Material  and  methods

Zebrafish  strains  and  husbandry  

AB*  zebrafish  and  mutant  strains  were  kept  in  a  14h/10  hours  light/dark  cycle   at   28°C.   Zebrafish   embryos   were   obtained   as   previously   described   [22].  

Generally,  2  male  and  2  female  zebrafish  of  the  desired  genotype  were  placed  in   a  breeding  tank  in  the  late  afternoon.  In  the  morning  when  the  dark/light  cycle   starts  female  zebrafish  spawn.  Eggs  were  collected  and  placed  for  24  hours  in  a   larvae  incubator  at  28°C.  Hereafter,  eggs  were  bleached  in  170  ml  system  water   containing  0.1  ml  of  5%  sodium  hypochlorite.  Next,  eggs  are  dechorionated  in  a  1   mg/ml   solution   pronase   (Roche).   Subsequently,   zebrafish   are   kept   during   four   days   at   28°C   before   being   placed   in   the   nursery   system.   The   following   mutant   zebrafish   were   used:   Cx41.8tq270/tq270,   Cx41.8^t1/t1,   Cx41.8^t1/t1Cx45.6^{-­‐/-­‐},   flk1:eGFP,   Cx41.8tq270/tq270   flk1:eGFP,   Cx41.8^t1/t1   flk1:eGFP   and   Cx41.8^t1/t1Cx45.6^{-­‐/-­‐}  

flk1:eGFP  zebrafish.  

Zebrafish  genotyping  

For   genotyping   genomic   DNA   was   extracted   from   fin   clips.   In   short,   a   small   (2mm  x  2mm)  fin  clip  fragments  were  incubated  for  10  min  at  98°C  in  100  μl  50   mM  NaOH.  Subsequently,  the  basic  solution  was  quenched  with  20  μl  0.5M  TRIS   (pH  8).  Thereafter,  obtained  DNA  was  diluted  by  adding  100  μl  H2O,  and  2  μl  of  

of   the   PCR   mix   and   temperature   cycles   are   given   in   Table   2   and   Table   3,   respectively.   For   the   Cx41.8   tq270   and   tq1   mutation,   PCR   products   were   genotyped  (Fasteris,  Switzerland)  using  the  forward  primer  and  blasted  against   the  WT  zebrafish  sequence  for  mutation  detection.  For  genotyping  of  Cx45.6^{-­‐/-­‐},  a   restriction  digestion  was  used.  In  fact,  the  knock-­‐out  mutation  introduces  a  7bp   deletion  located  at  a  BsrD10  restriction  site  resulting  in  a  275bp  PCR  fragment   for   WT   zebrafish   and   a   268bp   PCR   fragment   for   Cx45.6-­‐/-­‐  zebrafish.   The   PCR   products   were   incubated   with   the   BsrD10   enzyme   for   30   min   at   65°C   and   the   reactions   were   stopped   by   heating   the   sample   to   80°C   for   20   min.   Effective   digestion   for   WT   zebrafish   was   controlled   through   agarose   gel   electrophoresis   resulting  in  a  221bp  fragment.  For  a  step-­‐by-­‐step  lab  protocol  see  Table  4.  

FACS  sorting  of  flk1:eGFP+  endothelial  cells  and  connexin  detection    

GFP+  endothelial  cells  were  obtained  from  adult  flk1:eGFP  transgenic  zebrafish   tails.   In   short,   tail   fragments   were   dissociated   using   a   0.5   mg/mL   liberase   (Roche)  solution  at  33°C  for  90  min  and  resuspended  in  0.9x  PBS-­‐1%  fetal  calf   serum   (FCS).   ECs   were   sorted   on   basis   of   the   GFP   fluorescence   and   remaining   cells  (GFP-­‐)  were  used  as  negative  control.  Dead  cells  were  excluded  by  SYTOX-­‐

red   (Life   Technologies)   staining.   Approximately   800   GFP+   cells   were   lysed   to   obtain  RNA  using  the  NucleoSpin  RNA  II  kit  (Macherey-­‐Nagel)  according  to  the   manufacturer’s  instructions.  After  extraction,  RNA  was  used  to  obtain  cDNA  with   the   QuantiTect   Reverse   Transcription   Kit   (Qiagen)   following   manufacturer’s   protocol.  PCR  was  performed  using  the  ABI  StepOne  Plus  detection  system  with   SYBERgreen   gene   expression   assays   (KAPA   Syberfast,   Kappa   Systems)  

and   normalized   to   ef1α   using   the   ∆∆CT   method   see   table   5.   CT   values   for   undetermined  samples  were  arbitrary  set  to  40.    

