Sample Talk

Report
Learning to swim:
What fish can teach us about CMDs
Jim Dowling, M.D., Ph.D.
University of Michigan
Congenital Muscular Dystrophy Family Conference
August 14-15, 2010
Meet the zebrafish
•
•
•
Small, fresh water aquatic vertebrate
Lifespan 1-2 years
Independently swimming by day of life 3
Why zebrafish??? (part 1)
It’s a numbers game…
Breeding age = 3
months
Average litter size
= 8-13
Time between
litters = 20 days
Breeding age = 2-3 months
Average clutch size = 100-300
Time between clutches = 1 week
Breeding age =
16+ years
Average litter size
=1
Time between
litters = 9-12
months minimum
Why zebrafish??? (part 2)
•
Crystal clarity!
– Zebrafish are optically
translucent allowing for
live imaging of muscle
and heart
Why zebrafish??? (part 3)
•
•
Invertebrate style genetics
– Large number of offspring
– Can easily introduce
DNA/RNA
– Can do saturating
mutagenesis screening
Vertebrate style genome
– Genome at least as complex
as ours
– Genome sequenced as part
of the NIH genome project
– All known muscular
dystrophy genes are found
in the zebrafish genome
(Steffen et al., 2007)
Zebrafish as a model for muscle disease
•
Muscle development begins at 20
hpf and is completed by 48 hpf
– i.e. very quickly!!!
•
Fish muscle shares many features
with human muscle
– Slow and fast twitch fibers
– Limb and trunk muscle
– Contains the common muscle
structures
•
What’s different?
– Slow and fast fibers in separate
compartments
– Significantly more trunk than limb
muscles
– Some reagents do not work in fish
Zebrafish as a model for muscle disease
(cont)
•
Obvious phenotypic
consequences from
muscle dysfunction
– Impaired swimming
•
Abnormal muscle
observable in live fish
– Can see it with
conventional microscopy
•
Dowling et al. (2008) Circulation Resçearch
Histopathologic changes
that reflect human
muscle pathologies
– (dystrophic pattern in
dystrophies, for example)
Zebrafish as a model of muscle disease
Control embryos (3 days old)
Myopathy embryos (3 days old)
Zebrafish models of CMDs
•
How do we make zf models of CMDs?
•
What models currently exist?
•
What have they been used for in the past?
– In other words, what have we learned from them?
•
What can we use these models for in the future?
– How can they help patients with CMDs?
•
Additional thoughts and future directions
How do we make zebrafish CMD
models?
•
Two ways to make a model
1.
Transient models
–
–
–
2.
Morpholino knockdown
Mutant transgene expression
Effect lasts 2-5 days
Stable models
– Chemically induced or
spontaneous mutant
– Dominant transgenics
– Direct gene targeting
–
–
–
Gene trap
TILLING
ZF method
What CMD models current exist?
•
Transient Models (morpholino based)
– LAMA2 (MDC1A)
– FKRP
– dystroglycan
– COL6A1/A3 (UCMD)
– SEPN1 (RSMD)
•
Stable
– LAMA2 (candyfloss)
– RYR1 (relatively relaxed)
Zebrafish model of MDC1A
•
•
ENU induced mutation called candyfloss
Identified/characterized by Currie’s group
– (Hall et al., 2007)
•
•
•
Clinical
– Onset at 3 days
– Progressive weakness
– Death by 2 weeks
Genetic
– Point mutation in LAMA2 leading to
premature stop codon
– Absent LAMA2 staining
Histopathologic
– Progressive myofiber injury and
degeneration
– Fragility at the myotendinous junction
Zebrafish model of UCMD
•
Experiment from my group
– (Telfer et al., 2010)
•
•
•
Clinical
– Severely reduced motor function
at 24 and 48 hpf
– Obvious morphologic
abnormalities
Genetics
– Morpholino mediated knockdown
of COL6A1 (exon 9) and
COL6A3 (exon 13)
– Models two common dominant
mutations
Histopathology
– Myofiber disorganization and
sarcolemmal breakdown
– mitochondrial swelling, increased
apoptosis
– Reduced and disorganized
collagen VI staining
Zebrafish models of
dystroglycanopathies
•
Experiments from two groups
– Thornhill et al., 2009
– Kawahara et al., 2010
•
Clinical
– Reduced motor function
– Morphologic changes at 24 and 48
hpf
•
Genetic
– Morpholino knockdown of FKRP
(x2 groups: Straub, Kunkel)
•
Histologic
– Pathology in muscle, eye and
brain
– Reduced laminin binding
– Reduced glycosylated
dystroglycan
Zebrafish model of core myopathies (RSMD): ryr
•
Identified at UM by John Kuwada
and colleagues
•
Spontaneous mutant with abnormal
swimming (named relatively relaxed)
•
Have impaired excitation-contraction
coupling
•
Found to have a homozygous recessive
mutation in ryr1b
•
Mutations cause significantly reduced
levels of RYR1 in fast muscle
•
Genetically and histologically a model
for recessive core myopathies
– RYR1 and SEPN1 related myopathies
Hirata et al. 2007
What did we learn from these models?
