Download Study of Developmental Biology using Zebrafish

Study of Developmental
Biology using Zebrafish
Major advantages of zebrafish
as a vertebrate model
Where is zebrafish in the world
Natural habitats of zebrafish
still water (currents, 0 m–sec to 0.1 m–sec) at 27°C to 34°C and pH 7.9–8.2;
widths of water bodies ranged from 1 to 12 m, and depths ranged from 16 to 57
cm; water was relatively clear (transparent to 35 cm).5-6 months for sexual
maturation.
Engeszer RE, et al. Zebrafish, 4:21-40, 2007
1. Rapid development
=28d human embryo
Movie of Zebrafish
Embryogenesis
2. High reproductivity
y
A few hundreds of
eggs per female
y
y
Laying weakly
y
External
fertilization and
development
y
Transparent
embryos for easy
observation
Controllable laying
time
3. Small size and easy raising
4. Well-developed genetic and
embryonic technologies
y
y
y
Mutagenesis
Transgenesis
Parthenogenesis and
androgenesis
y
y
y
Lineage tracing
Cell transplantation
Nuclear transplantation
Major Research Directions
y Gastrulation/embryonic induction
y Segmentation/circadian rhythmicity
y Neural patterning and neurobiology: AP
patterning, brain development, eye, axonal
guidance, neurodegeneration, behavior,and
etc.
y Organogenesis: heart, liver, pancreas,
blood, vasculature, gonads, fin, and etc.
y Cell convergency and extension
y Germ cells: specification, migration
y Disease models: cancers, DiGeorge
syndrome, Bardet-Biedl syndrome, and etc.
y Toxicity estimation
y Evolution
Mutagenesis
y
Cloning of novel genes functioning
in a developmental pathway
y
Demonstration of interactions
between genes
y
Starting materials for microarry,
proteomics, drug screening, and etc.
y
Animal models for human diseases
Approaches for creating mutants
y Chemical mutagenesis, e.g., N-ethyl-Nnitrosourea (ENU)
y Insertional mutagenesis, e.g., DNA
microinjection, proviral insertion, and
transposon insertion
y Physical mutagenesis, e.g., X-ray and
r-ray
Mutagenesis with N-ethyl-Nnitrosourea (ENU)
=
O
N
H3C-CH2-N-C-NH2
=
O
Alkylating agent
Sites of action: O6 guanine
O4 thymine
N3 adenine
PO4
y Large-scale mutagenesis
y Identification of important
genes
DNA Lesions after ENU treatment
Mutation
E.coli Drosophila Big Blue Mouse
mouse germline
A.T - T.A 1%
A.T - G.C 18%
G.C - A.T 74%
G.C - C.G 1%
A.T - C.G 3%
G.C - T.A 1%
Other
3%
(small deletion)
13%
13%
67%
3%
3%
3%
0%
33%
7%
38%
3%
3%
11%
5%
41%
42%
7%
5%
2%
2%
1%
In zebrafish, 1-3 mutations per gene can be obtained per
1000 mutagenized genomes
Steps for identifying recessive mutants
WT
WT
Present status of ENU mutagenesis
y It is widely used to obtain new mutations
y Tissue- or pathway-specific mutagenesis
using transgenic materials
y Gene-specific mutagenesis
y Identify mutants with peculiar defects,
e.g., sight, circadian rhythm, and behavior
New tool: Targeted mutagenesis
Cecelia Moens, Fred
Huthinson Cancer
Research Center,
WU, Tilligen, Inc.
Weinholds E et al. Science, 297:99-102, 2002
Retroviral insertional mutagenesis
y Single-copy insertion；
y High integration efficiency；
y Easy cloning of mutant genes
y 600 mutants have been generated and
300 mutant genes have been identified
(as said on July 2004)
Retroviral insertion
正常胚胎
X 基因
完整的 X 基因使胚胎正常发育
突变胚胎
病毒基因组
X 基因被病毒基因组破坏后胚胎异常发育
Enlarged
ventricle
Long
fin
reduced circulation,
edema around heart, bent
body, jaw does not form
Small size
Apart eyes,
looped heart
(A) Ten-week-old wild-type (top) vs. dominant mutant hiD862 with long fins. (B)
Wild-type (left) vs. bubble brain at day 2. (Arrowhead) Region of enlarged ventricle
in mutants. (C) Wild-type (top) vs. two no knack mutant embryos at day 4. (D)
Nineweek-old wild-type (top) vs. a nearly normal sibling, one of ∼10% of the
homozygotes that survived. (E) Wild-type (top) vs. hi37 mutant at day 4.
Abnormal liver and swim bladder
small head
No pigment
Edema around eyes
Mottled eyes
(F)Wild-type (top) vs. hi43 at day 5. (Arrowhead) Liver that is abnormal in the
mutant. (G) Closer view of wild-type (top) vs. hi43 liver region. (H) Wild-type (top) vs.
hi63 mutant at day 3. (I) Wild-type (left) vs. hi96 mutant embryo at day 4.
(Arrowhead) Unusual edema with pooled blood around eye. Edema around body of
mutant is also visible. (J) Wild-type (left) vs. bleached blond mutant at day 4. (K)
Closer view of eyes of bleached blond at day 4 showing mottled appearance.
