Transcriptional  targets  of  Eph  receptor  and  ephrin  signalling   in  the  zebrafish  hindbrain       Hannah  Amy  Stanforth       University  College  London   and   The  Francis  Crick  Institute   PhD  Supervisor Dr  David  Wilkinson   :       A  thesis  submitted  for  the  degree  of   Doctor  of  Philosophy   University  College  London     February 2018 Declaration   I  Hannah  Amy  Stanforth  confirm  that  the  work  presented  in  this  thesis  is  my  own.     Where  information  has  been  derived  from  other  sources,  I  confirm  that  this  has  been   indicated  in  the  thesis.   2 Abstract   In  vertebrates,  there  is  a  large  family  of  Eph  receptor  tyrosine  kinases  and  their   ephrin  ligands,  which  have  complex  and  varied  roles  during  development  and  in  adult   homeostasis.  The  most  researched  role  of  Eph  receptors  and  ephrins  is  in  control  of   cell  migration  through  the  regulation  of  the  actin  cytoskeleton  and  cell  adhesion.   More  recently,  it  has  been  found  that  in  some  tissues  Eph-­ephrin  signalling  also  leads   to  changes  in  gene  transcription,  for  example  to  control  cell  differentiation.  In  the   zebrafish  hindbrain,  Eph  receptors  and  ephrins  are  expressed  segmentally  in  the   rhombomeres  in  a  complementary  pattern  with  respect  to  their  binding  partner.   Signalling  via  this  pathway  induces  a  unique  cell  population  to  arise  at  rhombomere   borders,  known  as  the  boundary  cells.       In  order  to  understand  more  about  Eph  receptor  and  ephrin  function  in  the  hindbrain,   RNA-­sequencing   was   carried   out   on   dissected   hindbrains   of   zebrafish   with   endogenous  Eph-­ephrin  signalling  and  fish  that  lack  Eph-­ephrin  signalling.  The   transcriptional  profiles  were  then  compared  to  identify  potential  downstream  targets,   which  were  verified  using  RT-­qPCR  and  in  situ  hybridisation.  This  identified  four   genes  regulated  downstream  of  Eph-­ephrin  signalling  that  are  markers  of  progenitor   cells   and   neural   differentiation.   When   Eph-­ephrin   signalling   is   disrupted   the   expression  of  these  genes  alters,  and  the  expression  pattern  of  one  gene,  mdka,   was  consistent  with  loss  of  hindbrain  boundary  cells.     To  investigate  this  observation  further,  the  expression  of  progenitor  and  neurogenic   markers  was  determined  when  Eph-­ephrin  signalling  was  disrupted.  This  supported   previous  studies  which  found  that  Eph-­ephrin  signalling  is  required  for  formation  of   boundary   cells   and   that   boundary   cell   loss   results   in   ectopic   neurogenesis.   In   addition,  it  was  found  that  ectopic  neurogenesis  was  accompanied  by  the  depletion   of  nestin-­expressing  neural  progenitor  cells  at  later  stages  of  development.  Together   these  findings  support  previous  work  showing  that  hindbrain  boundary  cells  are   essential  for  restricting  neurogenesis  to  neurogenic  zones  adjacent  to  the  boundaries.       3 Impact  Statement   Eph-­ephrin  signalling  is  important  for  normal  development  as  it  is  active  in  many   tissues  and  regulates  a  variety  of  cellular  processes.  It  has  been  well  characterised   that  signalling  results  in  regulation  of  the  actin  cytoskeleton  and  cell  adhesion  leading   to  cell  responses  such  as  cell  migration.  This  ensures  that  cell  populations  are   organised  at  the  correct  location  as  well  as  forming  boundaries  between  different  cell   populations.  In  addition,  Eph-­ephrin  signalling  has  also  been  shown  to  regulate   transcription   in   many   tissues.   In   the   hindbrain   Eph-­ephrin   signalling   prevents   intermingling  between  segments  as  well  as  being  responsible  for  the  induction  of  a   distinct  cell  population  that  arise  at  the  borders  of  segments.  As  little  is  known  about   the  genes  that  are  regulated  by  Eph-­ephrin  signalling,  identifying  these  will  help  to   contribute   to   understanding   hindbrain   development.   Understanding   Eph-­ephrin   signalling   is   also   important   as   when   signalling   is   mis-­regulated   this   can   have   detrimental  consequences.  During  early  development,  this  can  result  in  the  incorrect   formation  of  head  structures  as  signalling  is  responsible  for  guiding  the  neural  crest   cells  to  the  correct  location.  Regulation  of  Eph-­ephrin  signalling  is  also  important  later   in  life  and  a  mouse  model  for  Alzheimer’s  disease  shows  that  Eph  receptors  are   expressed  at  the  incorrect  levels.  Finally,  Eph-­ephrin  signalling  is  mis-­regulated  in   many  types  of  cancers,  and  leads  to  changes  in  cell  migration.     In  this  project,  the  role  of  Eph-­ephrin  signalling  in  regulating  gene  expression  was   investigated  in  the  context  of  hindbrain  development.  