•
Candyfloss (MDC1A)
– LAMA2 important for the myotendinous junction
– Like mice, LAMA2 zebrafish have an early onset, severe phenotype
•
UCMD model
– Confirmed mitochondrial proton pore hypothesis (and confirmed
potential therapeutic role for molecules that act at the MPP)
– Unlike mice, UCMD zebrafish have an early onset, severe phenotype
•
FKRP morphants
– Demonstration of ability to model dystroglycanopathies in the zebrafish
– Defined key techniques for future experimentation
•
Core myopathy models (SEPN1 and RYR1)
– Confirmed key role of excitation-contraction coupling in muscle function
– Established/strengthened linked between SEPN1 and RYR1 related
myopathies
How can we use these models in the
future?
•
Identification of new/novel aspects of disease
pathogenesis
•
Identification of disease biomarkers
•
Platform for rapid identification/development
of novel therapeutics
Therapy development in zebrafish
models of CMDs
•
Two stable genetic models of
CMDs in zebrafish
– Candyfloss (MDC1A)
– Relatively Relaxed (ryr)
•
Morpholino models
recapitulate clinical, genetic,
and pathologic features of
CMDs
•
No new therapies yet
developed in these models
– Verification of efficacy of CsA
in UCMD model
•
Zebrafish have excellent
potential for MTS and HTS
Telf er et al., 2010
Drug screening in the zebrafish
•
ZF and drug screening
–
–
–
–
•
Large number of offspring
Frequent mating
Easily absorb drugs in media
Translucent body plan plus
many GFP markers
Muscle specific phenotypes
for drug screens
– Birefringence
– Motor function
– Other targets? (for example,
cardiac phenotypes)
HTS and birefringence in zebrafish
•
Birefringence
– Property of muscle when
placed under polarized light
– Abnormal birefringence is a
measure of impaired myofiber
integrity and/or organization
– Abnormal birefringence
reported in all muscular
dystrophy ZF yet described
•
Birefringence and CMDs
– Abnormal in candyfloss
(LAMA2)
– Abnormal in MO knockdown
of COL6A1 (UCMD model)
– Abnormal in MO knockdown
of FKRP (dystroglycan model)
FKRP morphants (Kunkel)
Drug screen of birefringence in zebrafish
Embryos are
fertilized and
collected
De-chorionate
embryos
Place in 24 well dish
with one drug in each
well (16 embryos per
drug)
Allow exposure to
drug for 3 day s
(change drug each
day)
Examine
birefringence at day
of life 4
Secondary analysis of
positive hits (dose
response; post-sympt
Rx; survival; etc)
Re-test positive hits
with a large sample
size
Positive hit = no fish
with abn
birefringence at day
of life 4
Evaluate
birefringence at days
of life 5-7
Wash away durg
Birefringence in UCMD model +/- cyclosporin A
An example of a successful screen using
birefringence with a DMD model
3 day washout
Drug 44: 0/16 with sap
Drug 44: 2/16 with sap
Drug 50: 4/16 with sap
Drug 50: 4/16 with sap
Drug screening and motor function in zebrafish
•
CMD models in
zebrafish all have
abnormal motor
function
– Impaired touch
evoked escape
response (starting at
3 dpf in candyfloss,
earlier in MO
models)
– Impaired swimming
in all models
3 day old control
3 day old ryr
Antioxidant treatment strategy
Identify ryr embryos (DOL3)
Place in 12 well dish with antioxidant
or placebo (DMSO). Replace solution
daily
Record daily swim speeds
Determine survival
(lethality for ryr at 12-14 days)
Noldus Zebrafish System
Proof of principle:
Antioxidant treatment in ryr zebrafish
Untreated
CTL
CTL
ryr
ryr
Antioxidant treated
Future Directions- Zebrafish Models
•
What do we have
– Excellent model of MDC1A and RYR1
– Transient models of UCMD and ZKRP
dystroglycanopathy
• Not really amenable to large scale drug screening
– Sound, quantitative assays for drug screening
•
What do we need
– Standardization of phenotypes and assays
– “stable” models for UCMD, dystroglycanopathies,
SEPN1, and laminopathies
A few parting ideas…
•
What validation is (or should be) required for
novel concepts regarding pathogenesis that are
developed in the zebrafish?
•
What are the step(s) (or what should they be)
between drug discovery in non-murine models
and clinical trial?

similar documents