Transgenesis
Microinjection of linearized plasmid DNA
Promoter/enhancers
Test cDNA
pA
1-10% transgenic rate; Inducible systems
available
Infection with recombinant retrovirus
15-50% transgenic rate
Examples of transgenic fish
GAL4/UAS
Inducible
Transgenic
System
y Avoid embryonic
lethality
y Spatiotemporal
control of
expression
Scheer N, Campos-Ortega JA.
Mech. Dev., 80 (1999) 153–158
GAL4
expressing line
UAS target
gene line
New tool: Transposon-based
transgenesis/Gene trapping
Tol2 transposon was
found in medaka
y High transgenic
efficiency (>50%)
y Identify genes
expressing in a
tissue-specific
manner
y Provide tissuespecific markers
y Create mutants
Kawakami K, et al. Dev Cell. 2004 Jul;7(1):133-144
New tool: In vitro cultured sperms
for transgenesis
yEasy handling
yTransgenic rate: 5%
Kurita K, et al. Proc Natl Acad Sci USA.
2004 Feb 3;101(5):1263-1267
Uses of transgenic fish
y Dissection of cis-regulatory elements
y In vivo anatomy/ongenesis of organs
y Isolation of tissue-specific cells
y Mutagenization of specific pathways
Cell fate mapping of early blastomeres
by single-cell injection
Injecting dye into
one cell of 8-cell
embryo
Observing
distribution of
labeled cells at 24 hpf
Cell fate mapping of late blastula
by single-cell injection
Study of cell fate by labeling
and transplantation
Useful tool for
addressing cell
autonomous or
nonautonomous
Fate map of 8-cell stage
zebrafish embryo
Anterior
Posterio
r
Dorsal
Left
Right
Ventral
Strehlow D and Gilbert W. Nature, 361:451-453, 1993
Fate map of 16cell stage embryo
NOTO, notochord; HATCH, hatching
gland; OLF, olfactory epithelium; TEL,
elencephalon;BLOOD, blood; HEART,
heart; MES, mesencephalon;
RHOM,rhombencephalon; YOLK,
epithelium over the yolk; NEPH,
pronephric ducts; LLENS, left lens;
RLENS, right lens; DFIN, epithelium over
the dorsal fin region; VFIN, epithelium
over the
ventral fin region; LRET, left retina; RRET,
right retina; SPI, spine; HEAD, epithelium
over the head region; ALSOM, muscle
cells of the anterior left somites; ARSOM,
muscle cells of the anterior right somites;
DTRU, epithelium over the dorsal trunk;
VTRU, epithelium over the ventral trunk;
MLSOM, muscle cells of the middle left
somites; MRSOM, muscle cells of the
middle right somites; LOTO, left otocyst;
LGILL, left primordial gill slits; PLSOM,
muscle cells of the posterior left somites;
PRSOM, muscle cells of the posterior
right somites; ROTO, right otocyst; RGILL,
right primordial gill slits; LATRU,
epithelium over the lateral trunk; Number
of injections indicates the number of times
each designated blastomere was injected.
Total number of injections is 112.
Strehlow D, et al.
Development,120:1791-8, 1994
Fate map of zebrafish gastrulas
tb
eye
dien
hb mb
Study of induction by
transplantation
(A) Schematic representation of the
operations. The labeled YC, from
which the blastoderm had been
removed, was transplanted on top
of the animal-pole region of
unlabeled embryos. (B–C) Induction
of gsc expression by the
transplanted normal YC. Four
figures were obtained from the
same specimen. (B) Ectopic gsc
expression (arrowheads) was
observed near the donor YSL of the
YC. (C) After avidin-biotin
peroxidase staining, ectopic
expression (arrowheads) was
observed in the unlabeled host cells.
Mizuno T et al. Mech. Dev., 81 (1999) 51–63
Identification of tissue- or
organ-specific genes
y From easily dissectible tissues/organs, e.g.,
heart, fin, eyes
y From sorted cells with a label, e.g., in
transgenic fish
y mRNA differential display or subtractive
cloning
y Screen by whole-mount in situ hybridization
Thisse group, Laboratoire de Biologie Moleculaire du Developpement,
INSERM U368, France, has analyzed 10,089 cDNAs and identified 2,605
cDNAs with a restricted expression pattern.
Morpholino knockdown
Advantages:
亚磷酸二酰胺键
High specificity
Stable
Non-toxic
Disadvantages
Costly
Low successful
ratio
Block of translation with MOantisense oligo
Nasevicius A, Ekker SC. Nat Genet, 26(2):216-220, 2000.
Splice morpholinos
RNAi in zebrafish
Long dsRNA causes nonspecific
mRNA degradation
GFP mRNA
GFP mRNA + dsGFP
GFP mRNA + dspouII
pouII mRNA + dsGFP
pouII mRNA + dspouII
Antisense
GFP probe
Antisense
pouII probe
pouII mRNA
Gene-specific mRNA degradation
induced by siRNA
y Not activate PKR/RNase
L pathways.
y Applicable in
invertebrates and
vertebrates.
y High throughput
screening.
y High cost for synthetic
dsRNA
y Vector-driven siRNA has
not been available yet
Cloning and gene knockout in zebrafish
Cultured embryonic cells
transfection
Target construct
selection
Nuclear transfer
Lee K et al. Nat. Biotechnol.,
Aug;20(8):795-9, 2002
Generation of haploid individuals