouse moved both fore- and hind limbs from one square to another. The unpaired one-tailed
student’s t-test was used to determine the significance.
Human post-mortem tissue Paraffin-embedded midbrain sections from PD patients (n=8)
and age-matched control individuals (n=4) were provided by the UK Parkinson’s Disease
Society Tissue Bank at Imperial College. For histological analysis, sections were first
deparaffinised by heating 20 min at 60°C followed by incubation in Xylene and rehydrated in
an ethanol series. Antigen retrieval was performed by a 45 min incubation in Target Retrieval
Solution (DAKO) at 100°C. Sections were then blocked in PBS - 0,25% TritonX-100 - 10%
donkey serum for 1 hour and incubated with anti-Sox6 (Sigma, 1:50) in blocking buffer
overnight at 4 °C. Sections were then washed and incubated with biotinylated anti-rabbit
secondary antibody (Jackson Immunoreseach, 1:200) for 1 hour at room temperature, washed,
and incubated with ready-to-use ABC complex solution for 1 hour at room temperature. SG
substrate or 3,3-diaminobenzidine (Vector Laboratories) was added to visualize the
peroxidase reaction, and identification of dopamine neurons was ascertained by the
visualization of neuromelanin pigments. The intensity of Sox6 levels in neuromelanin
positive cells was measured by densitometry in both control samples (n=4) and PD patient
samples (n=8) using ImageJ software. Of each specimen 2-6 images were taken and in total
145 cells from the control samples and 323 cells from the PD patient samples were analysed.
The intensity values are shown in arbitrary units (AU) where a value of 255 was set as a
maximum value and 0 as a minimum value. The difference in intensities between control and
PD samples was determined in two different ways. One way was to compare average intensity
levels in all individual cells between the control (n=145) and the patient samples (n=323). The
second way was to take the average level from each sample and compare control (n=4) with
PD samples (n=8). The one-tailed unpaired students ttest was used to determine significance.
In situ hybridization In situ hybridizations on sectioned embryos were performed as
previously described (Schaeren-Wiemers and Gerfin-Moser, 1993). cDNA of Epha5 for DIG-
RNA probe synthesis (Roche) was obtained from FANTOM2 RIKEN collection or from
Open Biosystems. Sections were analyzed and photographed using Eclipse E1000K (NIKON)
microscope equipped with a digital camera (Spot2, Diagnostic Instruments Inc.) using 10x
magnification.
Antibodies The following antibodies have been used mouse TH (chemicon); rabbit TH
(PelFreez), Lmx1a, Otx2, Raldh1, Sox6, Nolz1 (Sigma), Calbindin (Swant), Nurr1 (Santa
Cruz), Corin (Gift from Malin Parmar); Guinea Pig Sox6 (gift from J.M), Pitx3 (J.E), Lmx1a
(J.E.); Goat Otx2 (R&D); Sheep TH (PelFreez) Rat Dat (Chemicon).
Supplemental references Kehr, J. (1999). Monitoring chemistry of brain microenvironment: biosensors, microdialysis
and related techniques. Chapter 41. In: Modern techniques in neuroscience research. (Eds. U.
Windhorst and H. Johansson) Springer-Verlag GmbH., Heidelberg, Germany. 1149-1198.
Kehr, J., Yoshitake, T. (2006). Monitoring brain chemical signals by microdialysis. In:
Encyclopedia of Sensors, Vol. 6. (Eds. C.A. Grimes, E.C. Dickey and M.V. Pishko)
American Scientific Publishers, USA. 287-312.
Zhao, S., Maxwell, S., Jimenez-Beristain, A., Vives, J., Kuehner, E., Zhao, J., O'Brien, C., de
Felipe, C., Semina, E., Li, M. (2004). Generation of embryonic stem cells and transgenic mice
expressing green fluorescence protein in midbrain dopaminergic neurons. Eur. J. Neurosci.
19(5), 1133-1140.