Whole  mount  in  Situ  hybridization  (WISH)  staining  and  analysis  

WISH  was  performed  on  48  hours  post-­‐fertilization  (hpf)  embryos  fixed  with  4%  

paraformaldehyde  (PFA).  Digoxygenin  labeled  probes  were  synthesized  with  the   RNA   labeling   kit   (SP6/T7;   Roche).   The   RNA   probes   were   generated   through   linearization  of  TOPO-­‐TA  vectors  (Invitrogen)  that  contained  the  PCR-­‐amplified   cDNA   sequence   (Table   6).   WISH   was   performed   as   previously   described   [23];  

details   are   given   below.   Embryos   were   imaged   in   100%   glycerol   using   an   Olympus  MVX10  microscope.  

WISH  extended  protocol  

• Probe  preparation  

Primers  against  the  coding  sequence  of  Cx41.8  and  Cx45.6  were  designed  using   the  Primer3  online  software  resulting  in  an  amplicon  of  829bp  for  Cx41.8  and  of   789bp   for   Cx45.6   (see   Table   6).   PCR   was   performed   using   REDTaq   DNA   polymerase   (Sigma).   Next,   the   obtained   PCR   product   was   ligated   inside   the   TOPO-­‐TA   vector   (Invitrogen)   and   transformed   inside   Mach1   bacteria   (Life   Technologies).   Optimal   transformation   was   assessed   through   IPTG   and   X-­‐Gal   screening   of   the   colonies   on   100   μg/ml   ampicillin-­‐containing   agar   petridishes.  

Subsequently,   positive   colonies   were   picked   and   amplified   overnight.   The  

Biolabs),   linearized   vectors   were   controlled   for   integration   of   the   cDNA   using   agarose   gel   electrophoresis   and   sent   for   sequencing   to   confirm   introduction   of   the   desired   sequence   and   direction   using   a   M13   forward   primer.   Once   integration   of   the   right   cDNA   was   confirmed   a   Midiprep   (GenoMed)   was   performed   to   collect   a   considerable   amount   of   vectors.   Then,   the   vector   was   linearized  (EcoRI)  and  precipitated  overnight  at  4°C.  In  short,  10  μg  of  vector  in   100  μl  solution  was  washed  twice  with  Phenol:choloroform.  Subsequently,  800   μl   of   a   solution   containing   100%   EtOH,   50μl   3M   NaAc   and   1   μl   of   2   mg/ml   glycogen   was   added   to   the   supernatant   containing   the   vector   and   stored   overnight   at   -­‐20°C   for   precipitation.   DIG-­‐labeled   probes   were   synthetized   through   incubation   of   the   linearized   vector   with   DIG-­‐RNA   labeled   mix   (Roche)   with  SP6  transcriptase  for  the  Cx41.8  TOPO-­‐TA  (plus/plus)  and  T7  transcriptase   for   Cx45.6   TOPO-­‐TA   (plus/min)   for   2   hours   at   37°C   followed   by   probe   purification  using  the  High  Pure  PCR  Cleanup  Micro  kit  (Roche)  according  to  the   manufacturer’s  protocol.  Finally,  synthesis  and  probe  purification  were  checked   by  agarose  gel  electrophoresis.    

