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					• a change in distribution of expression in segment polarity
mutants suggested a co-ordinate mechanism of control...
• a close correlation between Wnt and En expression domains
was also observed in vertebrate brain development….
• What was mediating the cell-cell signaling??
• a change in distribution of expression in segment polarity
mutants suggested a co-ordinate mechanism of control...
• a close correlation between Wnt and En expression domains
was also observed in vertebrate brain development….
• What was mediating the cell-cell signaling??




    Hh cloned in 1992: shown to be confined to
    engrailed expressing cells
• a change in distribution of expression in segment polarity
mutants suggested a co-ordinate mechanism of control...
• a close correlation between Wnt and En expression domains
was also observed in vertebrate brain development….
• What was mediating the cell-cell signaling??




    Hh cloned in 1992: shown to be confined to
    engrailed expressing cells
  Brief summary of cell signaling
  pathway discussed in article:

en gene activated in cells
having high levels of eve, ftz
or prd…repressed by high
levels of opa, odd, slp
Pattern is
maintained by
intercellular
signaling...
 Cloning and characterization
            of Hh
• Cloning led to probes to follow its
  expression
• expression observed in many
  developing structures
  – wing, leg, eye discs
  – germ cell migration, gonad
  – abdomen and tracheal system
 Cloning and characterization
            of Hh
• Genes isolated from numerous
  organisms (C. elegans the exception)
• Several related genes in vertebrate
  species (Indian, Sonic, Desert; teleosts
  have undergone additional duplication
  for Shh and Ihh classes)
• Shh shown to be expressed in several
  key signaling centers in vertebrate
  development: notochord, floor plate,
  ZPA
 Cloning and characterization
            of Hh
• Like in flies, ectopic expression studies
  in vertebrates suggested that Shh is
  important in sending a morphogenic
  signal
• Signaling can be short or long range,
  direct or indirect, and concentration
  dependent…
 Cloning and characterization
            of Hh
• Article primarily focuses on three of the
  many roles Hh can regulate…
  – short range signaling in Drosophila
    (segmentation)
  – long range signaling in Drosophila (wing
    disc formation)
  – long range patterning of neural tube in
    vertebrates
                    Short Range Hh Signaling
• The first two target genes identified for Hh signaling were wg and ptc
• The spatial regulation of these genes in response to Hh activity suggested
that Hh effects were restricted to nearby cells
• signal for ptc seems to be distributed symmetrically… not a monotonic
gradient?
  Short range Hh
    signaling
• a continuous
denticle belt is
observed if
anterior-posterior
information is
lost…
• since Wg also
encodes a known
morphogen, wg
expression could
also control
signaling...
   Short Range Hh Signaling
• Other target genes of Wg and Hh have
  been identified and are believed to
  contribute to the unique denticle pattern
  for each row of cells
• Serrate (encodes Notch ligand)
  – repressed by Wg and Hh in adjacent cells
  – narrow stripes of Rhomboid (EGFR ligand
    processing) expression are established by
    Ser/Hh induction…
                               Ser expressed
Wg expressed

                     Hh          Ser repressed




                                          Denticle formation
               Rho expressed              repressed
   Long Range Hh Signaling
• Hh exerts long range effect through
  regulating Dpp expression
• Hh restricted to posterior compartment
  of wing disc
• dpp confined to narrow strip of cells at
  compartment boundary
• gradient of dpp activity spreads in both
  directions
   Long Range Hh Signaling
• Hh attenuates activity by
  downregulating Dpp receptor and also
  participates in signaling
• target genes dpp, col (transcription
  factor), en and ptc are induced in the
  wing by different levels of Hh activity
                                  Ci import into
                                  nucleus
      Hh


      dpp
Ptc, col expression


                                         Dpp
                                         gradient




     Long Range Hh Signaling in the Wing Disc
   Long Range Hh Signaling
• In vertebrates, development of the
  neural tube is also controled by Hh
  signaling from the notochord and the
  ventral floor plate
• Shh forms a gradient and specifies
  various ventral cell identities
• ectopic, cell autonomous expression
  changes pattern predictably
• extends into regions of the brain...
     Long Range Hh Signaling in the Vertebrate
                  Neural Tube
                                     Expression of
                    Different        different
                    levels of Ci     homeodomain
                    homolog          proteins in
                    activators       response to
                    and              Shh gradient
                    repressors



