DB3002 Developmental Genetics

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DB3002 Developmental Genetics

• J Martin Collinson
• School of Medical Sciences
• University of Aberdeen
• m.collinson_at_abdn.ac.uk
• Tel F55750

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What is a developmental gene?

• Surprisingly difficult question to answer.
• A gene that is expressed and required during
  embryogenesis.
• Excludes genes that are required in all cells for
  survival e.g. respiratory enzymes.
• Developmental genes are involved in patterning
  the embryo, specifying cell types and controlling
  morphogenesis.

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What is a developmental gene?

• Random examples
• Sonic hedgehog signalling molecule expressed,
  among other places, in midline of CNS and is
  responsible for patterning (inhibits eye
  formation in midline mutation leads to
  cyclopia).
• Gli3 transcription factor expressed in
  developing limb - mutation leads to extra digits.

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Scary genetics
Gregory Mendel was an Australian Vicar who bred
smooth pigs with wrinkled pigs and counted how
many smooth and wrinkled babies they had. He
then repeated the experiment with green pigs and
yellow pigs and hence invented the science of
genetics. However Im not sure I believe the bit
about green pigs. A genuine exam answer,
University of Nottingham how many mistakes can
you find and do you award a mark for the critical
analysis at the end?
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Basic genetics

• Genotype the genetic composition of an organism
  its DNA sequences
• Phenotype the physical appearance or
  physiology/function of the organism.
• Mutations changes in genotype may change the
  phenotype.
• (But not all unusual phenotypes are caused by
  mutation e.g. environmental insult)

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Mutation

• Mutations - accidental changes in DNA
  sequence, occur all the time in all cells
  (usually repaired).
• Mutations in important genes are potentially
  lethal e.g. cyt b highly conserved
• Mutations in less important DNA sequences may
  have no effect (neutral).
• Some mutations give an interesting developmental
  phenotype T brachyury, short tail.
• required for notochord
• formation and mesoderm
• Some mutations cause disease DF508 mutation in
  the CFTR gene is a 3 bp deletion leading to
  protein misfolding in this important chloride
  channel and is the most common cause of cystic
  fibrosis.

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Mutation

• Alleles different versions of the same gene,
  controlling e.g. blood group (A, B, O), hair
  colour, eye colour. There may be many different
  alleles of a particular gene within populations
  of a species.
• Wild-type a normal, functional allele
• Null an allele that, due to mutation, has
  lost all function or is deleted.
• hypomorph an allele that retains some function.
• neomorph an allele that, due to mutation,
  leads to a new function.
• The mixture of normal, null, hypomorphic, neutral
  alleles of genes and other DNA sequences within a
  population is collectively referred to as
  polymorphism.
• Wild populations are generally polymorphic lots
  of variation. Inbreeding reduces polymorphism.
• Polymorphism is what natural selection works on,
  to drive the evolution of species hence
  developmental genetics is fundamental to
  understanding evolution.

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Mutation

• Genetic change is heritable.
• Mutation happens in all our cells all the time,
  but only mutations in germ cells (eggs and sperm)
  will be carried to the next generation. Most
  congenital disease is inherited, not the result
  of a new mutation. Its your parents who really
  screw you up.
• Mutation in somatic (non-germ) cells leads to
  mosaicism e.g blotchy pigmentation (trivial) or
  cancer (serious)

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Mutation

• Recessive alleles of genes only see effects
  when organism is carrying this allele on both the
  maternal and paternal chromosomes. E.g. albinism
  c, null mutation in the tyrosinase gene
  required for production of pigment melanin, only
  manifests in homozygous state.
• Dominant alleles see full effect when dominant
  allele is present on only one chromosome (e.g. in
  heterozygous state)
• E.g. C/C fully pigmented. C/c fully
  pigmented. c/c fully albino.
• Semi-dominant alleles heterozygotes are
  somewhere in between the homozyogus wild-types
  and homozygous mutants.
• E.g. Transcription factor Pax6 Pax6/
  normal eyes, Pax6-/- (homozyogus for null
  alleles) no eyes. Pax6/- (heterozygotes
  small eyes).

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Maternal effect mutation

• Sometimes your parents really do screw you up.
• Maternal effect mutants gene product is
  required in mum for correct development of
  offspring.
• E.g. stella, a mouse gene expressed in germ cells
  and oocytes. Stella-/- females are normal, but
  after mating their embryos die before blastocyst
  stage.
• Stella protein is deposited by maternal tissues
  into the oocytes amd is required for early
  development

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Maternal effect mutation

• stella/- x stella/-

stella/
stella/-
stella-/-
1 2 1
All normal (because mothers had stella)
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Maternal effect mutation

• Male stella-/- x female stella/

All stella/-
All normal (because mother had stella)
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Maternal effect mutation

• Male stella/ x female stella-/-

All stella/-
All dead (because mother had no stella)
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Developmental genetic strategies

• Forward genetics
• Traditional genetics, pre-molecular biology.
• Starts with an interesting developmental
  phenotype, e.g. extra toes.
• Works back to find the mutated gene responsible
  for that phenotype.
• In the old days, this analysis would aim to name
  the gene, determine by genetic mapping where it
  was on the chromosomes, and determine by
  complementation and epistatic analysis (see
  later) which other genes it interacted with or
  controlled to do its job (elucidation of genetic
  pathways).
• Nowadays, we can also sequence everything to see
  what the gene actually is and what it might be
  doing (e.g. signalling molecule, transcription
  factor).