• In  Situ  Hybridization  

Embryos  were  fixed  overnight  at  4°C  in  4%  PFA,  washed  twice  in  PBT  (1x  PBS   0.1%   Tween   20)   and   dechorionated.   Dehydration   was   performed   overnight   in   methanol   at   -­‐20°C.   Subsequently,   embryos   were   rehydrated   stepwise   in   methanol/PBT  (3:1,  1:1,  1:3)  till  a  solution  of  100%  PBT  was  reached.  Next,  the   48  hpf  embryos  were  incubated  for  15  min  with  proteinase  K  (10  μg/ml  in  PBT)   and  washed  with  1x  PBT  to  stop  the  reaction.  Post-­‐fixation  was  performed  in  4%  

PFA   for   20   min   and   embryos   were   rinsed   5   times   for   5   min   with   1x   PBT   and  

buffer,   50   μg/ml   heparin,   500   μg/ml   tRNA,   0.1%   Tween   20,   9   mM   citric   acid).  

Hybridization  was  performed  in  identical  buffer  containing  50  to  100  ng  of  the   desired  probe  overnight  at  70°C.  Subsequently,  embryos  were  washed  at  70°C  a   series   of   buffers   (for   10   min   in   75%   hybridization   buffer,   25%   SSC   2x   SSC;   10   min  in  50%  hybridization  buffer,  50%  SSC  2x  SSC;  10  min  in  25%  hybridization   buffer,  75%  SSC  2x  SSC;  10  min  in  2x  SCC;  and  finally  2  times  30  min  in  0.2x  SSC).  

An   additional   series   of   washing   step   were   subsequently   performed   at   room   temperature,  i.e  5  min  in  75%  0.2%  SSC,  25%  PBT;  5  min  in  50%  0.2%  SSC,  50%  

PBT;  5  min  in  25%  0.2%  SSC,  75%  PBT;  5  min  in  PBT;  and  finally  1  hour  in  PBT   with   2   mg/ml   BSA,   2%   sheep   serum.   Next,   embryos   were   incubated   at   4°C   overnight   with   alkaline   phosphatase-­‐coupled   anti-­‐digoxigenin   antiserum   at   a   concentration  of  1/5000  in  PBT-­‐buffer  containing  2%  BSA  and  2%  sheep  serum.  

Finally,  embryos  were  washed  6  times  for  15  min  in  PBT  at  room  temperature.  

The  detection  was  performed  in  alkaline  phosphatase  reaction  buffer.  Upon  color   development  the  reaction  was  stopped  in  1x  PBS  4%  PFA  at  4°C.  Embryos  were   imaged  using  an  Olympus  MVX10  microscope.  

Statistics  

Results   are   presented   as   mean   ±   SEM.   Unpaired   Student’s   t-­‐tests   were   used   to   compare   differences   between   groups.   Differences   with   a   P   <   0.05   were   considered  statistically  significant;  *,  P  <  0.05.  

4.3.1. Cx41.8  and  Cx45.6  are  expressed  in  ECs  of  zebrafish  

To  investigate  whether  Cx41.8  and  Cx45.6  mRNA  is  present  in  the  endothelium   of   zebrafish,   flk1:eGFP   zebrafish   were   fin   clipped   and   cells   were   dissociated   using  liberase.  ECs  expressing  eGFP  were  separated  from  non-­‐endothelial  eGFP-­‐

negative  cells  by  FACS  sorting  (Figure  1A).  mRNA  was  isolated  from  the  FACS-­‐

sorted   samples   and   the   expression   of   the   Cx41.8   and   Cx45.6   was   assessed   by   qPCR.  We  found  Cx41.8  (Figure  1B)  and  Cx45.6  (Figure  1C)  transcripts  in  ECs  of   zebrafish,  whereas  these  mRNAs  were  almost  absent  in  eGFP-­‐negative  cells.  Of   note,  the  very  low  level  of  Cx41.8  in  the  eGFP-­‐negative  cells  may  originate  from  a   few  melanophores  present  in  the  fin  clip  [19].  