This  has  unveiled  novel  genes   that  are  regulated  downstream  of  Eph-­ephrin  signalling,  and  based  on  their  function   imply   that   signalling   is   regulating   the   progression   of   neurogenesis.   Further   investigation  has  shown  that  Eph-­ephrin  signalling  is  important  for  patterning  of   neurogenesis  via  the  induction  of  boundary  cells.  These  findings  will  contribute  to   understanding  hindbrain  development  and  the  role  that  Eph-­ephrin  signalling  plays   in  regulating  hindbrain  neurogenesis.     During  this  PhD,  I  have  generated  gene  expression  data  by  RNA-­sequencing  to   identify  transcriptional  targets  of  Eph-­ephrin  signalling.  Whilst  I  was  able  to  validate   several  of  these  target  genes,  this  list  still  holds  many  possibilities  for  avenues  of   further  research.  One  approach  could  be  to  investigate  the  significance  of  altered   4 expression  of  other  genes  found  by  RNA-­sequencing  analysis.  In  addition,  the  quality   control  method  designed  for  selecting  hindbrains  with  low  contamination  from  tissues   surrounding  the  hindbrain  continues  to  be  used  in  the  lab  for  future  RNA-­sequencing   experiments.   Finally,   during   this   PhD   I   have   communicated   my   research   by   presenting  posters  at  several  meetings,  including  the  18th  International  Congress  of   Developmental  Biology,  the  Young  Embryologist  Meeting  and  student  symposia  at   the  Francis  Crick  Institute.         5 Acknowledgements   Firstly,  I  would  like  to  thank  David  for  giving  me  the  opportunity  to  carry  out  this  project   in  his  lab  and  for  his  continued  support  and  guidance  throughout  the  course  of  my  PhD.   I  would  like  to  thank  the  rest  of  the  members  of  the  Wilkinson  Lab,  past  and  present,  for   making  the  last  four  and  a  half  years  enjoyable,  for  celebrating  the  successes,  and   sharing  the  frustrations,  of  science.  In  particular,  I  would  like  to  thank  Jordi  for  his  advice,   suggestions,  as  well  as  the  use  of  his  fish  lines,  one  of  which  was  invaluable  to  the   success  of  this  project.  I  would  also  like  to  thank  Qiling  for  sharing  her  wealth  of   knowledge  about  zebrafish  alongside  being  a  caring  lab-­mate  and  an  excellent  cook.  I   must  thank  Probir,  who  not  only  analysed  the  RNA-­sequencing  data  collected  during  this   project,  and  was  also  patient  in  dealing  with  the  many  questions  that  came  with  this  task.   Thank  you  to  the  High  Throughout  Sequencing  facility,  who  sequenced  all  the  samples   in  this  project  and  the  Crick  Aquatics  staff  for  looking  after  the  fish  that  I  have  used.  I   would  also  like  to  thank  my  thesis  committee:  Vassilis  Pachnis,  Francois  Guillemot  and   Mike  Gilchrist  for  their  advice  and  useful  discussions.     I  would  like  to  thank  my  family  and  friends  for  their  support  over  the  course  of  my  PhD.   Thank  you  to  my  parents  for  continually  paying  an  interest,  their  belief  in  my  capabilities   and  for  their  consistent  encouragement  throughout.  Laurence,  my  husband-­to-­be,  for   having  more  faith  in  me  than  I  do  in  myself,  for  celebrating  all  the  small  victories  along   the  way  and  for  looking  after  me  during  this  process,  therefore,  I  cannot  thank  you   enough.  Thank  you  to  Ben  and  Jade  for  their  support,  understanding  and  checking  in  on   how  things  are  going.  I  would  also  like  to  thank  my  grandparents,  for  always  asking   irrespective  of  not  knowing  exactly  what  I  was  doing.  Thank  you  to  my  friends,  scientist   and  non-­scientists,  for  their  motivation  and  ever-­increasing  interest  in  fish,  especially   Katie  for  her  supporting  audio  notes.  Thank  you  to  the  Forman’s  for  sending  snacks  in   the  post.       Finally,  thank  you  to  a  few  groups  of  people.  Firstly,  I  would  like  to  thank  my  fellow  Crick   students  who  have  attended  ‘Breakfast  Club’  over  the  past  four  years  to  provide  a   wonderful  support  network  over  a  few  hash  browns.  Thank  you  to  the  Crick  Running   Club,  who  have  made  Thursday  lunchtimes  fun  and  kept  my  mind  clear  during  my  writing   up.  Finally,  thank  you  to  London  Heathside  AC  who  have  kept  me  moving  and  lowered   my  stress  levels  in  the  final  phases.     6 Table  of  Contents   Abstract  ..............................................................................................................  3   Impact  Statement  ..............................................................................................  4   Acknowledgements  ...........................................................................................  6   Table  of  Contents  ..............................................................................................  