PowerPoint Presentation

							Segment Polarity
    Genes
              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
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.
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.
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.
        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 doesn’t contain any hydrophobic
  residues).
  A series of elegant experiments show that this arrangement ensures
  that hedgehog only acts on the adjacent cell and doesn’t diffuse to
  act on cells further away (i.e. it is spatially restricted).
The engrailed, hedgehog, wingless Circuit



                     Wg

                Wg
                           en
                          Hh


                     Hh
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 83–471 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 β-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 274:255.]
  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.
  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 doesn’t 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.
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 21:1243
       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.
Figure 23-17. The Wingless (Wg) signaling pathway. In the absence of Wg (or Wnt in vertebrates), the kinase GSK3 constitutively
phosphorylates β-catenin. Phosphorylated β-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 β-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. 329:209.]
Combining Both Pathways
           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 10=hour 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 β-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.)
Further Reading: Hammerschmidt, M. (1997) The
world according to hedgehog. TIG 13, 14-20.