http://flymove.uni-muenster.de/Processes/Segmentation/SegmentGes.html
WT
wg
Mirror image duplication
A>
P
A>
P A>
<A A>
<A A>
loss of
P
P
WT
en
anterior
posterior
WT
en
http://flymove.uni-muenster.de/Processes/Segmentation/SegmentGes.html
CHRISTIANE NUSSLEIN-VOLHARD
Max Planck Institute Tuebingen, Germany
Saturation mutagenesis:
-identify mutations in all possible genes
involved in embryo patterning
-saturation reached at ~5 mutations per gene
Saturation mutagenesis:
-identify mutations in all possible genes
involved in embryo patterning
-saturation reached at ~5 mutations per gene
tossing marbles into bins
How many bins are there?
Saturation mutagenesis:
-identify mutations in all possible genes
involved in embryo patterning
-saturation reached at ~5 mutations per gene
15 marbles tossed into bins
5
5
Probability of missing
fourth bin in 15 trys:
5
Saturation mutagenesis:
-identify mutations in all possible genes
involved in embryo patterning
-saturation reached at ~5 mutations per gene
15 marbles tossed into bins
5
5
Probability of missing
fourth bin in 15 trys:
5
0.75 15 = 0.013
Saturation mutagenesis:
-identify mutations in all possible genes
involved in embryo patterning
-saturation reached at ~5 mutations per gene
15 marbles tossed into bins
5
5
Probability of missing
fourth bin in 15 trys:
5
~99%
probability
there are only
3 bins
0.75 15 = 0.013
dorsal
ventral
anterior
posterior
Genes that control pattern and polarity in the embryo:
Maternal genes:
1. anterior-posterior
-bicoid
-nanos
2. terminal
-torso
3. dorsal ventral
Zygotic genes:
1. Gap genes
2. Pair-rule genes
3. Segment polarity genes
4. Homeotic genes
early
late
Triple mutant: nanos, torso bicoid, torso bicoid, nanos
double:
double:
-no information double:
-only bicoid -only nanos -only torso
Maternal genes:
Bicoid
Hunchback
Gap genes:
Hunchback
Kruppel
Knirps
Giant
Pair-rule genes:
Even-skipped
Ftz
Runt
Prd
Odd-skipped
Segment polarity genes:
Engrailed
Wingless
Decapentaplegic
Hedgehog
Homeotic genes:
Ubx
abdA
abdB
Antp
WT
knirps
Knirps
expression
phenotype
Kruppel
Mirror image
duplication
expression
phenotype
Maternal genes:
Bicoid
Hunchback
Gap genes:
Hunchback
Kruppel
Knirps
Giant
Pair-rule genes:
Even-skipped
Ftz
Runt
Prd
Odd-skipped
Segment polarity genes:
Engrailed
Wingless
Decapentaplegic
Hedgehog
Homeotic genes:
Ubx
abdA
abdB
Antp
Pair rule: Even-skipped
expression
phenotype
Hairy Runt
Eve Ftz
Segment polarity: patched
WT
wg
Maternal genes:
Bicoid
Hunchback
Gap genes:
Hunchback
Kruppel
Knirps
Giant
Pair-rule genes:
Even-skipped
Ftz
Runt
Prd
Odd-skipped
Segment polarity genes:
Engrailed
Wingless
Decapentaplegic
Hedgehog
Homeotic genes:
Ubx
abdA
abdB
Antp
Lecture 3…
WT
en
WT
wg
http://www.ucalgary.ca/UofC/eduweb/virtualembryo/D_m_segment_I.html
giant
Gap genes
kruppel
knirps
Even-skipped
Pair-rule stripes
bicoid
hunchback
knirps
Knirps
expression
phenotype
BICOID bindings sites in the Hunchback and Knirps
enhancers
Hunchback
Knirps
Ma et al. (1996) Development 122 (4): 1195.
The EMBO Journal (1998) 17, 5998–6009
The EMBO Journal (1998) 17, 5998–6009
Cell. 2007 Jul 13;130(1):153-64.
Cell. 2007 Jul 13;130(1):141-52.
Is cooperativity sufficient?
Spatial Bistability Generates hunchback Expression
Sharpness in the Drosophila Embryo
Francisco J. P. Lopes1,2,3*, Fernando M. C. Vieira3,4, David M. Holloway5,6,7, Paulo