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Epistasis some proper genetics
Imagine a genetic pathway the product of gene a
activates expression of gene b which in turn
upregulates gene c, which leads to activation of
some developmental process e.g. production of
segment 3. All three gene products vital, so
homozygous mutation in any one of them leads to
loss of segment three.
An animal that is b-/- has no segment three.
Injecting extra a has no effect, because the
genetic pathway is still blocked at the level of
b (c not turned on). However injecting active
c product rescues a b-/- animal get segment
3. a is epistatic to b, b is epistatic to c. By
doing these complementation assays, the order in
which genes work towards a developmental endpoint
can be worked out
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Epistasis some proper genetics
Eg. Sonic hedgehog (Shh), Smoothened (Smo), Gli3
are three genes involved in vertebrate CNS and
limb patterning. Mutation in Shh can be rescued
(in part) by introduction of constitutively
active Smoothened or Gli3. Mutation in Smo can
be rescued by active Gli3, but not by additional
Shh. Mutation in Gli2 cannot be rescued by extra
Smo or Shh. Hence genetically infer Shh Smo Gl
i3
We now know that Shh is the signalling molecule,
Smo is part of its downstream receptor complex,
and Gli3 is a transcription factor downstream of
Smo that activates genes required to perform the
cellular differentiation events in response to
Shh signals.
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Forward genetics - Mutagenesis screens
A hit n hope approach to deriving new mutations.
The male animal is subjected to a mutagen, e.g.
radiation, or chemical mutagens such as
ethylnitrosurea (ENU) or ethylmethyl sulphate
(EMS).
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Mutagenesis screens
A screen for dominant mutations Mate mutated
mice with wildtype females. Screen babies for
mutations. Those that are heterozygous for a
dominant gene will show a phenotype.
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Mutagenesis screens
A screen for recessive mutations Some of the F1
progeny of the mutagenised mice might LOOK
normal, but be heterozygous for a recessive
mutation. Have to breed a litter of progeny then
do brother-sister matings to get -/- mice.
/-
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x
F3 or B0
/-
/-
/-
-/-
/-
/
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Mutagenesis screens
At this point, you can start to screen/sequence
your mutant mice to determine what mutation has
been created in which gene, and hence find the
gene responsible for, e.g. yellow mice.
E.G genetic (linkage) mapping studies. If you
find that the mutation is reliably inherited with
a genetic marker such as a pink eye gene (made
up example), then your mutation is closely linked
to that gene. Do this in more detail elsewhere.
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Developmental genetic strategies

• Reverse genetics
• Genetics facilitated by modern sequencing and
  cloning technology.
• Starts with a potentially interesting gene, e.g.
  a newly discovered transcription factor.
• Deliberately mutate or manipulate the gene to
  see what it does during development.
• Commonly use transgenic technology (artificial
  introduction of new or mutated genes), very often
  by gene knockout, to look for an interesting
  mutation.

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Developmental genetic strategies

• Reverse genetics gene knockout
• e.g. 1989 -discovery of a DNA-binding
  transcription factor Msx1 that is widely
  expressed during face development.
• Reverse genetic strategy to determine its
  function knock the gene out to create Msx1-/-
  mice
• Msx1-/- mice had cleft palates showed Msx1
  important for fusion of palatal shelves during
  development.
• Satokata, I., Maas, R.  (1994) Msx1 deficient
  mice exhibit cleft palate and abnormalities of
  craniofacial and tooth development. Nat.
  Genet. 6, 348 - 356

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Developmental genetic strategies

• Reverse genetics transgenic misexpression
• Discovery of a transcription factor Foxe3 that
  is expressed in lens epithelium but not in lens
  fibre cells.
• Introduce a transgene that leads to Foxe3 being
  expressed in lens fibre cells they develop a
  lens epithelial phenotype (leads to cataract)

Landgren et al., 2008. Invest.Ophthalmol. Vis.
Sci. 494269 4277
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Making the transgenic
Isolate cDNA coding for the protein you want to
express
Incorporate the promoter elements you want to
drive expression
Ensure correct RNA processing
Poly(A) signal (SV40 processing and termination
sequence)
Cryaa promoter
Foxe3 ORF
Intron
Inject this into fertilised eggs.
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Foster mother gives birth to transgenic pups
see which ones are expressing the transgene
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Summary

• Developmental genes are those that are required
  for patterning the embryo, tissue
  differentiation, and morphogenesis (maybe about
  5000 genes, or 10-20 of all our genes).
• Mutations in these genes can be studied as the
  basis of developmental genetics and evolutionary
  change.
• Traditional, scary, genetics involving test
  crosses, 9331 ratios, and gene mapping
  involving breeding pedigrees and looking for
  linkages, is still a big part of the process.
• Cloning and genome sequencing technology has
  revolutionised genetics opens up fields of
  reverse genetics by allowing direct manipulation
  of gene sequences.