Download C. elegans Life Cycle

PowerPoint to accompany
Genetics: From Genes to Genomes
Fourth Edition
Hartwell ● Hood ● Goldberg ● Reynolds ● Silver
Reference
C
Prepared by Malcolm Schug
University of North Carolina Greensboro
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Caenorhabditis elegans:
Genetic Portrait of a Simple
Multicellular Animal
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Fig. C.1
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Summary of Reference C
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Overview of C. elegans as experimental organism
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Genetic dissection of several developmental processes
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Description of genome
Life cycle and anatomy
Precise patterns of cell lineage throughout development
Techniques of genetic and molecular analysis
Specification of early embryonic blastoderms
Role of programmed cell death
Timing of decision during larval development
Comprehensive example on use of genetics to probe a
signaling pathway that helps control development of the
hermaphrodite vulva
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Nuclear Genome of C. elegans
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97 Mb – very small (2/3 size of Drosophila)
Six chromosomes (I-VI) – about same size
No defined centromere – holocentric
Detailed physical map of cosmids and YACs
Recombination rates vary considerably
1 gene every 5 kb
19,000 genes
Proteins match homologous sequences of other organisms
20% proteins carry out core biological functions
Rest involved in processes required only in multicellular
organisms
Repetitive sequences – e.g., 7 kinds of TEs
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Gene Expression
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70% of mRNAs have one of two spliceleader sequences trans-spliced onto the 5
end of the message
Conserved among all nematode genera
examined
25% of adjacent genes are transcribed as
operons
Trans-splicing with splice-leader sequences
produces single-gene mRNAs
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Gene expression
Fig. C.3
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Life cycle of C. elegans
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Hermaphrodite
First 40 germ cells
that enter meiosis
in each arm of
gonad develop into
~150 sperm
deposited into
spermatheca
 Sexual fate switches
and rest of germ
cells are oocytes
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Fig. C. 4
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Mating
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Male lies next to female and slides fan-shaped tail
along its body until it contacts the vulva
Inserts specialized structures at base of its tail into
vulva
Sperm move from male’s tail through vulva and
into uterus
Sperm then migrate to spermathecae
Gain advantage over resident sperm and produce
outcrossed progeny
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Development from Zygote to Adult
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3 days
Embryonic development begins in uterus
Hermaphrodite lays her eggs 2 hours after fertilization
Blastoderms – large cells produced by early cleavage
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Gastrulation - At 28 cell stage, two gut precursor cells
begin to move to embryo interior
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Unequal cleavages in early development
Until 28 cell stage all blastoderms contact surface of embryo
Three germ layers produced (endoderm, mesoderm, ectoderm)
Later, cell division ceases while cells differentiate
Contractile proteins squeeze embryo into wormlike shape
14 hours after fertilization, egg hatches
L1 larvae is 250mm long and has 558 cells
Next 50 hours – L2, L3, L4 stages of development
separated by molts
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Embryonic Development in C. elegans
Unequal cleavage
gastrulation
Figure C.5a
wormlike shape
before hatching
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C. elegans Life Cycle
Fig. C.5b
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Laboratory Culture
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Grow on agar plates with lawn of E. coli for food
Transfer with flattened platinum wire
Adults reproduce for 3-4 days
Adults live up to 2 weeks after reproduction
Dauer larva
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Arrest development at L3 if starved
Can live up to 6 months
Given food they reinitiate development
Can arrest aging in response to food deprivation
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Anatomy of an Adult
Cross section
Fig. C.6a
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Linear Diagrams Show the Life
History of Every Somatic Cell
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Researchers focus on small part of cell lineage to study
particular developmental process
Partially metameric
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Fig. C.6b
Composed of repeating patterns of cell groups with similar functions
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Cell Lineage Diagram
Fig. C.7a
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Lineage Diagram of Early Cleavages Give Rise to
Six Founder Cells Which Each Give Rise to Various
Features of Adult
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Genetic Analysis and Recombinant
DNA Technology
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Diploid organism
Males have single X chromosome (XO)
Hermaphrodites are XX
Males arise spontaneously via
nondisjunction of X during hermaphrodite
gametogenesis
Once males present, mating generates more
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After fertilization, half gametes result from
sperm that carry no X = XO males
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Mutant Isolation
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EMS induces high forward mutation rate
Hermaphrodite heterozygotes for mutation will produce
¼ homozygous mutant progeny by self-fertilization
Severe effect mutations recovered because of selffertilization (e.g., complete paralysis of body wall muscles)
1600 genes on genetic map from mutant screens
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Examples
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Dumpy (Dy) – abnormal body shape
Long (Lon) – abnormal body shape
Unc – uncoordinated
Phenotypes capitalized and not italicized
Genes and alleles are in lower-case italics
Protein products are all in upper case (e.g., DPY-10)
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DNA Transformation
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Fig. C.8
Inject DNA into distal
syncytial gonad of
hermaphrodites
Irradiation promotes
integration of
transgenes into
genome
Reporter constructs
show transgenes
GFP is commonly used
reporter gene
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RNAi
