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.