7   Table  of  figures  ................................................................................................  10   List  of  tables  ....................................................................................................  12   Abbreviations...................................................................................................  13   Chapter  1.   Introduction  .............................................................................  14   1.1   Eph  receptor  and  ephrin  signalling  ...................................................  14   1.1.1   Classes  and  domain  structure  of  Eph  receptors  and  ephrins  ..........  14   1.1.2   Eph-­ephrin  signalling  activation  .......................................................  17   1.1.3   Downstream  signalling  from  Eph  receptors  and  ephrins  .................  19   1.1.4   Eph-­ephrin  internalisation  and  cleavage  .........................................  23   1.1.5   Roles  of  Eph-­ephrin  signalling  .........................................................  23   1.2   Hindbrain  development  .......................................................................  25   1.2.1   Hindbrain  specification  and  segmentation  .......................................  25   1.2.2   Hindbrain  boundary  cells  .................................................................  29   1.2.3   Eph  receptors  and  ephrins  during  zebrafish  hindbrain   development  ...............................................................................................  30   1.3   Transcriptional  regulation  by  Eph  receptor  and  ephrin  signalling  .  32   1.3.1   Direct  versus  indirect  transcriptional  regulation  ...............................  33   1.3.2   Transcriptional  regulation  via  other  signalling  pathways  .................  33   1.3.3   Activation  of  transcription  factors  by  Eph  receptors  and  ephrins  ....  34   1.3.4   Cell  fate  specification  in  Ciona  ........................................................  34   1.3.5   Survival,  proliferation  and  differentiation  of  neural  progenitors  .......  34   1.3.6   Keratinocyte  development  ...............................................................  35   1.3.7   Bone  remodelling  ............................................................................  36   1.4   Aims  ......................................................................................................  36   Chapter  2.   Materials  and  Methods  ............................................................  38   2.1   Solutions  and  Reagents  ......................................................................  38   2.1.1   General  Solutions  ............................................................................  38   2.1.2   Fish  Husbandry  ...............................................................................  38   2.1.3   Zebrafish  strains  ..............................................................................  39   2.1.4   Genotyping  ......................................................................................  39   2.1.5   Microinjection  with  morpholino  oligonucleotides  .............................  43   2.1.6   Heat  shock  ......................................................................................  43   2.2   Analysis  of  gene  expression  ..............................................................  43   2.2.1   Whole  embryo  and  hindbrain  tissue  dissections  .............................  43   2.2.2   RNA  extraction  ................................................................................  44   2.2.3   cDNA  synthesis  ...............................................................................  44   2.2.4   Reverse  transcriptase  –  quantitative  polymerase  chain  reaction  (RT-­ qPCR)  ........................................................................................................  44   2.2.5   RT-­qPCR  primer  sequences  ...........................................................  45   2.2.6   Statistical  significance  .....................................................................  46   2.3   In  situ  hybridisation  .............................................................................  47   2.3.1   Cloning  new  in  situ  hybridisation  probes  .........................................  47   7 2.3.2   Synthesising  in  situ  hybridisation  probes  ........................................  49   2.3.3   In  situ  hybridisation  solutions  ..........................................................  50   2.3.4   In  situ  hybridisation  protocol  ............................................................  51   2.4   Whole  mount  immunofluorescence  ...................................................  52   2.4.1   Whole  mount  immunofluorescence  protocol  ...................................  52   2.5   RNA-­sequencing  ..................................................................................  53   2.5.1   Library  preparation  ..........................................................................  