Download Meiotic markers of gonad development in zebrafish

SPUR program 2007
Cloning of zebrafish genes
involved in meiosis
Carmen M. Rivera García
Postlethwait Lab
August 3rd , 2007
Different mechanisms of sex determination
Genetic
Sex chromosomes
?
Environmental
Temperature
unknown
XX
XY
humans
turtles
zebrafish
Mechanism of sex determination unknown
No sex chromosomes found
Possible genetic sex determination influenced by environment ?
Juvenile hermaphrodites with separate sexes in adults
Zebrafish are juvenile hermaphrodites
embryos
juveniles
adults
Juvenile
hermaphrodites
females
(Takahashi et al., 1977
Uchida et al., 2002)
(ovaries)
Gonad
development
?
males
Oocytes in
all juveniles
Our GOAL:
Understand the
developmental processes
that make the juvenile
gonad to become ovary or
testis
(testes)
Meiosis is an essential process
during gonad development
Our APPROACH: Study of meiosis in zebrafish gonads
Meiosis in zebrafish
female
male
2n
2n
(diploid)
(diploid)
ovaries
testes
Ovary section
Testis section
meiosis
gonads
egg
meiosis
sperm
n
n
(haploid)
(haploid)
2n
(diploid)
Experimental Strategies
1) Look for candidate genes:
Search articles in Pubmed (NCBI) to find genes involved in meiosis in mouse
2) Selection of 4 genes to clone in zebrafish:
oct4: octamer-4
sycp3: synaptonemal complex protein 3
atm: ataxia telangiectasia mutated
dmc1: dosage suppressor of mck1 homolog
Mouse genes involved in meiosis
germ cell
(gonads)
oct4
oct4 is highly expressed in gonads.
Is a transcription factor essential for
the maintenance of totipotency in
embryonic stem cells.
(Bullejos and Koopman, 2004)
Mouse genes involved in meiosis
germ cell
(gonads)
oct4
enter
meiosis
oct4
When germ cells enter in meiosis
oct4 expression is being extinguished
(Bullejos and Koopman, 2004)
Mouse genes involved in meiosis
germ cell
oct4
(gonads)
enter
meiosis
oct4
formation of synaptonemal complex
sycp3
recombination
nodule
Protein axes
(lateral elements)
sister
chromatids
sycp3 encodes a component of the
synaptonemal complex, a meiosisspecific structure essential for
synapsis of homologous
chromosomes.
(Dobson et al.,1994; Lammers et al., 1994)
Mouse genes involved in meiosis
germ cell
oct4
(gonads)
enter
meiosis
oct4
formation of synaptonemal complex
sycp3
recombination
nodule
atm
Protein axes
(lateral elements)
sister
chromatids
atm is associated with sites along
the synaptonemal complex which
are thought to be involved in
meiotic recombination.
(Plug AW et al., 1997)
Mouse genes involved in meiosis
germ cell
oct4
(gonads)
enter
meiosis
oct4
dmc1 is a meiosis-specific gene
required to repair double-strand
breaks and pairing between
homologous chromosomes
(Katrin et al,2004)
formation of synaptonemal complex
sycp3
recombination
nodule
dmc1
atm
sister chromatids
Protein axes
(lateral elements)
sister
chromatids
Experimental Strategies
3) Design of primers:
mouse
database
blast
zebrafish
database
(NCBI)
(NCBI)
Reciprocal blast
should give us the
original mouse gene
Blast is a method to
compare gene sequences
Experimental Strategies
3) Design of primers:
mouse
database
blast
zebrafish
database
(NCBI)
(NCBI)
Reciprocal blast
should give us the
original mouse gene
4) Cloning of genes
Gene
nucleotide
sequence
from
zebrafish
Primer
design
for PCR
PCR is a
method to get
multiple copies
of our gene of
interest
Results
Amplification of zebrafish meiotic genes by PCR
oct4
100bp
ladder
O
T
sycp3
C-
100bp
ladder
O
T
atm
C-
100bp
ladder
O
T
dmc1
100bp
ladder
C-
O
600bp
600bp
600bp
700bp
Fragment size:
Fragment size:
Fragment size:
Fragment size:
778 bp
619 bp
601 bp
614 bp
O = ovary
T = testis
C - = negative control
Cutting out bands to clone genes
C-
Results
Cloning of genes (I)
Ligation of Bands and Transformation
Plasmids
E.coli cells
(TOPO vector)
ligation
transformation
PCR
fragments
(insert)
Amp r
Screening of colonies with insert
Pick colonies
Grow colonies
in LB+Amp
LB + Amp
Cells
grown
in Amp
plates ON
1h 37˚C
PCR
Results
Cloning of genes (II)
Selection of plasmids containing insert
PCR using
T7 and M13r
primers
800bp
763 bp
T7
M13r
Insert 600bp
Grow colonies that
have plasmid+insert
Plasmid with Insert
LB + Amp
Minipreps
Sequencing
of plasmids
Shaker
ON
37˚C
Lysis of
bacterial
cells
Recovering
of plasmid
oct4 Results
Sequencing of plasmids using primers from vector to know insert orientation
We have cloned oct4 gene
from zebrafish
blastn
Clones sequenced using T7 and M13r primers
5’
3’
Orientation of oct4 clones
T7
oct4 T1 (#6.1)
T7
oct4 T1 (#6.2)
T7
oct4 O1(#7.1)
T7
oct4 O1 (#7.2)
atm Results
Sequencing of plasmids using primers from vector to know insert orientation
We have cloned atm gene
from zebrafish
blastn
Clones sequenced using T7 primer
5’
3’
Orientation of atm clones
T7
T7
atm O1 (#5)
atm O1 (#6)
dmc1 Results
Sequencing of plasmids using primers from vector to know insert orientation
We have cloned dmc1 gene
from zebrafish
blastn
Clones sequenced using T7 primer
5’
3’
Orientation of dmc1 clones
T7
T7
dmc1 T1
dmc1 T2
T7
dmc1 O1
T7
dmc1 T3
T7
T7
dmc1 O2
dmc1 O3
sycp3 Results
Sequencing of plasmids using primers from vector to know insert orientation
Have we cloned sycp3 gene
from zebrafish ?
blastn
Clones sequenced using T7 primer
Orientation of sycp3 clones
T7
T7
scyp3 O1 (#10)
scyp3 O1 (#9)
T7
scyp3 T1 (#11)
T7
scyp3 T1 (#12)
Results
Have we cloned sycp3 gene from zebrafish ?
Putative
zebrafish
Sycp3
blast
mouse
database
(NCBI)
The gene that we have cloned is sycp3
Conclusions
 We have cloned 4 zebrafish genes with putative roles in meiosis
 Our results show that all cloned genes are expressed in adult
ovary and testis in zebrafish
Is the expression
pattern novel in
zebrafish?
Is the expression
pattern during
meiosis novel in
zebrafish?
No
No
Yes
atm
No
No
Yes
dmc1
No
Yes
Yes
sycp3
Yes
Yes
Yes
cloned
zebrafish
gene
New zebrafish
sequence ?
oct4
Future work
Analysis of expression of cloned genes by in situ hybridization
dmc1 expression in adult zebrafish gonads
Adult Ovary
Adult testis
20X
20X
Acknowledgments
Postlethwait lab
John Postlethwait
Adriana Rodríguez-Marí
Sara Wyckoff
Ruth BreMiller
and all people in the lab
SPUR program
Peter O’Day
Beth Roy
Summer Program for Undergraduate Research