Hedgehog  and  larval  development

Whilst  the  role  of  Hh  signalling  in  midline  development  can  be  explained  in   cysticercoids  and  adult  worms,  the  situation  is  less  clear  during  larval  

development.  Whilst  Hh  signalling  is  likely  to  be  involved  in  the  patterning  of   the  larval  midline,  exactly  what  cell  type  Hmic-­Hh  is  expressed  by  is  unclear.  

Larval  tapeworm  nerves  are  presumed  to  develop  laterally  and  do  not  form   until  later  stages  of  development.  Therefore,  given  the  expression  of  Hmic-­

Hh  along  the  midline  of  H.  microstoma  larvae,  it  is  highly  unlikely  that  Hmic-­

Hh  is  associated  with  nervous  tissue,  or  even  neural  precursors.  

Oncospheres  also  lack  a  brain  and  so  anterior  expression  cannot  be  linked   with  this  structure.  

The  expression  of  other  Hh  factors  in  larvae  prevents  a  meaningful  

interpretation  of  the  potential  role  of  Hh  signalling  during  larval  development.  

The  posteriorised  expression  of  many  of  these  factors  during  mid  

development  and  later  observation  within  the  cyst  tissue  could  indicate  a   potential  role  in  guiding  AP  axis  formation.  However,  Hh  signalling  could  also   be  playing  many  roles  simultaneously.  As  such,  further  analysis  is  still  

required.  

4.3.4  General  conclusions  and  future  directions    

Overall,  the  presence  and  expression  of  Hh  factors  in  H.  microstoma  shows   canonical  Hh  signalling  to  be  present  and  conserved  within  tapeworms.  

Expression  in  adults  suggests  that  the  pathway  is  involved  in  two  processes   simultaneously  –  neurogenesis  and  proglottisation.  Based  on  expression   patterns,  it  is  unlikely  that  Hh  signalling  is  involved  in  strobilation,  as  Hmic-­Hh   turns  on  before  this  process  begins  and  no  polarised  expression  of  Hmic-­Hh   (or  other  pathway  factors)  is  observed  in  individual  segments.  Therefore,   Hmic-­Hh  does  not  act  as  a  segment  polarity  gene  as  it  does  in  D.  

melanogaster  (or  other  arthropods).  The  repeated  expression  of  Hmic-­Hh  in   every  segment  is  more  likely  due  to  the  segmented  nature  of  the  tapeworm   nervous  system.  Hh  signalling  is  active  during  proglottisation  (that  begins   almost  instantly  in  the  neck)  and  is  likely  to  be  guiding  both  proglottisation   and  organogenesis.  Future  work  is  required  to  further  understand  Hh   signalling  in  larvae  and  the  use  of  now-­known  neuronal  markers  in  

planarians  will  aid  in  confirming  that  tapeworm  neurons  express  Hmic-­Hh.  

Ultimately  the  development  of  robust  functional  tools  is  required  to  confirm   the  role  of  Hh  signalling  in  H.  microstoma.  

Chapter  5  

The  Wnt  pathway  in  Hymenolepis  microstoma  

Elements  of  this  chapter  were  published  as  part  of:  ‘Comparative  analysis  of   Wnt  expression  identifies  a  highly  conserved  developmental  transition  in   flatworms’,  BMC  Biology,  2016.  U.  Koziol,  F.  Jarero,  P.D.  Olson  &  K.  Brehm  

5.1  Introduction    

The  Wnt  pathway  (Fig.  5.1)  is  a  highly  conserved  cell-­cell  signalling  system   that  controls  many  developmental  processes,  including  cell-­fate  

determination,  polarity,  patterning,  morphogenesis,  cell  proliferation,  

migration  and  apoptosis  (Cadigan  and  Nusse,  1997;;  Martin  and  Kimelman,   2009;;  Petersen  and  Reddien,  2009).  Wnt  signalling  controls  anteroposterior   (AP)  axis  patterning  in  many  metazoans  (Petersen  and  Reddien,  2009)  and   the  segmentation  of  many  arthropods,  annelids  and  vertebrate  

somitogenesis.  As  such,  the  pathway  is  likely  to  control  AP  patterning  in   tapeworms  and  is  a  strong  candidate  with  which  to  investigate  strobilation.  

Historically,  Wnt  ligands  have  been  categorised  to  transduce  three  discrete   pathways  depending  on  whether  or  not  they  lead  to  the  activation  of  b-­

catenin.  These  are  the  canonical  b-­catenin  dependant  pathway  and  the  non-­

canonical  planar  cell  polarity  (PCP)  and  Wnt/calcium  pathways.  However,   the  classification  of  these  different  pathways  (or  cascades)  may  be  artificial,   with  Wnt  signalling  proving  to  be  a  highly  complex  and  dynamic  system  in   which  cross-­talk  between  the  different  cascades  occurs  (van  Amerongen  and   Nusse,  2009).  These  authors  suggest  that  Wnt  signalling  should  not  be   thought  of  as  a  linear  pathway  and  instead  should  focus  on  context-­specific   interactions  between  Wnts  and  their  receptors.  Despite  this,  the  best  

understood  transduction  cascade  remains  the  canonical  pathway,  which  has   a  conserved  role  involved  in  the  specification  of  the  anteroposterior  (AP)  and   primary  axes.  This  chapter  focusses  on  the  role  of  b-­catenin  dependant  

signalling  in  the  development  of  H.  microstoma.  The  genes  of  the  pathway   have  already  been  characterised  (Riddiford  and  Olson,  2011)  and  this  

chapter  will  centre  on  the  expression  of  some  of  these  factors  through  in  situ   hybridisation  experiments  during  larval  and  adult  development.  

5.1.1  Wnt  discovery    

The  first  mammalian  Wnt  gene  (Int1)  was  discovered  in  the  early  1980’s   during  screens  identifying  tumour  growth  in  mice  (Nusse  and  Varmus,  1982).  

The  impact  of  Int1’s  discovery  was  overshadowed  by  a  rapid  influx  of  many   other  developments  in  cancer  biology  at  the  time  (Nusse  and  Varmus,  2012).  

Despite  this,  the  sequence  and  structure  of  Int1  was  characterized  (van   Ooyen  and  Nusse,  1984),  as  was  its  cDNA  sequence  (Fung  et  al.,  1985)   which  showed  no  homology  to  any  other  gene  at  the  time  (Nusse  and   Varmus,  2012).  Around  the  same  time,  screening  in  Drosophila  

melanogaster  identified  several  ‘segment  polarity  genes’  (so-­called  because   mutant  flies  presented  abnormal  segmental  patterning),  one  of  which  was  the   gene  Wingless  (Wg).  Wg  mutants  were  found  to  lack  segment  boundaries   (Nusslein-­Volhard  and  Wieschaus,  1980).  Previously  identified  in  earlier   developmental  studies,  Wg  mutations  resulted  in  wingless  or  haltere-­less   flies  and  other  developmental  deformities  in  the  mesothorax  (Sharma  and   Chopra,  1976).  Further  investigations  uncovered  Int1  and  Wg  to  in  fact  be   orthologous  genes  (Rijsewijk  et  al.,  1987).  As  interest  in  the  gene  took  hold,   other  groups  began  to  indicate  a  role  in  embryonic  axis  formation  (McMahon   and  Moon,  1989).  Early  knockouts  of  Int1  in  mice  caused  anteriorised