53   2.5.2   RNA-­sequencing  .............................................................................  53   2.5.3   Bioinformatic  analysis  of  RNA-­sequencing  data  .............................  54   Chapter  3.   Blocking  Eph-­ephrin  signalling  in  the  zebrafish  hindbrain  .  55   3.1   Introduction  ..........................................................................................  55   3.2   Morpholino  oligonucleotides  ..............................................................  56   3.2.1   Individual  morpholino  oligonucleotide  knockdown  ..........................  56   3.2.2   Combined  morpholino  oligonucleotide  knockdown  .........................  58   3.2.3   Hindbrain  morphology  phenotypes  ..................................................  59   3.3   Mutant  zebrafish  lines  .........................................................................  61   3.3.1   Individual  Eph  receptor  and  ephrin  mutants  ....................................  62   3.3.2   Double  Eph  receptor  and  ephrin  mutants  ........................................  63   3.4   Soluble  ephrin  ligands  ........................................................................  66   3.4.1   Optimisation  of  heat  shock  protocol  ................................................  67   3.4.2   Confirmation  of  soluble  ephrinB1a  over-­expression  ........................  69   3.5   Discussion  ............................................................................................  73   3.6   Conclusion  ...........................................................................................  75   Chapter  4.   Preparation  of  zebrafish  hindbrains  for  RNA-­sequencing  ..  77   4.1   Introduction  ..........................................................................................  77   4.2   Optimisation  of  sample  .......................................................................  78   4.3   Sample  selection  for  RNA-­sequencing  ..............................................  81   4.3.1   Identifying  samples  over-­expressing  soluble  ephrinB1  ...................  82   4.3.2   Identification  of  uncontaminated  samples  .......................................  82   4.4   RNA-­sequencing  ..................................................................................  84   4.4.1   Data  quality  and  alignment  ..............................................................  84   4.4.2   Differentially  expressed  genes  ........................................................  85   4.5   Discussion  ............................................................................................  95   4.6   Conclusion  ...........................................................................................  99   Chapter  5.   Validation  of  genes  regulated  downstream  of  Eph-­ephrin   signalling   100   5.1   Introduction  ........................................................................................  100   5.2   Validating  changes  in  gene  expression  by  RT-­qPCR  ....................  100   5.2.1   Validation  in  embryos  where  Eph-­ephrin  signalling  is  blocked  ......  102   5.2.2   Validation  in  Eph  receptor  and  ephrin  mutants  .............................  112   5.3   Validating  selected  genes  by  in  situ  hybridisation  ........................  114   5.3.1   Expression  of  candidate  genes  in  wild  type  embryos  ....................  114   5.3.2   Expression  of  candidate  genes  in  embryos  where  Eph-­ephrin   signalling  is  disrupted  ...............................................................................  115   5.4   Expression  of  target  genes  in  the  tail  ..............................................  124   5.5   Discussion  ..........................................................................................  125   5.6   Conclusion  .........................................................................................  127   8 Chapter  6.   Investigating  the  role  of  Eph-­ephrin  signalling  during   neurogenesis  in  the  hindbrain  .....................................................................  129   6.1   Introduction  ........................................................................................  129   6.2   Wild  type  expression  of  neural  markers  ..........................................  129   6.3   Expression  of  neuronal  markers  when  Eph-­ephrin  signalling  is   blocked  .......................................................................................................  133   6.3.1   The  effect  on  hindbrain  boundary  cells  when  disrupting  Eph-­ephrin   signalling  at  18  hpf  ...................................................................................  134   6.3.2   nestin  expression  when  Eph-­ephrin  signalling  is  blocked  .............  135   6.3.3   deltaD  expression  when  Eph-­ephrin  signalling  is  blocked  ............  136   6.3.4   neurog1  expression  when  Eph-­ephrin  signalling  is  blocked  ..........  