Gastrulation and Neurolation in

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					Gastrulation and Neurulation
     in Chick Embryos
      - The role of Churchill -

           Susitna Banerjee and Michelle Tiraborelli
A Summary of:
  Churchill, a Zinc Finger
  Transcriptional Activator,
  Regulates the Transition
  between Gastrulation and
  By: Guojun Sheng, Mario dos Reis , and
  Claudio D.
  In: Cell, 2003, Vol. 115, pp. 603-13.
 • Background Terms
 • Fibroblast growth factor (FGF)
 • Purpose
 • Churchill characteristics
Background Terms
• Gastrulation
  – embryonic epiblast -> germ layers
    (Mesoderm, Endoderm, Ectoderm)
  – cell movements
  – epiblast ingression -> mesoderm &
  – ectoderm subdivision -> future epidermal
    and neural areas
Fibroblast growth factor (FGF)
 neural plate
caudalization                 • all processes
                                at same time
                mesoderm        and same area
                & formation   • Q: How do
                                receiving cells
                                decide on right
+ other molecules
 neural induction
FGF and Neural Induction
 • FGF8 + other signals (2hrs) -> Sox 3
   and ERNI preneural genes
 • FGF8/Henson’s Node Signals (5hrs) ->
   sensitize cells to BMP antagonists ->
   Sox 3
 • Recall:
   – BMP --| neural fate
   – BMP antagonists --| BMP --| neural fate
General Questions:   Prediction:
• What acts as a     - Churchill (ChCh)
  switch between       gene is on after 5
  FGF’s roles of       hour organizer
  gastrulation and
  neural plate         exposure and has
  formation?           a role in
• How does the         separating 2
  switch work?         functions of FGF
Churchill Characteristics
                • encodes a zinc
                   finger protein
                 - DNA binding motif
                 - helix-turn-helix motif
                   of amino acids
                 - bound to a Zn atom
Churchill Characteristics
 • Activates Transcription:
 ChCh --> Zn finger protein --> activates
   genes (eg. Sip1)

 • Represses Mesoderm Induction:
 ChCh --| FGF --> mesoderm markers
   (Brachyury and Tbx6L)
ChCh Properties                 (from study)
 • induced by FGF
 • causes expression of
   protein-1 (Sip1)
 • regulates cell ingression
   during mesoderm
 • affects neural plate
 • sensitize cells to neural-
   inducing signals from
ChCh Predicted Properties
 • switch between gastrulation
   (mesoderm/endoderm formation) and

 • gate to separate 2 functions of FGF
   signalling (mesoderm formation, neural
Experimental Overview
 • Isolation of a gene involved in the
   induction of neural tissue (Churchill)
 • What is the product of this gene?
 • What is the function of Churchill (ChCh)
   and how is it involved in neurulation?
What genes are involved in the
induction of neural tissue?

• Differential screen
• Churchill (ChCh) is one of the genes
  expressed in chick embryos during
  neural plate formation
      - ChCh is a zinc finger protein
      - contains a conserved sequence
        found in many model organisms
What genes are involved in the
induction of neural tissue?
What genes are involved in the
induction of neural tissue?

 • Northern blot
   analysis confirms
   the presence of
   ChCh transcripts in
   the chick embryo
   (stages 4 – 13)
What genes are involved in the
induction of neural tissue?

 • In situ
   confirms ChCh
   expression in the
   prospective neural
   plate and
   throughout its
Is FGF signalling involved in the
induction of ChCh expression?

 • Hensen’s node
   - 4 hours of
   contact required
   for induction of
Is FGF signalling involved in the
induction of ChCh expression?
 • FGF4/FGF8 alone induces ChCh in a
   similar time frame as the node graft

 • ChCh is induced by FGF8 in the
   presence of BMP4  independent of the
   BMP pathway
Where does ChCh localize to in the
• ChCh is found in the nucleus of
  Xenopus cells after the onset of
     - myc-tagged ChCh mRNA
Where does ChCh localize to in the
 • Same results found in chick epiblast
   cells using electroporation of a tagged
   ChCh construct
What is Churchill’s function?

 • ChCh inhibits FGF signalling
   - phenotype of Xenopus embryo
   injected with ChCh is similar to the
   phenotype from injection of dominant-
   negative FGF receptor
   - ChCh represses the induction of
   brachyury (mesoderm marker) by
What is Churchill’s function?
What is Churchill’s function?
 • ChCh is a DNA
   binding protein
   - binds the
   consensus sequence
   - similar to binding
   sequences of
   other Zn finger
   proteins but ChCh
   is specific for this
What is Churchill’s function
                • ChCh is a transcriptional
                  - ChCh fused to
                  - ChCh-VP16 inhibits Bra
                  expression in the
                  presumptive marginal
What genes are regulated by
 • Sip1 gene is a ChCh target
   - Sip1 is a direct transcriptional
   repressor of Bra
   - expression patterns of Sip1 and ChCh
   are very similar
   - human and mouse Sip1 genes contain
   ChCh binding sites in their regulatory
What genes are regulated by
• Electroporation of a fluorescein-labelled
  morpholino oligonucleotide against
  chick ChCh
  - introduced in stage 4 chick embryo
  - Sip1 expression absent by stages 7-9
  - neural plate development was
What genes are regulated by
Does ChCh affect cell movements?

