Extracellular signaling: Hedgehog

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Extracellular signaling Hedgehog

• From the fly cuticle to cancer-killing creams

Jeremy Reiter
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Todays Questions

• Is Hh a morphogen?
• Are signaling molecules conserved through
  evolution?
• What is DB?

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Turing (1952) The chemical basis of
morphogenesis. Phil Trans. Roy. Soc. 237,
37-72 Morphogen a form generating substance
expressed by some cells that moves through
surrounding tissue providing other cells with
information about their relative position.
Lewis Wolpert coined the term positional
information and defined the French flag
problem - by some mechanism, morphogens elicit
concentration-dependent responses from cells
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bicoid the first morphogen
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Patterning a multicellular system the fly embryo

• Cuticle can be smooth (arrowhead)
• Or with denticles (arrow)

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hedgehog affects cuticle pattern

• Nusslein-Volhard and Wieschaus - 1980

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How is pattern created?
Hints from similar phenotypes
wild type hh wg en
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en and wg are expressed in different cells
wg en

• en posteior to parasemental groove
• wg antereior to parasemental groove

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but regulate each other!
wt
en-/-
wt
wg-/-
wg en

• en a transcription factor
• wg a secreted protein
• How can a transcription factor regulate the
  expression of a nearby gene?

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The segment organizer
How does the en-expressing cell maintain Wg
expression next door?
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Hh is a secreted protein
Identification of Hh as a signaling protein
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Putting it all together How cuticle becomes
smooth or with denticles
Is Hh a morphogen? Theme signals can act through
other signals
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Hh - not just in the ectoderm

• Cuticle Wing imaginal disc

A P A
P
Hh En Dpp En
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Wing pattern reflects disc pattern

• Lineage analysis reveals that disc domains form
  distinct wing domains

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En-expressing cells make Hh here, too
En Hh

• Posterior domain of wing disc

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Different Hh targets expressed in different
domains
Hh Ptc Dpp

• A threshold effect?

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A model for Hh as a morphogen
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but its never that simple.
Hh Ptc Dpp

• Dpp is a dependent morphogen

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Dpp is responsible for some Hh effects

• Ectopic Hh Ectopic Dpp

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How do morphogens have different transcriptional
readouts at different concentrations?
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Different targets have different affinities for
effectors
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Is Hh really a morphogen?

• Are there alternative ways of having different
  effects at a distance?

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Can signals be sensed locally by distant cells?

• Cytonemes bring the cell to the signal

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Shocking news! Other animals have Hhs

• Conserved through cordate evolution

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Hedgehogs are in some interesting places
Neural tube Limb bud
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Vertebrate neural development 101

• Source of brain, spinal cord, peripheral nervous
  system
• Comprised of many cell types

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Patterned across dorsal-ventral axis

• How does this occur?

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Notochord is a source of patterning
notochord

• Van Straaten et al., 1987, 1988

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Could Shh be the notochord signal?
Shh Shh Ptc

• Secreted from notochord and floor plate
• Distributed in a gradient
• Induces a gradient of gene expression

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Shh is necessary
Dbx2

• wt Shh-/- wt Shh-/-

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and sufficient
FoxA2 Islet1/2

• Shh 7nM 2nM 0.7nM
• Added to neural tube explants for 24 hours

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Model of Shh patterning

• But is necessity and sufficiency proof that Shh
  is acting as a morphogen?

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The Hh pathway genetically
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What are the essential elements of any signaling
pathway?

• Signal production
• The signal itself
• Secreted or membrane-bound
• Receptor
• Transmembrane (except for lipid soluble ligands)
• Transducers/effectors
• Targets
• Genes or cellular components

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The Hh pathway genetically
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Dispatched releases Hh from producing cells
Shh
wild type Disp-/-
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Hh reception and transduction
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Truly strange biochemical processing
Intein like cleavage by C-terminal domain Only
known cholesterol-modified protein All signaling
functions thought to be done by N-terminal domain
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Truly strange cell biology
Smoothened must go to the vertebrate primary
cilium for pathway activation
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Approach alter Shh distribution without changing
amount

• Avoid problem of secondary signals

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Chimera a single organism of two distinct
genotypes
Ptc?loop2 Nkx2-2

• Loss of Nkx2-2 in the presence of Ptc?loop2
• Ectopic Nkx2-2 dorsal to Ptc?loop2

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Extending Hh distribution shifts pattern dorsally

• By only altering Hh reception, pattern shift is
  not due to increased Shh or secondary signal
• Strong indication that Shh is a morphogen

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The jump to human disease Mutations in SHH

• SHH-/
• Holoprosencephaly
• Midline defects

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Misactivation causes skin cancer

• Basal cell carcinoma Nevoid basal cell
  carcinoma
• syndrome
• Spontaneous activation Germline
  mutation

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and medulloblastoma

• And pancreatic adenocarcinoma, rhabdomyosarcoma,
  small-cell lung cancer, fibrosarcoma, etc.

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Chemotherapy of tomorrow?

• Genentech and Curis clinical trial with Hh
  antagonist skin cream

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Shh also expressed in the limb
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Vertebrate limb development
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Grafts suggest posterior limb bud is an organizer

• Saunders and Gasseling,
• 1968

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Two organizing centers in the limb

• AER and ZPA

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Shh can substitute for ZPA
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Much like Hhs role in fly wing

• Ectopic Hh

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No or reduced Shh result in patterning defects

• wild type reduced Shh Shh-/-

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A Wolpert gradient model for Shh function in the
limb

• Done deal, right?
• Shh is a morphogen in the limb

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Todays Cliff Notes

• Is Hh a morphogen?
• Sometimes no, sometimes yes
• Are signaling molecules conserved through
  evolution?
• Definitely
• What is DB?
• Encompasses interesting aspects of
  biochemistry, cell biology, evolution, how
  molecules behave in 3D tissues