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Genetic pathways of cell
death in mammals
Transgenesisto unravel
cell death mechanisms
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studying a death pathway
In knockout orTransgenic mice
(in development)
In cellular systems ofembryonic origin
In surviving individuals
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1. Transgenic technology
2. Knockout technology
4. Conditional knockout technology
6. Conditional transgenesis technology
3. Gene trap
The mutant mouse approach
5. Knockout-knockin technology
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By Knudson
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The apoptoticpathways
The apoptosome
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Control STS
Cyt c
Hsp 60
Nuclei
Merge
Cytochrome cReleasein apoptosis
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By Knudson
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Bcl- 2- like proteins:
Bad
ANTI-APOPTOTIC
PRO-APOPTOTIC
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The apoptoticpathways
The apoptosome
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2. Knockout technology to studycell death in CNS development
E8. 5 E9. 0 E1 1 . 0
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PCD within the proliferative ventricular zones a potential rolein regulating the
size of the
progenitor pool PCD of postmitotic neurons a mechanism to match
the neuron populationto its target fields
PCD specific for early brain regioni.e. the lateral edges of the hindbrainneural fold
essential fornormal neuraltube closure
MULTIPLE FUNCTIONSOF PROGRAMMED CELL DEATH
IN BRAIN DEVELOPMENT
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Several genes implicated in PCDduring brain development
K.O. mouse exhibits an apparently normal forebrain (FB)formation and shows a decreased PCD in specific neuronalsubpopulations
K.O. mouse shows a reduction of PCD in most peripheral
ganglia and in spinal cord. K.O. mouse has more motor neurons than its w.t.
counterpart
Bax:
E 12
From White et al.,1998
From Roth et al., 1999
Bax BclXL Apaf1 Casp9 Casp3
Jnk1/Jnk2
?
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Several genes implicated in PCDduring brain development
Jnk1 /Jnk2: dK.O. mouse exhibits the lack of closureof the hindbrain neural tube
w.t. Jnk1/J nk2 dKO
E 9.0
E 11 .5
Bax BclXL Apaf1 Casp9 Casp
Jnk1/Jnk2
?
From Kuan et al., 1999
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Several genes implicated in PCDduring brain development
BclXL: K.O. mouse shows an increased PCD in specific neuronalsubpopulations (postmitotic neurons of the developingbrain, spinal cord and dorsal root ganglia). However it dies at E 13for hematopoietic defects
Preplate (PP) andventricular zone (VZ) of the
developing cortical wall of
E12.5 embryos
From Kuan et al.,
2000
Bax BclXL Apaf1 Casp9 Casp3
Jnk1/Jnk2
?
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K.O. mouse exhibits forebrain overgrowth, mostly throughthe expansion of the neural progenitor cells population.
It also affects the postmitotic neurons
Apaf1:
E 16.5E 12.5
Several genes implicated in PCDduring brain development
From Cecconi et al., 1998
Bax BclXL Apaf1 Casp9 Casp3
Jnk1/Jnk2
?
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Caspase- 9: K.O. embryo exhibits severe brain malformationwith a large overproduction of forebrain progenitors
Several genes implicated in PCDduring brain developmen
E 10.5E 16.5
From Kuida et al.,1998
Bax BclXL Apaf1 Casp9 Casp3
Jnk1/Jnk2
?
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Several genes implicated in PCDduring brain development
K.O. mouse exhibits forebrain abnormalities (*) andincreased forebrain surface area. By preventing selected PCD in the early forebrain progenitor
lineage, caspase deletion causes an increase in forebrainfounder cells and leads to a convoluted cerebrum
Caspase- 3:
E 12.5
Lamina terminalis PN 16
From Rothet al.
,1999 From Haydar et al., 1999
Bax BclXL Apaf1 Casp9 Casp3
Jnk1/Jnk2
?
S l i li t d i PCD
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Several genes implicated in PCDduring brain development
dKO BclXL/Apaf1, BclXL/Casp9, BclXL/Casp3:compensative effect in the PCD of neuronal postmitotic cells, butthey still show severe malformations of brain. They die at E1 3 as
KO BclXL: epistatic and independent actions in neural PCD
dKO BclXL/Bax:compensative effect in the PCD of neuronal postmitotic cells, they
still die at E13 as KO BclXL: epistatic action in neural PCD
Epistatic and independent apoptotic functions
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Epistatic and independent apoptotic functionsof Apaf1 , Caspase- 3, Bax and BclXL in the
developing nervous system
Apaf1
From Kuanet al., 2000
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Several genes implicated in PCDduring brain development
Jnk1 /Jnk2: dK.O. mouse exhibits the lack of closureof the hindbrain neural tube
w.t. Jnk1/J nk2
dKOE 9.0
E 11 .5
Bax BclXL Apaf1 Casp9 Casp3
Jnk1/Jnk2
?