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Enzyme complex, dicer,
cleaves dsRNA into 22bp
fragments called trigger
RNAs or small interfering
RNAs (siRNAs)
siRNAs spread throughout
cells
siRNAs unwind and
complex with
complementary sequence
of endogenous mRNA
two fates of siRNA
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Fig. C.9
Destruction of mRNA
Reverse transcribed, cleaved,
more siRNA – amplification
of siRNA
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Cloning of C. elegans Genes
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Positional cloning – based on location in genome
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SNPs used as markers
Identification of gene
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Rescue mutant phenotype by injection into
hermaphrodites, each cosmid corresponding to interval
identified by positional cloning
Candidate gene approach using complete genome
sequence
Candidate genes can be tested by RNAi
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Identification of Mutations Caused by
Insertion of a Transposable Element
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Forward genetics
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Begin with mutant phenotype and isolate gene for molecular analysis
Reverse genetics
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Gene of possible developmental interest predicted from genomic DNA sequence
Assign function through PCR methods
Assign function through RNAi
Fig. C.10
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Genetic Mosaic Analysis
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Animals composed of wild-type and mutant cells
Helps determine focus of action for a gene
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Chromosomal fragments generated from X or g irradiation
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Cells that must be active to allow animal to develop normally
Fragments maintained as extrachromosomal free duplications in
stocks
Construct strain chromosomally homozygous for recessive
mutation
Introduce free duplication including wild-type allele of
same gene
If free duplication is lost, clone of mutant cells will arise
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A genetic mosaic
experiment
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Fig. C.11
Free duplication carries
wild-type allele of lin-12+ in
a mutant worm (lin-12-)
mrk+ on duplication, mrk- in
background provides
phenotypic marker
indicating loss of lin-12 free
duplication
Results of mosaic analysis
showing Z1.ppp or Z4.aaa
must have lin-12+ activity to
assume a VU shape
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C. Elegans Research in Postgenomic
Era
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Functional genomics
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Identification of all the genes expressed and all genes
required during various stages of development or in
response to physiological stimulus
Global analysis using DNA chips with ordered
microarrays of 19,000 predicted genes in genome
In situ hybridization patterns at various stages of
development using probes for every predicted gene
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Identification of Maternal Effect
Lethals
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Specification of early
embryonic
blastoderms
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Early cleavages depend
on maternally supplied
components
F1 hermaphrodites
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Fig. C.12
No vulva
Progeny eat them from
the inside
Surviving F1 have
mutant progeny that
arrest development
before hatching
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Cell-autonomous Determinants must be
Distributed to Cells in Which They Act
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Fig. C.13
PAR proteins help direct
embryonic polarity
(a) wild-type zygotes, P
granules initially
uniformly distributed, but
then localize to posterior
part of zygote
(b) shortly after
fertilization, PAR-2 is
found in posterior cortex
and PAR-3 in anterior
cortex
Maternal lethal mutations
that produce two-cell
embryos with no polarity
identify six par genes
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Figure C.14
Inductive Ssignals Control Cell Fates in Early
Embryogenesis
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PAR proteins only affect cells
that contain them
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Cell autonomous
Early development also depends
on control signals sent between
cells to partition
Intercellular signaling
determines fates of ABa and
ABp cells
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Contact between P2 and ABp
enables P2 to send signals to ABp
required for proper fate of Abp
lineage
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Programmed Cell Death - Apoptosis
Fig. C.15
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Control of Timing During Larval Development
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Heterochronic
mutations
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Result in inappropriate
timing of cell division
and cell-fate decisions
during larval
development
Switch genes – loss-offunction and gain-offunction mutations have
opposite temporal
effects
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Lin-14 loss-of-function
mutation cases retarded
development
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Fig. C.16
Alter timing and cell
cycle
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LIN-14 protein behaves as central clock
Concentration descends from high in L1
animals to a low in L4 animals
 Epistasis tests show lin-4 negatively regulates
expression of lin-14 by novel mechanism
 Lin-4 gene transcript is processed to produce a
smaller 22-nucleotide RNA complementary to 3
untranslated transcript of lin-14 mRNA
 RNAi machinery appears to control LIN-14
concentrations
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Using Genetics to Probe Development of
Hermaphrodite Vulva
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Mutant screens identify genes involved in vulva formation
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6 cells capable of giving rise to vulva (P3p – P8p)
P5p-P7p normally give rise to vulva in response to inductive signal from anchor
cell (AC)
P3p, P4p, P8p produce only additional hypodermal cells
Fig. C.17a
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Genetic screens identify genes involved in AC
induction of P5p, P6p, and P7p
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Fig. C.17b
Search for vulvaless mutants
Mutants with no vulva fertilize eggs, eggs hatch and
devour mother’s organs
Multivulva mutants form ectopic vulva-like
structures
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Signal Transduction Pathway Activated by
Binding of LIN-3 Ligand to LET-23 Receptor
Fig. C.17c
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