137   6.3.5   neuroD4  expression  when  Eph-­ephrin  signalling  is  blocked  .........  138   6.3.6   huC/D  expression  when  Eph-­ephrin  signalling  is  blocked  .............  139   6.4   Expression  of  neuronal  markers  in  Eph  receptor  and  ephrin   mutants  .......................................................................................................  140   6.4.1   HuC/D  expression  in  Eph  receptor  and  ephrin  mutants  ................  144   6.5   Discussion  ..........................................................................................  146   6.6   Conclusion  .........................................................................................  147   Chapter  7.   Discussion  .............................................................................  149   7.1   Identification  of  genes  regulated  downstream  of  Eph-­ephrin   signalling  in  the  hindbrain  ........................................................................  149   7.2   Direct  versus  indirect  consequences  of  blocking  Eph-­ephrin   signalling  ....................................................................................................  149   7.3   Is  soluble  ephrinB1a  blocking  or  activating  signalling?  ...............  151   7.4   Future  work  to  identify  direct  targets  of  Eph-­ephrin  signalling  in  the   hindbrain  .....................................................................................................  154   Reference  List  ................................................................................................  156   9 Table  of  figures   Figure  1  Eph  receptor  and  ephrin  protein  domains  ................................................  16   Figure  2  Cis  and  trans  interaction  of  Eph  receptors  and  ephrins  ...........................  18   Figure  3  Downstream  pathways  of  EphB-­ephrinB  signalling  .................................  22   Figure  4  Hindbrain  patterning  and  morphology  ......................................................  29   Figure  5  Eph  receptor  and  ephrin  expression  in  the  zebrafish  hindbrain  ...............  32   Figure  6  rfng  expression  after  MO  mediated  knockdown  .......................................  58   Figure  7  Morphological  differences  between  control  and  knockdown  embryos  .....  60   Figure  8  Hindbrain  boundary  phenotypes  of  individual  Eph  receptor  and  ephrin   mutants  ...................................................................................................................  63   Figure  9  Generation  of  double  homozygous  mutant  embryos  ...............................  64   Figure   10   Hindbrain   boundary   phenotypes   of   double   Eph   receptor   and   ephrin   mutants  ...................................................................................................................  65   Figure  11  Heat  shock  protocol  for  over-­expressing  soluble  ephrinB1a  ..................  68   Figure  12  Hindbrain  boundary  phenotypes  after  soluble  ephrinB1a  expression  ....  69   Figure  13  Confirming  soluble  ephrinB1a  expression  by  in  situ  hybridisation  .........  71   Figure  14  Confirming  soluble  ephrinB1a  expression  by  immunofluorescence  .......  72   Figure  15  The  effect  of  ephrinB1a  expression  on  EphB4a  .....................................  73   Figure   16   Comparison   of   gene   expression   in   whole   embryos   and   hindbrain   dissections  ..............................................................................................................  79   Figure  17  Expression  profiles  of  genes  representing  contamination  in  wild  type   hindbrains  ...............................................................................................................  81   Figure  18  Expression  of  soluble  ephrinB1a  in  hindbrains  used  for  RNA-­sequencing ................................................................................................................................  82   Figure  19  Gene  expression  profiles  of  hindbrains  used  for  RNA-­sequencing  ........  83   Figure  20  Volcano  plot  of  differentially  expressed  genes  .......................................  86   Figure  21  Significantly  differentially  expressed  genes  between  control  hindbrains  and   hindbrains  expressing  soluble  ephrinB1a  ...............................................................  88   Figure  22  Heatmap  of  the  50  most  statistically  significant  differentially  expressed   genes  ......................................................................................................................  90   Figure  23  Correlation  of  gene  expression  with  ephrinB1a  across  RNA-­sequencing   samples  ..................................................................................................................  95   Figure  24  Expression  of  soluble  ephrinB1a  in  individual  whole  embryos  .............  102   10