From Kuan et al.,
1999
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3. The Gene Trap Strategy
Example: Apaf1
A t i i d d i A f1 / b
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Apoptosis is reduced in Apaf1-/- embryos
The Apaf1-/- phenotype
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The Apaf1 / phenotype
Onset Organ Description
e11.5 CNS Open brain and/or spina bifida
e13.5 CNS Forebrain overgrowth
e14.5 Eye Retina overgrowth, lens reduction, lens
mispolarization, accumulation of hyaloidendothelial cells
Lack of fusion of palatal shelvesPalate
Skull Absence of skull vault, absence of the
basisphenoid ossification centre
Limb Persistence of interdigital webs
Brain Differentiation into 2 layers of thechoroid plexus,expansion of the neural progenitor cellspopulation,thickening of the hindbrain
Inner ear Malformation of the otic vesicle
Th A f1 / h t
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The Apaf1-/- phenotype
PCD i di l li b d l
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PCD in distal limb development
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Lack of apoptosis physiologiacl Apoptosis
PCD in interdigital webs
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Apaf1-/-
wt
Palate formation
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Retina hyperplasyin Apaf1 -/- embryos
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The apoptoticpathways
The apoptosome
4 The Conditional knockout technology
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4. The Conditional knockout technologyExample: caspase-8 and Lck-Cre
Ph t i th T
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Phenotype in the Tcell lineage:
1. Marked increase in
the number ofperipheral T cell
2. Impaired T cellresponse
Caspase-8 isrequired for
CD95/Fas but notmitochondrial-mediated apoptosisin the T-cell lineage
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Caspase-8 isrequired for T cells
homeostasis
5 Fine tuning of phenotypes: the knockout knockin technology
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Specific knockout-
knockinof Cytochrome cKAform leads to
Specific inhibition ofapoptosome formation inKA/KA mice
5.Fine tuning of phenotypes: the knockout-knockin technology
All l f C h b t i bili bi d A f1
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All roles of Cytochrome c but its capability to bind Apaf1
are preserved
The phenotype of Apaf1 knockout is surprisingly more severe
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The phenotype of Apaf1 knockout is surprisingly more severe
than in KA/KA mice
Additional
roles for
Apaf1 ?
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6. The conditional (over)expression strategy. IExample: Apaf1
*
This mouse expresses Cre Recombinasejust in the developing cortex
cortical
promoter
Cre
Recombinase
pA
This mouse expresses GFP in all tissue
CMV prom.
-act ehn.
loxP
siteGFP pA Apaf1 cDNA IRES LacZ pA
loxP
site
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6. The conditional (over)expression strategy.IIExample: Apaf1
Apaf1Apaf1 ++
This mouse (over)xpresses Apaf-1 and --galgal in the cortex, and GFP in the others tissues
In the cortex, LacZ translation is not dependent from Apaf1 translation because of the
IRES box
Apaf1 cDNA IRES LacZ pA
--galgal
loxP GFP pA loxP Apaf1 cDNA IRES LacZ pA
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REDUNDANCY IN CELL DEATH
1. Among members of the same gene family
2. Among cell death alternative pathways
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By Knudson
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By Knudson
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By Knudson
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Apaf1 mutantIW undergo celldeath by a
nonapoptoticpathway
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The cellulardeathmorphotype
mimicksnecrosis
The apoptotic
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The apoptoticpathways
The apoptosome
AIF DAPI
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AUTOPHAGY: complex catabolic
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Widely used autophagy pathways
during development: lessons fromyeast to nematodes
A role for autophagy in
stress-induced development
AUTOPHAGY: complex catabolicprogram for lysosomaldegradation of proteins and other
subcellular constituents
CELLDEATH
Beclin-1 (Atg6 homolog) mutant embryos show early
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Beclin 1 (Atg6 homolog) mutant embryos show earlylethality
Bak-/-/Bax-/- dKO cells undergo anyway cell death by
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Bak /Bax dKO cells undergo anyway cell death byautophagy
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1. The fog(Apaf1 hypomorph) mouse exhibits anabnormal brain architecture
2. The main features of the phenotype are:extraventricle, inflammation (gliosis), abnormal
cortical layering, cortical hyperproliferation, enlargedchoroid plexus
3. The fogbrains show fried-eggcells, a typicalhallmark of oligodendrogliomas AND neurocytomas:
autophagy
The mutant mouse approach
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1. Transgenic technology
2. Knockout technology
4. Conditional knockout technology
6. Conditional transgenesis technology
3. Gene trap
The mutant mouse approach
5. Knockout-knockin technology
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The Gene Disrupters:
R.A. Flavell, T.W. Mak,
R. Hakem, P. Gruss,A. Strasser, J. Penninger,
K.A. Roth, C.B. Thompson,T. Knudson, N. Heintz,
many others and myself
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studying a death pathway
In knockout orTransgenic mice(in development)
In cellular systems ofembryonic origin
In surviving individuals
Laboratory of Molecular Embryology Dulbecco Telethon Institute
D f Bi l U i i f T V R I l
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Department of Biology, University of Tor Vergata, Rome, Italy
Elisabetta Ferraro
Marco CorvaroDaniela De Zio
Luigi Giunta
Giovanni Marchetti
Daniele Soroldoni
Maria TrignettiEmanuele Zaina
Elisa Tino
Francesca Fausti
University of Roma TreSandra Moreno
CollaboratorsKevin A. Roth, Marjo Salminen, Mauro Piacentini,