Segment Polarity Genes

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Segment Polarity Genes
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Segment Polarity Genes

• Represents the final phase in the process laying
  down the segmentation plan.
• Take control as cellularisation of the embryo is
  completed and gastrulation commences.
• Encode a diverse array of proteins (compared to
  gap and pair-rule genes).
• Transcription factors engrailed, gooseberry.
• Protein kinases fused, shaggy.
• Secreted signalling proteins wingless,
  hedgehog.
• Membrane receptors frizzled.
• Notes (1) Cellularisation is complete and
  further patterning depends on intercellular
  signalling.
• (2) Expression of co-ordinate, gap and
  pair-rule genes fades away thus new mechanisms
  for regulating wingless and engrailed are required

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Expression of Segment Polarity Genes

• Expressed in a variety of patterns in the newly
  cellularised embryo.
• Expressed throughout the embryo fused, shaggy,
  dominant, armadillo.
• Expressed in segmentally reiterated stripes
  wingless, engrailed, hedgehog, patched,
  gooseberry.
• Will outline hedgehog, wingless and engrailed
  circuit.
• Pair-rule genes (already expressed in a periodic
  pattern) establish the segment polarity gene
  expression in every parasegment.
• e.g. en is activated by Ftz or Eve in each
  parasegment, wingless is repressed by Ftz or Eve
  in each parasegment.

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Defining the Anterior and Posterior Borders of
Parasegemnts

• The anterior border of each parasegment is marked
  by a stripe of cells expressing engrailed (en).
• Wingless (wg) sets the posterior border of each
  parasegment.
• The interface between the expression domains of
  en and wg sets the border between parasegments.
• Initial expression of en and wg is controlled by
  pair-rule gene products but both continue to be
  expressed long after pair-rule stripes have
  faded.
• The wg-en repeating pattern is dependent on
  interactions between neighbouring wingless- and
  engrailed-expressing cells.
• e.g. in embryos lacking wg, en expression
  disappears prematurely and vice versa. Therefore
  cell communication and positive feedback
  mechanisms must maintain en and wg expression in
  adjacent cells.

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How is Communication Between En and Wg Achieved?

• Engrailed encodes a homeodomain protein (a
  transcription factor), wingless encodes a
  secreted peptide, a member of the WNT family.
• Wingless is secreted from the cells that make it,
  binds to its receptor on posterior cells and
  tranduces signals to the nucleus to maintain
  transcription of engrailed.
• A second signal must be invoked to explain the
  maintenance of wingless expression by engrailed
  expression.
• Another segment polarity gene, hedgehog (hh), is
  expressed in the same cells as engrailed.
  Mutations in hedgehog cause loss of wingless and
  engrailed expression.
• Findings imply that hedgehog is part of the
  genetic circuit linking wingless and engrailed
  expression.

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How Does Hedgehog Function?

• The hedgehog gene encodes a novel membrane-linked
  inductive ligand important in local patterning of
  many tissues.
• The primary translation product contains a signal
  peptide that is cleaved to produce a 45 kDa
  polypeptide precursor.
• Cleavage of this secreted precursor produces a 20
  kDa N-terminal fragment associated with the
  plasma membrane and with inductive activity plus
  a 25 kDa fragment.
• The N-terminal fragment becomes tethered to the
  membrane via a hydrophobic cholesterol moiety (it
  doesnt contain any hydrophobic residues).
• A series of elegant experiments show that this
  arrangement ensures that hedgehog only acts on
  the adjacent cell and doesnt diffuse to act on
  cells further away (i.e. it is spatially
  restricted).

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The engrailed, hedgehog, wingless Circuit
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Figure 23-12. Processing of Hedgehog (Hh)
protein. Removal of the N-terminal signal peptide
from the initial translation product yields the
45-kDa Hh precursor consisting of residues 83471
in the original protein. Nucleophilic attack by
the thiol side chain of cysteine 258 (Cys-258) on
the carbonyl carbon of glycine 257 (Gly-257)
forms a thioester intermediate. The C-terminal
domain then catalyzes formation of an ester bond
between the ß-3 hydroxyl of cholesterol and
glycine 257, cleaving the precursor into two
fragments. The covalently attached cholesterol
moiety tethers the N-terminal signaling fragment
to the membrane. Adapted from J. A. Porter et
al., 1996, Science 274255.
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Components of the Signalling Pathway

• Genetic studies identified a gene, smoothened
  (smo) that acts in the Hh signalling pathway and
  is a transmembrane protein.
• Mutants of smo suggested that Hh bound to it
  (i.e. similar phenotype to hh-), but biochemical
  studies did not support this.
• Cells over-expressing another membrane protein,
  Patched (Ptc), were found to bind Hh.
• These findings and genetic data suggest that Ptc
  inhibits Smo function and this is blocked by Hh
  binding to Ptc.
• The steps following signalling by Hh from the
  membrane through the cytoplasm to transcription
  factors is still not fully elucidated, but
  genetic studies suggest a complex set of
  proteins.

