The Drosophila life cycle: ten days of eating, drinking, and mating

1
The Drosophila life cycle ten days of eating,
drinking, and mating
2
Some of what we have learned from flies

• Genes are on chromosomes genetic maps
  (Sturtevant and Morgan).
• X-rays and DNA alkylating reagents are mutagens
  (Muller).
• Genes can be positionally cloned. cDNA and
  chromosomal libraries (Hogness).
• Behavior (circadian rhythm, mating, learning and
  memory) is genetically programmed (Benzer).
• Development is genetically programmed (Lewis,
  Wieschaus and Nusslein-Volhard).

3
The first physical and genetic maps of an organism
Drosophila melanogaster A model metazoan.
The centimorgan 1 recombination
How we know genes are on chromosomes Correlate
physical changes in chromosome with
mutations. Genetic map distance 1
recombination 1 centimorgan. In situ
hybridization.
Polytene chromosomes
After TH Morgan, Nobel lecture, 1933
4
Drosophila genetics and understanding stem cells

• Basic features and advantages of using Drosophila
  as a model for human health and disease.
• Using fly genetics to address a basic biological
  question how do two different cells arise from a
  common precursor?
• Asymmetric segregation of determinant molecules
  at cell division defines cell fate.

5
Summary of tools available for dissecting
biological pathways in Drosophila

• Genetic
• Loss of function genetics
• Chemically generated mutants
• Chromosomal deficiencies
• Transposable elements
• Gene knockout by homologous recombination.
• Mosaic analysis making clones of mutant cells
  in a normal tissue.
• Gain of function genetics
• Dominant mutants
• Chromosome duplications
• Tissue specific expression systems
• Biochemical

6
Summary of tools available for dissecting
biological pathways in Drosophila

• Molecular
• A physical map that corresponds to the genetic
  map Fully sequenced, genetically marked genome.
  Excellent cytogenetics. Genome is arrayed on
  overlapping Bacterial Artificial Chromosome
  clones.
• ESTs (Expressed Sequence Tags) characterized for
  all stages of the life cycle.
• Antibodies and transposable elements to mark gene
  products, cells and cell morphology/subcellular
  structure.
• Stable single copy transformation.
• Targeted, tissue specific expression systems.
• dsRNA interference. Injecting of double stranded
  RNA causes sequence specific degradation of
  corresponding mRNA.

7
Understanding cellular differentiation using
Drosophila genetics

• Cell lineage cells result from a defined series
  of cell divisions.
• Cell fate/identity position, morphology, gene
  expression profile.

Shaft Sf Socket So Sheath Sh Neuron N Glial
cell G
8
The external sensory organ a well marked
neuronal lineage
How are neurons made? Answering this basic
question is useful for understanding cell growth
and differentiation, and ultimately for
manipulating cell fate decisions in
therapeutically relevant contexts.
9
Mutants that cause cell fate transformations
Example musashi causes duplication of hair
(shaft) and socket cells at expense of neurons.
WT
msi
After Okabe et al., Nature (2001) 211 94-98.
10
How do you isolate mutants that affect cell
fate in Drosophila? An F2 screen and then an
assay.
Mutagen
F0
/
/
X
Sco/CyOGFP Sco results in missing bristles on
the back and CyOGFP causes curly wings and
embryonic expression of GFP.
F1
m/
X
Single males
3 virgin females carrying dominantly marked Sco
chromosome and a dominantly marked CyO balancer
for corresponding chromosome .
Balanced stocks even lethals and steriles are
maintained as heterozygotes. If m/m is viable
and fertile, homozygous mutant stocks can be
obtained.
F2
X
m/CyOGFP
m/CyOGFP
11
Characterization of embryonic lethals

• Most mutants of interest, such as those that
  cause cell fate transformation, may be lethals.
  Flies cant live without neurons.
• All lethal stocks contain only m/CyOGFP (all
  curly winged flies).
• Screen embryos from lethal stocks by
  histological methods. Use balancers marked with
  GFP to distinguish m/m (GFP-) from
  Balancer/Balancer or m/Balancer embryos (GFP)