Next,  we  used  WISH  to  detect  Cx41.8  and  Cx45.6  mRNA  in  whole  mount  48  hpf   zebrafish.   Therefore,   in   situ   probes   were   designed   directed   against   the   two   connexins.   First,   we   analyzed   MACH1-­‐bacteria   containing   the   TOPO-­‐TA   vector   expected   to   include   the   sequence   of   Cx41.8   and   Cx45.6.   Figure   2A   shows   after   extraction  of  the  vector  from  4  positive  colonies  that   2   TOPO-­‐TA  Cx41.8  and  4   TOPO-­‐TA   Cx45.6   MACH1   bacteria   colonies   contained   the   right   insert   of   829bp   and  789bp  for  Cx41.8  and  Cx45.6,  respectively.  Of  note,  the  upper  band  present   in   all   colonies   represents   the   linearized   empty   vector.   Next,   to   assess   the   insertion   direction   of   the   gene   in   the   TOPO-­‐TA   vector   2   positive   vectors   were   sequenced   using   the   M13   Forward   primer.   Subsequently,   the   obtained   sequences   were   blasted   against   the   WT   sequence   of   Cx41.8   and   Cx45.6.   We   found  that  the  sequence  of  Cx41.8  was  inserted  in  the  plus/plus  and  Cx45.6  in   the   plus/min   direction.   Indeed,   to   obtain   an   optimal   blast   result   the   reverse  

purified  probes  showed  one  discrete  band  accounting  for  a  pure  probe  (Figure   2C).  WISH  was  performed  on  WT  48  hpf  zebrafish  embryos  using  the  designed   DIG-­‐labeled   probes.   At   this   developmental   stage,   Cx41.8   mRNA   was   mostly   detected  in  the  heart  (Figure  2D)  and  Cx45.6  was  detected  in  the  heart,  lateral   dorsal  aorta  (LDA),  dorsal  midline  junction  (DMJ)  and  pectoral  fin  buds  (PFB)  of   the  zebrafish  larvae  (Figure  2E).  

4.3.2. Genotyping  of  Cx41.8tq270/tq270,  Cx41.8^t1/t1  and  Cx41.8^t1/t1Cx45.6^{-­‐/-­‐}

zebrafish.  

To   study   the   effect   of   Cx40   in   early   atherogenesis   we   intend   to   use   different   zebrafish   strains   mutated   for   the   Cx41.8   and   Cx45.6.   Zebrafish   harboring   the   Cx41.8tq270/tq270  (leading  to  decreased  Cx41.8  channel  function;  [19]),  Cx41.8^t1/t1   (containing  a  premature  stopcodon,  inducing  a  knock-­‐out  phenotype;  [19])  and   Cx41.8^t1/t1Cx45.6^{-­‐/-­‐}  (double  knock-­‐outs)  were  provided  by  Professor  Masakatsu   Watanabe,   Osaka   University,   Japan.   To   facilitate   the   visualization   of   the   vasculature   for   further   experiments   all   zebrafish   strains   were   crossed   with   flk1:eGFP   zebrafish   [24].   Offspring   were   screened   at   day   5   hpf   for   eGFP   fluorescent   vasculature   by   microscopy   (Figure   3A).   To   discriminate   between   Cx45.6  wild-­‐type  (Cx45.6^+/+),  heterozygous  (Cx45.6^+/-­‐)  and  homozygous  mutant   (Cx45.6^{-­‐/-­‐})   fish   we   used   an   approach,   which   was   based   on   the   fact   that   the   mutation   introduces   a   7bp   deletion   located   at   a   BsrD10   digestion   site.   Firstly,   using   designed   primers   we   amplified   a   sequence   of   275bp   containing   (or   not)   the  BsrD10  site  (Figure  3B).  Secondly,  the  275bp/268bp  fragment  was  exposed  

fragment  indicating  the  Cx45.6-­‐/-­‐  genotype  (Figure  3C,  lanes  1  and  2),  or  2  band   at   268bp   and   221bp   indicating   a   heterozygous   genotype   (Figure   3C,   lanes   3,   4   and  5)  or  the  DNA  from  WT  fish  was  completely  digested  resulting  in  1  band  at   221bp  (Figure  3C,  lane  6).  Finally,  the  t1  and  tq270  mutations  in  the  Cx41.8  gene   were  detected  by  sequencing.  The  t1  mutation  is  a  C>T,  resulting  in  a  TGA  codon   thus   introducing   a   premature   stopcodon   (Figure   3D).   The   tq270   mutation   is   a   A>T,  which  changes  the  codon  ATT  (isoleucine)  to  TTT  (phenylalanine)  resulting   in  a  decreased  channel  function  (Figure  3E)[19].