M. Bisch3, Alexander V.
Spirov1,2
PLOS Computational Biology (2008) 4:e1000184
BCD
Cooperative binding
n=~5
Hb
+
Positive feedback loop
Bi-stable dynamics
BCD
Cooperative binding
n=~5
Hb
+
Positive feedback loop
BCD
Cooperative binding
n=~5
Hb
No feedback
No feedback – no bi-stability
lowering Hill co-efficient shifts curve, but still bi-stable
http://www.ucalgary.ca/UofC/eduweb/virtualembryo/D_m_segment_I.html
Giant
Kruppel
Giant
Giant expression
Wild Type
bicoid
Kruppel
Mirror image
duplication
expression
phenotype
Knirps
expression
phenotype
Pair rule: Even-skipped
expression
phenotype
giant
Concentration
dependent
effects of
kruppel
Hunchback
Bicoid
Fishhook
knirps
Even-skipped
WT eve expression
Stripe 2- enhancer lacZ
Stripe 3+7 enhancer lacZ
Gap gene regulation of stripe 3
WT
kni
kni tor
hb
kni hb tor
Knirps defines boundaries of stripe 3 and 7
Stage 14
Later
cellularized
Bicoid binding site
deleted
Expression partially
restored by compensating
removal of Giant repressor
site
Deletion of GIANT binding sites expands band
Even-skipped expression
Lecture 2
-segment polarity
-wing polarity
-evolution of development
WT
wg
anterior
posterior
WT
en
A
Segment
P
A
P
A
Parasegment
P
Parasegments
Even
prd
runt ftz
Odd
prd
runt
wg
eve
Even
prd
runt ftz
en
Segments
denticals
Hairy Runt
Eve Ftz
bicoid
hunchback
kruppel
giant
eve
hairy
Even
runt
Odd
Parasegments
Even
Parasegments
Even
prd
runt ftz
Odd
prd
runt
wg
eve
Even
prd
runt ftz
en
Segments
denticals
Hedge hog
Segment polarity: patched
Hh is a short range signal
En mRNA
Hh protein
en
ci
wg
ptc
hh
ptc
wg
In the absence of the hh
peptide wg is repressed
by the ptc signalling
pathway
Cells on the en side secrete
hh peptide, but lack the hh
receptor encoded by ptc
hh
p p p p
wg
en
wg
frizzled
dsh
Armadillo
(beta-catenin)
TCF
gene
phenotype
vertebrate ortholog
Wingless (Wnt1)
segment polarity, many others
Wnt1
DWnt2
Pigment cells gonads; Kozopas 1998
Wnt7
adult muscle Kozopas 2002
Trachea (with wg) Llimargas 2001
DWnt3/5
Axon Guidance (through Derailed)
Wnt5
Yoshikawa 2003
DWnt4
Cell Movement in ovary Cohen 2002
Wnt9
Dorsoventral specificity
of retinal projections (Sato 2006)
DWnt6
DWnt8
Wnt6
Antagonist Dorsal,
no ortholog
immunity phenotype
(Gordon et al, 2005; Ganguly et al, 2005)
DWnt10
Wnt10
Gene
Phenotype of Knockouts or other functions 19 mammalian Wnts
Wnt1
midbrain, cerebellu; neural crest derivatives; hymocyte number
Wnt2
placental defects Monkley, 1996
Wnt2b/13
retinal cell differentiation Kubo, 2003, Kubo, 2005
Wnt3
early gastrulation defect; Axis formation; Hair growth; medial-lateral retinotectal topography; hippocampal neurogenesis
Wnt3a
vestigial tail; neural crest; hippocampus;Segmentation oscillation clock; left right asymmetry
Wnt4
kidney defects; sex determination; side-branching in mammary gland; number of thymocytes
migration of steroidogenic adrenal precursors into the gonad Jeays-Ward 2003
Anterior-posterior guidance of commissural axons.
Wnt5a
truncated limbs, truncated AP axis, reduced number proliferating cells Yamaguchi 1999
Distal lung morphogenesi; chondrocyte differentiation, longitudinal skeletal outgrowth;
Inhibits B cell proliferation and functions as a tumor suppressor
Defects in posterior growth of the female reproductuve tract
Wnt5b
Wnt6
Wnt7a
limb polarity; uterine patterning during the development of the mouse female reproductive tract
Delayed maturation synapses in Cerebellum
Wnt7b
Placental developmen; lung hypoplasia; macrophage-induced programmed cell death
Wnt8a
Wnt8b
Wnt9a
Joint integrity
Wnt9b
mesenchymal to epithelial transitions
Wnt10a
Wnt10b
decreased trabecular bone; myogenic and Adipogenic program; overexpression inhibits adipogenesis
Wnt11
Ureteric branching defects; cardiogenesis
Wnt16
anterior
posterior
WT
en
ptc
ptc
en
en
ptc
dpp
hh
ptc
dpp
In the absence of the hh
peptide dpp is repressed
by the ptc signalling
pathway
Cells on the en side secrete
hh peptide, but lack the hh
receptor encoded by ptc
hh
dpp
en
ptc
ptc
en
MAD signaling factor is activated by the dpp receptor:
phosphorylation state of MAD forms a signaling gradient
Conservation of Hedgehog
haltere
ANTp loss of function transforms leg to antenna-like appendage
Antennapedia
Wild type
Antp
c.a. 1949
Ubx mutant
?
?
Evolution of development
Dragon fly
fly
crustacean
Ubx
abd A
~550 MYA
Segment polarity: patched
prd
ftz
runt
ptc
wg
eve
ptc
en
En
het
en-D
En
en-D
DNA
replication
En
en-D
Mitotic
Cross-over
en-D
en-D
Mutant
daughter
cell
Creating genetic mosaic flies by mitotic recombination
anterior
posterior
WT
en
Hunchback is sensitive to ~10% changes in BICOID protein concentration
1. The gradient is approximately (but not necessarily
exactly) an exponential decay in intranuclear (and
cytoplasmic) concentration that is established rapidly
(less than 90 min).
2. Bcd diffuses relatively slowly (D = 0.3 mm2/s) in the
cortical cytoplasm containing the nuclei.
3. The Bcd gradient is stable over nuclear cycles 10–
14, when the number of nuclei is growing by a factor
of two with each division and Bcd is concentrated
and released from nuclei in a dynamic process. In
particular, the initial postinterphase concentration
in nuclei in successive cycles is constant to at least
10%.
4. Bcd is not simply trapped in nuclei; rather, it is in dynamic
equilibrium between influx and efflux with the
cytoplasm and, possibly, intranuclear degradation.
5. The spatial shape of the Bcd gradient (the length
constant for an exponential decay) scales with embryo
length over a factor of five range in lengths in
different dipteran species (Gregor et al., 2005).