Screening for mutants that affect a complex
developmental process is a first step towards
identifying the components and individual
biochemical steps
12
How to read fly genetics notation
Drosophila has four chromosome pairs an X or
first sex chromosome, a second and third
autosomal chromosome, and a fourth very small
chromosome. Sex is determined by the ratio of X
chromosomes to autosomes (chromosomes 2 and 3),
not by the presence of a Y chromosome. However,
in practice, this means that XY flies are male
and XX flies are female. Genes are often
referred to by their location on polytene
chromosomes Divisions 0-20 fall on the X, 21-40
fall on the left arm of chromosome 2 (2L), 41-60
fall on the right arm of chromosome 2 (2R), 61-80
fall on the left arm of chromosome 3 (3L), and
81-100 fall on the right arm of chromosome 3
(3R). Bands are further subdivided by 3-7
letters 21A, 21B, etc.
13
How to read fly genetics notation

• In a fly cross, the slash or line symbol /
  separates the genotypes of each member of the
  chromosome pair and the semicolon symbol
  separates the genotypes of different chromosome
  pairs. You generally have to know the
  chromosome that a particular gene is on, but the
  genotypes of each pair is listed in order (X234),
  with any wild-type chromosome pairs generally
  omitted.
• Recessive alleles are lower case.
• Dominant alleles begin with a capital letter.
• Superscripts denote when the allele was obtained
  or the number of the allele.
• For example w/w px/px Sb/ means females
  flies homozygous for white on the X and plexus on
  the second, and heterozygous for Stubble on the
  third. white and plexus are recessive, and Sb is
  dominant.

14
Making and maintaining lethal (or sterile)
strainssuppressing recombination using
balancers and identifying flies that carry
recessive lethals

• l / l is dead. Cannot maintain l/l as a stock.
• l/ is viable, but eventually takes over the
  stock.
• l1 / l2 is viable, but eventually takes
  over the stock.
• Recombination between the wild type alleles
  eliminates the lethals.
• Suppose the l2 chromosome cannot recombine with
  l1 .
• Then the only gametes produced will be l2 and
  l1 .
• Recombination is naturally suppressed in males,
  but must be suppressed using a balancer in
  females.
• Stable, balanced lethal stock!

15
Balancer chromosomes

• l1 / l2 The stock is now balanced, but how do
  we tell the l1 chromosome from the l2
  chromosome? Answer add a dominant marker (D) to
  one chromosome. l1 / l2 D.
• Balancers contain multiple inversions, a dominant
  marker, and a recessive lethal. Consequently,
  they cannot recombine with normal chromosomes and
  they are not viable as homozygotes. Balancer
  stocks are maintained with a dominant visible,
  recessive lethal on the other chromosome.

16
Reading notation

• Balancer chromosomes are available for each
  chromosome FM6 or FM7 for the X, CyO or SM6 for
  the second, and TM3 or TM6 for the third.
• Check out Flybase on the World Wide Web to
  understand mutants and their phenotypes or
  Lindsley and Grell, The genetics of Drosophila
  melanogaster.
• For more details, check http//fly.bio.indiana.edu
  /nomenclature.htm

17
What do you do with your mutants?

• Arrange them into complementation groups by
  pairwise crosses. m1/Balancer x m2/Balancer is
  m1/m2 viable? If m1/m2 is viable, then m1 and m2
  lie in different genes. If m1/m2 is dead, then
  m1 and m2 are allelic. How many genes can be
  mutated to produce the phenotype of interest?
  This experiment provides an indication of whether
  the screen has reached saturation.
• Map them.
• Clone the affected genes.

18
Screens being careful

• Are you obtaining desirable mutants? Can you
  recover new alleles of known mutants?
• Saturation? Are you recovering multiple alleles?
• Independence? Are mutants products of separate
  mutagenic events?
• Address by removing mutagenized males from
  females after 3 days. Sperm cells are hit, but
  sperm stem cells that were hit have not yet
  produced sperm.
• Are single gene mutations responsible for the
  phenotype?
• Address by cleaning up mutant chromosomes with
  recombination and by obtaining multiple alleles.

19
The numb mutant cell fate transformation.
After Zhong et al, Neuron (1996)
20
The numb mutant neurons into support cells
Neurons and sheath cell nuclei marked with
anti-prospero (green) anti-Cut marks all es
organ nuclei
21
Mapping the numb mutant by recombination.
Strategy is to break up the chromosome into
intervals using genetic markers. As always, the
distance between markers is proportional to the
frequency of obtaining recombinants between them.
Distance in centimorgans.
22
Mapping the numb mutant by recombination.

• Recombination produces a series of chromosomes
  that have exchanged portions of the marked
  chromosome for portions of the numb chromosome.