Motoneurons
specified through
signaling

     Shh
Hh biosynthesis and structure
• Undergoes proteolytic cleavage
  catalyzed by C terminal of protein
• N terminal fragment is signal
• autocleavage linked to covalent
  coupling of cholesterol to C terminus of
  N fragment: Hh-Np
• results in membrane retention
• further lipid modification (palmitoylation)
  of most N-terminal cysteine
Hh biosynthesis and structure
• palmitoylated form required?
  unpalmitoylated only active an high
  concentration?
• Accumulates in membrane
  microdomains (membrane rafts)
  important in intracellular sorting
    Summary of Hh function
• Involved in many activities- cell fate
  specification, cell proliferation/survival
• Signaling can be short (embryonic
  ectoderm) or long-range (wing
  disc/vertebrate nervous system/limb)
  and concentration dependent
• Hh is processed; N terminal region
  active and modified via lipid attachment
• Presumably this modification is related
  to mediating long/short term signaling
            Hh reception
• Still not completely understood
• Ptc is the Hh receptor (sterol-sensing
  domain protein, SSD; implicated in
  membrane trafficking)
• single gene in flies, two receptors in
  vertebrates
• Hh and Ptc colocalize to intracellular
  vesicles (suggests internalization-
  lysosome targeting)
            Hh reception
• Smo (smoothened) is essential for Hh
  signaling
• Smo is a G-protein coupled receptor
  (but has no known activity and not
  inhibited by classic inhibitors)
• Smo also appears to not interact with
  signaling molecule
• Patched inhibits Smo function:
  interaction with Hh eliminates inhibition
Conventional Model:




But: There is no strong evidence for colocalization at the
cell surface...
and Smo overexpression not sufficient to activate
expression
            Hh reception
• The implication of several proteins
  involved in intracellular trafficking has
  put forward another model
• In the absence of Hh signaling Ptc
  receptor will mediate sorting in the Golgi
  to intracellular vesicles leading to
  degradation or inactivation of Smo
  receptor: does not reach membrane
There is also some evidence
Hh can signal by a Ptc                  Hh
independent                             binding
mechanism…another Hh                    removes
binding protein?                        Ptc




                         Modified or
                         specifically
                         targeted?
Smo
inactivated
or
degraded
Hh Secretion and Distribution
• Cholesterol moiety helps explain short
  range signaling…held to membrane
• Hh-Nu, the unmodified form, can diffuse
  farther from site of synthesis
• A ptc-related protein, Dispatched (Disp)
  is required for the controlled release of
  Hh-Np; interestingly, this mutant has
  effects during embryonic development,
  implying a role in short term signaling
  as well as long term...
Hh Secretion and Distribution
• Mechanism of Disp function unclear
• lipid moiety required for controlled release
• The lipid modification also appears
  important for mobility once released from
  source
• Hh-Np is subject to some restraining
  influence once it is released from cell
  membrane
 Hh Secretion and Distribution
• The modified form moves in a more
  restrictive manner, mediated by
  interactions with Ptc in neighboring cells:
  Ptc sequesters Hh-Np
• Since Ptc is a target gene of Hh, this also
  creates a negative feedback mechanism
• the structural features responsible for Hh-
  Np:Ptc interactions are unclear
    Hh Secretion and Distribution
• In vertebrates, the Shh-Nu protein, behaves
  completely differently than the situation in
  Drosophila!
• For Shh-Nu, loss of modification limits range
• Reason for this difference? Model….
  – Shh is subject to an additional restraining influence:
    an Hh-binding protein Hip1, with no fly counterpart;
    it also, like Ptc, may prevent diffusion
  – If Shh-Nu bound to Hip with greater affinity than its
    lipid counterpart, its range might be limited
   Hh Secretion and Distribution
• Transfer of Hh-Np is mediated in Drosophila
  by tout velu (ttv (all-hair); glycosaminoglycan
  transferase)
• movement may be controlled by the
  production of a proteoglycan that mediates
  cell-cell transfer
• ttv is required in Hh receiving cells- required
  for Ptc presentation?
• What if proteoglycan and Hip compete for Hn
  -N binding, and cholesterol is needed for
  former interaction?
 Differential
 target gene
 activation

