Drosophila Embryological evidence, and maternal effect genes

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Lecture 3

• Drosophila Embryological evidence, and maternal
  effect genes

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Lecture 3

• Aims
• Demonstrate Drosophila has polar centres
• Introduce the maternal effect genes
• Describe the two groups
• Anterior
• Posterior
• Terminal

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Drosophila embryos have obvious A/P polarity

• Secretes a cuticle, marked with denticle belts,
  easy to score segments

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Five parts Acron, Head, Thorax, Abdomen, and
Telson
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Score cuticle defects after transplanting
cytoplasm or pricking embryos

• Prick anterior
• lose head and thoracic segments
• Transplant posterior to anterior
• as above
• Prick posterior
• lose abdominal segments and pole cells, but not
  telson!
• Transplant anterior to posterior
• suppress posterior development and duplicate
  thoracic segments

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• Prick anterior
• lose head and thoracic segments
• Transplant posterior to anterior
• as above
• Prick posterior
• lose abdominal segments and pole cells, but not
  telson!
• Transplant anterior to posterior
• suppress posterior development and duplicate
  thoracic segments

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Maternal effect mutants

• Genetic screens performed by Nusslein-Volhard and
  Wieschaus (1980s)
• Female mutants laying defective eggs, as scored
  by cuticle pattern
• 3 groups
• Anterior
• Posterior
• Terminal

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Anterior Group

• bicoid - is the key
• exuperentia
• exuperentia-like
• swallow
• staufen

Localisation in the Oocyte
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bicoid

• allelic series of severity
• transplantation
• rescues in anterior
• duplicates anterior structures in the middle of
  embryos
• changes in fate map with increased gene dosage

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Allelic series of severity
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Transplantation
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To the anterior - rescues To the middle -
duplicates
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Gene dosage and fate map
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bicoid cont.....

• mutations mapped to 3rd Xme
• gene cloned by complementation
• Transcription factor and translational inhibitor
• Bicoid is the anterior determinant
• How is the RNA localised?

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bicoid localisation

• mRNA localised to the anterior pole of late
  oocytes and embryos.
• Protein translated in the fertilized embryo -
  short half-life
• Distribution in other anterior mutants altered.
• 3UTR of bicoid message required

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Localised in oocyte
RNA in situ to bicoid
Antibody to Bicoid
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Other anterior mutants

• Swallow, Staufen - RNA binding proteins
• Exuperentia - binds microtubules, shown by GFP
  fusions
• Exuperentia-like isolated as binding to bicoid
  3-UTR, also binds to Exuperentia

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Swallow?
Lc8/Dynein
Staufen
Microtubules
BLE1
Exuperentia-like
Exuperentia
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Refs
Mancebo et al 2001 BSF Binds Specifically to
the bicoid mRNA 3' Untranslated Region and
Contributes to Stabilization of bicoid mRNA.
Molec. Cell. Biol. 21(10) 3462--3471 Cha et
al. 2001 In vivo analysis of Drosophila bicoid
mRNA localization reveals a novel
microtubule-dependent axis specification pathway.
Cell 106(1) 35--46
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Arn et al. (2003) Dev. Cell 4(1) 41--51

• A biochemical analysis
• purified a protein complex
• identified proteins
• demonstrated involvement in directed
  translational control

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Used Domains IV and V of 3UTR Previously shown
to contain enough info for localisation
Gel Mobility Shift assay with ovary extracts
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Component Identification
Protein Probe Proteins Westerns

• Modulo
• Swallow
• PABP
• Smooth
• Nod

Native Prep Gel
SDS PAGE
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Form a complex
Co-immunoppt with anti-Mod
Mod, PABP and Smooth are RNA binding
proteins Nod is a kinesin motor protein And
Swallow is?
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SwallowGFP Enriched in anterior of Oocyte Is
largely immobile Possibly an anchor for The
bicoid message?
Stephenson (2004) Localization of swallow-Green
Fluorescent Protein in Drosophila oogenesis and
implications for the role of swallow in RNA
localization. Genesis 39 (4) 280-287
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Posterior group mutants

• 10 mutants first isolated
• no clear determinant - all have similar
  phenotypes, missing abdominal segments
• Transplantation used to find the determinant

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All missing abdominal segments If they all
look the same which is the determinant?
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Take cytoplasm from each mutant and see if can
rescue the other mutants - the one that cant
must be the determinant
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Nanos is the posterior determinant

• cytoplasm from mutant nurse-cells or oocytes
  rescues all but nanos
• nanos cytoplasm rescues nothing
• pumilio cytoplasm rescues everything!!!
• determinant is not localised or moved properly

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RNA in situ to nanos
Antibody to Nanos
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The key players are Oskar Nanos
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• Genes regulating oskar mRNA localization
• cappuccino
• mago nashi
• orb (female sterile mutation rather than a
  maternal effect)
• spire
• staufen

• Genes acting downstream of Oskar
• nanos
• pipsqueak
• pumilio
• tudor
• vasa

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Oskar is a key player in nanos localisation

• All but nanos and pumilio are also germ plasm
  determinants
• Stepwise localisation of products
• oskar mRNA has to be translated for Vasa to
  localise
• oskar translation strictly controlled
• Bruno and Aubergine

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Double posterior embryos can be produced by
either directing oskar RNA to the anterior pole
by fusing the message to bicoid 3UTR or the use
of BicD mutant mothers
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Meosin
Par4
oskar translation
Gustavus
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Meosin
Bruno
Prevents oskar translation
Aubergine
Par4
Augments translation of oskar
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Key refs
Cha et al . Kinesin I-dependent cortical
exclusion restricts pole plasm to the oocyte
posterior. Nat Cell Biol 2002 Aug4(8)592-8 Van
zo NF, Ephrussi A. Oskar anchoring restricts
pole plasm formation to the posterior of the
Drosophila oocyte. Development 2002
Aug129(15)3705-14 Long and short Osk
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........and Nanos is?

• translational repressor
• e.g. of bicoid (and hunchback) binds to NREs in
  3UTR via Pumilio
• Evolutionary homologues
• Dipterans, Xenopus, C.elegans?
• Next time will see it can be completely
  dispensable!!!!!

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Summary Anterior determinant - Bicoid Posterior
determinant - Nanos Previous experiments on
ligatures suggested the existence of
Morphogens Bicoid is a good candidate for such
a molecule