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Components of the Signalling Pathway

• Fused (Fu) a serine-threonine kinase
• Costal-2 (Cos-2) a microtubule-associated
  protein
• Cubitus interruptus (Ci) a transcription factor
• In the absence of Hh, Ptc inhibits Smo and the
  three protins (Fu, Cos-2 and Ci form a complex
  and bind to microtubules in the cytoplasm
  resulting in protease cleavage of Ci. The Ci
  fragment translocates to the nucleus and inhibits
  target gene expression.
• In the presence of Hh,Smo is not inhibited, the
  complex doesnt bind microtubules and an
  alternatively cleaved Ci translocates to the
  nucleus where it binds to a transcriptional
  co-activator, CBP.
• One of the target genes up-regulated is ptc
  therefore this gene is not only a component of
  the pathway, but its expression is controlled by
  Hh.

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Figure 23-13. A model of the Hedgehog (Hh)
signaling pathway. (a) In the absence of Hh, the
Ptc protein inhibits Smo at the cell surface. A
complex containing the Fu, Cos-2, and Ci proteins
binds to microtubules. An as-yet unidentified
protease cleaves Ci generating Ci75, a
transcriptional repressor. (b) In the presence of
Hh, Ptc inhibition of Smo is relieved. The Fu,
Cos-2, and Ci proteins are not associated with
microtubules. Ci is cleaved by another protease,
yielding a fragment that acts in conjunction with
CBP to promote transcription. In the presence of
Hh, transcription of ptc is markedly
up-regulated. Adapted from L. V. Goodrich and M.
P. Scott, 1998, Neuron 211243
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The Wingless Signalling Pathway

• Wingless binds to a surface receptor called
  Frizzled (7 membrane-spanning domains).
• Binding of Wg to Frizzled activates an internal
  protein, Dishevelled.
• Dishevelled inhibits glutamine synthase kinase 3,
  GSK3, which in unstimulated cells phosphorylates
  ß-catenin, a transcription factor.
• When ß-catenin is phosphorylated it is targeted
  for rapid degradation.
• In stimulated cells Dishevelled is activated,
  GSK3 is inhibited and ß-catenin accumulates. It
  can then translocate to the nucleus where it
  forms a complex with a DNA-binding protein called
  TCF to activate transcrition of target genes.

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Figure 23-17. The Wingless (Wg) signaling
pathway. In the absence of Wg (or Wnt in
vertebrates), the kinase GSK3 constitutively
phosphorylates ß-catenin. Phosphorylated
ß-catenin is degraded and, hence, does not
accumulate in cells. Binding of Wg to its
receptor Frizzled recruits Dishevelled to the
membrane. Dishevelled, in turn, inhibits GSK3. As
a result, unphosphorylated ß-catenin accumulates
in the cytosol, translocates to the nucleus,
binds via its C-terminus to the transcription
factor TCF, and activates transcription of target
genes. Adapted from T. C. Dale, 1998, Biochem.
J. 329209.
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Combining Both Pathways
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Engrailed Expression Throughout Life
Figure 21-66. The pattern of expression of
engrailed, a segment-polarity gene. The engrailed
pattern is shown in a 5-hour embryo (at the
extended germ-band stage), a 10-hour embryo, and
an adult (whose wings have been removed in this
preparation). The pattern is revealed by an
antibody (brown) against the Engrailed protein
(for the 5 and 10hour embryos) or (for the
adult ) by constructing a strain of Drosophila
containing the control sequences of the engrailed
gene coupled to the coding sequence of the enzyme
ß-galactosidase, whose presence is easily
detected histochemically through the blue product
of a reaction that it catalyzes. Note that the
engrailed pattern, once established, is preserved
throughout the animal's life. (Courtesy of Tom
Kornberg and Cory Hama.)
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Further Reading Hammerschmidt, M. (1997) The
world according to hedgehog. TIG 13, 14-20.