The recombinants
al aristaless (aristae are antennae) dp dumpy
(wings are small) b black (back is black) cn
cinnabar (eyes are cinnabar) px plexus (wing
veins have spurs and star patterns)
al al dp al dp b al dp b cn dp b cn px
b cn px cn px px
23
Mapping the numb mutant by recombination

• Three issues
• 1) identify what portion of the multiply marked
  chromosome is in the recombinants and distinguish
  the recombinant from the test chromosome.
• Recover recombinants over an al dp b cn px
  multiply marked chromosome that also contains a
  dominant marker.
• 2) Prevent further recombination.
• Recover recombinants in males (no
  recombination)
• 3) identify recombinant chromosomes that contain
  numb.
• Take males that contain the recombinant
  chromosomes and test for whether they carry numb
  by complementation.

24
Mapping the numb (nb) mutant by recombination.
Numb maps between dp (dumpy) and b (black)
25
Mapping the numb mutant by crosses to chromosomal
deficiencies.
Deficiency (Df) chromosomes are missing defined
chunks of a chromosome. A large Df stock
collection ( cytologically and genetically
mapped) exists. Notation Df(chromosome
arm)Name. Chromosome arms 1 (X has a centromere
at the end), 2L, 2R, 3L, 3R, 4
nb/CyO X Df(2L)30AC/CyO
CyO/CyO dead Df/CyO alive nb/CyO alive What about
nb/Df? If you get no straight winged flies, (all
flies have balancer) then nb lies in the Df
After T.H. Morgan, Nobel lecture, 1933.
26
Identifying the right deficiency
27
Using recombination, map nb to 29-31 region of
polytene chromosome (arm 2L)
Overlapping deficiencies provide still finer
resolution. Genetic definition of a
deficiency contains at least two complementation
groups.
Polytene divisions 1-20 are X, 21-40 are 2L,
41-60 2R, 61-80 3L, 81-100 3R 101-104 4.
28
A key use for deficiencies defining the null
(complete loss of function) phenotype without
molecular analysis

• Genetic test place numb alleles over Df(2L)30AC
  and compare the severity of numb/Df and numb/numb
  phenotypes.
• If numb/Df and numb/numb phenotypes are similar
  in severity, then the numb allele is a functional
  null. Reducing gene dosage by putting the
  mutation over a deficiency has no effect if the
  mutation is already a null.
• Are numb/Df embryos worse phenotypically than
  numb/numb? If so, allele is not a null.

29
Cloning the numb mutant.

• You could clone numb by chromosome walking. The
  genome is sequenced, and arrayed on
  minichromosomes and DNA markers are available.
• Microdissecting the 30B region from polytene
  chromosomes, making a library, characterizing
  transcripts.
• Plasmid rescue tag the numb locus by making a
  numb mutant that results from the insertion of a
  transgene. Use the transgene DNA to isolate
  flanking chromosomal DNA.

30
Cloning the numb mutant by plasmid rescue
A DNA insertion (green) disrupts the nb locus
(mRNA shown as arrow). nbPlacW fails to
complement nb1.
Plasmid rescue Cut chromosomal DNA at
restriction sites (blue), ligate to itself,
isolate DNA from locus (magenta) using drug
resistance and bacterial origin of replication
encoded on transposon DNA (green).
31
How do you know that you have cloned numb?

• DNA is altered in numb mutants in ways that alter
  predicted protein (could affect coding region in
  conserved residues, could put stop codons in).
  Need multiple numb alleles.
• Rescue numb phenotype using cDNA and transgene.
  Complementation test.

32
Using Numb as a molecular marker for asymmetric
cell division.

• Numb is a novel 557 aa protein that contains a
  phosphotyrosine binding domain.
• How it works is not obvious from the structure
  ask where it is located in cells in a neuronal
  lineage.
• Raise an antibody! Use protein null as a
  negative control.

Nuclear marker for neuronal precursor
After Rhyu et al., Cell (1994)
Numb
Merge
33
Summary

• Model asymmetric segregation of Numb at mitosis
  produces asymmetry in cell fates.
• Evidence Numb is asymmetrically segregated.
• Numb is required for neuronal cell fate.
• Drosophila provides a powerful system in which to
  study many developmentally and physiologically
  important processes how the body plan is laid
  out, the basis of cancer, sleep, diabetes,
  anesthesia, learning and memory, synaptic
  transmission, cell migration etc.
• Classical and molecular genetics make it possible
  to make and map mutants, to clone the affected
  genes, and to introduce modified genes into the
  germline.