Summary of disp
and ttv effects
when mutant in
Drosophila
    Nuclear Signal Transduction
• Major target of Smo activity is cubitus
  interruptus (ci)
• the Ci TF is both a repressor and an activator
• genetic analysis of Ci expression in mosiacs
  led to suggestion of dual function
  – some Hh targets lose expression in Ci mutants
    (e.g. ptc)
  – some Hh targets seemed activated in absence of
    Ci (e.g. dpp): although at a lower level than normal
    domain close to compartment border
   Nuclear Signal Transduction
• The repressor form of Ci is a proteolytic
  cleavage product
• Hh works by inhibiting proteolytic cleavage
• PKA and Costal-2 implicated in cleavage
  mechanism (mutants accumulate Ci
  independent of Hh activity)
• Cos-2 binds microtubules in a Hh dependent
  manner, suggesting sequestration of
  cleavage
    Nuclear Signal Transduction
• Rate limiting step seems to be recruitment to
  microtubules; removal of Ci from microtubule
  network could inhibit cleavage
• slmb gene priming needed for proteasome
  mediated cleavage
• does phosphorylation stimulate cleavage?
  (PKA sites needed for cleavage, not
  sequestration) Does it also attenuate Ci
  activity via import?
                                                         high Hh
                          Moderate                       signal
     Serine-              Hh signal                 Phosphoryl-
     threonine                                      ates ?
     kinase




                                                    Release and
                                                    nuclear
                                                    activty?

               Dpp, Hh

Truncated Ci
                                                                   CBP/p300:
                         SuFu binding: no cleavage but
                                                                   coactivator with
                         import inhibited and
                                                                   HAT activity
                         transcription attenuated
    Vertebrate Ci homologs
• 3 Ci-related proteins
• to analyze function, they were
  expressed in Drosophila imaginal discs
  and compared to known activities of Ci
  – Gli1 and Gli2 Hh dependent activators
  – Gli2 is Hh dependent repressor
  – cleavage of Gli2 and Gli3 generate
    repressor forms of each protein
     Vertebrate Ci homologs
• Role in vertebrates?
  – Gli1: an activator of Hh target genes, does
    not undergo proteolytic cleavage and does
    not require CBP for coactivation; GL1 is not
    a required gene
  – Gl2: full length protein can stimulate in
    response to Hh signal. Loss of amino
    terminal leads to activation that is Hh
    independent (Su(fu) binding domain)
    mutants suggest role in neural tube.
     Vertebrate Ci homologs
• Role in vertebrates?
  – Gl2: repressing activity is uncertain. Will
    undergo cleavage in tissue culture, and an
    artificial C-terminal truncated form will
    repress reporter gene expression
  – Gl3: acts as a repressor of Hh target genes
    and Shh. Cleavage similar to Drosophila
    Ci, lacking amino terminal. Evidence for
    activation function in culture, which is Hh
    dependent and potentiated by CBP
     Vertebrate Ci homologs
• General Conclusions?
• Individual characteristics of three
  proteins show general characteristics of
  Ci
• do they perform subsets of Ci function?
     Vertebrate Ci homologs
• General Conclusions?
• Individual characteristics of three
  proteins show general characteristics of
  Ci
• do they perform subsets of Ci function?
Vertebrate limb
development
• apical ectodermal ridge:
programs series of reactions
with underlying mesenchyme
• governs proximal-distal
outgrowth/proliferation (FGFs)
• differentiation
Zone of Polarizing Activity: ZPA
• small region of mesodermal tissue at posterior margin
• appears to send a signal that provides information for
limb development
Shh
localizes
to the
ZPA (in
situ
hybridizat
ion)
Effect of
ZPA
transplant
can be
mimicked
by Shh
secreting
cells
Feedback between cells
mediate these inductive
interactions
1) Shh induced by FGF-8
2) Shh activates FGF-4 in
posterior AER
3) FGF-4 recruits
mesenchyme cells into
progress zone and
maintaining Shh
4) Wnt 7a needed to
maintain D/V axis
(expressed dorsally)

				
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posted:6/4/2013
language:English
pages:47
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