 • Epiblast cells near the primitive streak
   were electroporated with different ChCh
      - ChCh-IRES GFP
      - ChCh VP16 IRES-GFP
      - IRES GFP (control)
      - ChCh MutVP-IRES-GFP (control)
Does ChCh affect cell movements?

 • Cells expressing control constructs
   showed normal movement
 • Cells expressing ChCh/ChCh VP16 did
   not ingress through the primitive
   streak or move out to form mesoderm
Does ChCh affect cell movements?
Does ChCh affect cell movements?

 • At stage 4+ down regulation of ChCh
   results in mesoderm formation
    - Electroporation with labelled MOs
    - control cells developed into neural
    - ChCh-MO cells developed into
      paraxial mesoderm
Does ChCh affect cell movements?
Does ChCh sensitize cells to neural
inducing signals?
 • Nonneural epiblast
   loses competence
   between stages 4 - 4+
 • With electroporated
   ChCh-IRES-GFP, the
   neural marker (Sox2) is
   induced in the stage 5
Summary of Results
 • ChCh expression is induced after 4 hrs
   exposure to the organizer (signalling by FGF)
 • ChCh acts as a transcriptional activator
      - promotes expression of Sip1
 • ChCh function leads to the repression of Bra
   and Tbx6L (mesodermal genes)
      ChCh ---> Sip1 ---l Bra, Tbx6L
 • ChCh is required for the regulation of cellular
   movements during gastrulation
 • ChCh is involved in sensitizing epiblast cells to
   neural induction
 • ChCh involved in all parts of
 - germ layer formation
 - cell movement and ingression
 - subdivision of ectoderm into future
   epidermal and neural areas
ChCh separates FGF Signals
                   • ChCh
                     function in
                     and neural
ChCh and Mesoderm
• ChCh and Sip made at anterior end of
  primitive streak when ingression to
  make mesendoderm stops
• ChCh defect -> cells still come towards
  primitive streak but mesendoderm
  formation is blocked
• no ChCh -> continuous ingression
Other work showed that
 • binds to active Smad
   1/5 (BMP targets)
 • binds to active Smad
 • stops activin-
ChChSip & mesendoderm
1) end ingression movements through
   primitive streak
2) change responses of cells to BMP
3) block mesendoderm induction by
   TGF/activin/nodal + FGF
ChCh Roles in Neural Induction
 Other research:
 • at least 13 hrs of node exposure to
   maintain expression of neural markers
   ERNI, Sox3, Otx2
 • 5 hr node exposure:
   Chordin --| BMP and get stable Sox 3
ChCh Roles in Neural Induction
 Why 5 hr for cells to become sensitive
   to BMP?
 • ChCh induction by node or FGF needs
   4 hrs
 • then Sip 1 is made
 • Sip1 associates with P-Smad1
   - Smad1 needed for BMP-4 action
   - Sip1 may be a sensor for the status
   of BMP signalling
ChCh Roles in Neural Induction
ChCh Roles in Neural Induction
 • Sip1 may play a role in getting stable
   Sox3 transcription?
 • Two possibilities of Sip + P-Smad1:
   - control activation and repression
   actions of Sip1 and regulate Sox3
   - activates proteins that affect Sox3
   mRNA turnover
ChCh and neural cell formation
 • blocks epiblast ingression through
   primitive streak  neural plate cells
   stay in ectoderm
 • regulates Sip1 -> neural cell formation
 • maintain/ epiblast competence to
   induction signals from node/FGF
 • recent study shown that Sox2 (neural
   gene) has several binding sites for Sip1
Germ Layer Decisions


   Neural vs               Neural vs
   epidermal ?             mesodermal?

 Epidermal                Mesodermal
ChCh may act with cofactors
 • ChCh misexpression far from the normal area
   does not induce Sip1 even though Sip1 has
   many ChCh binding sites
 • ectopic ChCh in chick or frog embryos does
   not carry out normal function
 • in Xenopus ChCh localizes to nucleus only just
   before gastrulation
 • ChCh + VP16 activator domain  wt ChCh
 • 1 Zn finger may bind to DNA, the other with
ChCh activation is likely
 • 4 hr lag time
 • 480 bp homologous sequence in
   human, mouse, rat ChCh homologues
   just upstream of coding region
   - has binding site for 12 TF
   (5 present in early embryos)
Possible Future Work
 • determine if several paths converge and
   cause slow induction of ChCh by FGF
 • what cofactors bind ChCh
 • how Sip1 is involved in Sox3
 • what are the unknown node signals that
   affect preneural gene formation (ERNI,
   Sox 3)
 • ChCh is induced by FGF
 • ChCh causes expression of Smad-interacting-
   protein-1 (Sip1)
 • Sip1 stops mesendoderm ingression and
   mesoderm formation
 • Sip1 sensitize cells to neural-inducing signals
   from organizer
 • ChCh separates 2 functions of FGF signals
   (mesendoderm formation & neural