Download Transplant Pit Talk

Genetic Mosaic/Chimeric Analysis in the Zebrafish
Mosaic:
An organism consisting of cells of more than one
genotype (derived from the same individual)
Chimera:
An organism consisting of cells derived from more than
one individual
What is chimeric analysis used for?
1. Determining cell autonomy or non-autonomy
2. Identifying later functions for genes with essential
early functions
3. Identifying maternal functions for essential genes
Cell Autonomous Action
A gene is required in the cells that exhibit the mutant phenotype
Wild-type
Gbx mutant has no cerebellum
r3
Wt >wt
gbx- >wt
Gbx mutant cells are excluded
from the cerebellum
Cell Non-autonomous Action
A gene is required in cells other than those that exhibit the mutant
phenotype
Phenotype: motor axon guidance
Jing et al., Neuron 2009
How do you make a chimeric zebrafish embryo?
In the fly: genetic tricks: flp-mediated mitotic recombination
creating homozygous mutant clones.
In the mouse:
1) aggregation of blastomeres from pre-implantation
embryos
2) Cre-mediated tissue-specific recombination (“tissueautonomy”)
In the zebrafish:
1) Transplantation of cells between wild-type and mutant (or
morphant) embryos.
2) Cell-type specific expression of a dominant-negative
protein
3) Cell-type specific rescue of a mutant phenotype
Making chimeras by transplantation
1) Transplanting cells between embryos at the blastula stage
-Easier, faster
-Largely untargeted (mesendoderm vs. ectoderm; germ cells)
2) Transplanting cells between embryos at the gastrula stage
-Requires a compound scope
-Cells can be targeted to specific tissues
-Takes advantage of the gastrula fate map
Neural fates can be mapped at shield stage
Woo and Fraser, 1996
Blastula Chimera Analysis
Instructors:
Jim Fadool
Andres Collazo
Michael Granato
Why do blastula chimera:
- it’s the ‘perfect’ experiment for a grant proposal- each outcome is informative
- often reviewers will ask for it, so why not do it right away
- despite that this is a powerful approach, only few papers include such data
-presumed to be a ‘complicated’ ‘high tech’ ‘labor intensive’ experiment,
not for small labs
Purpose of the lab: to show you that this is a powerful technology, that
does not require much…..
but....
-need to use ‘null’ mutations; cells from hypomorphic alleles might behave
like wildtype cell when transplanted into a wildtype host, suggesting a
cell non-autonomous role even if the gene acts only cell autonomously.
What will we do today:
-use Pronase to dechorionate donors and hosts
( stop when chorions start blistering & start dropping out of chorions)
- place & orient donors and hosts into single well agar molds
donors
hosts
6
‘deep’
‘shallow’
-pull in 120 or as many as possible donor cells
(don’t suck in YSL nuclei layer at the interface)
-select area to transplant into and inject 10-40 donor cells
don’t poke through YSL nuclei layer at the interface
carefully remove needle to ensure that cell stay in embryo
-Move on horizontally to next host and repeat
-Move on to next row and start over
-When done, carefully place the entire plate&lid into 28.5 incubator
25
Donors:
H2A-GfP (ubiquitous, nuclear GFP); RhD
Isl1-GfP (motoneurons, cytoplasmatic GFP); RhD
mnx1-GfP (motoneurons, cytoplasmatic GFP) ;RhD
Brn3:GFP (RGC, cytoplasmatic
GFP;
Ath5:GFP (RGC, cytoplasmatic
GFP;
Hosts: Wildtype
RhD
RhD
At the end of the day carefully transfer
postepiboly embryos into 60 mm dish & keep at 28.5
Tips during transplanting:
-orient all embryos before placing the transpl. needle into the holder
- before taking up the first embryos, make sure the meniscus is steady
If not tighten all connections- the system has to be airtight in order to work
- keep meniscus low and in eyesight!
- do not ‘suck’ in donor cells with to much pull, slowly!
- don’t transplant >40 cells along margin.
- analysis of cell transplants: tomorrow 9am-11:30 pm!
Germ line replacement chimeras
• A tool for identifying maternal functions for essential genes
- mRNA and protein accumulate during oogenesis
- zygotic transcription begins only hours prior to gastrulation
• also useful for studying double or triple mutant phenotypes, eg RNA-Seq
Example: One-eyed pinhead (oep)
WT
Zoep
MZoep
• essential co-factor for Nodal signaling
• required for induction of endoderm and patterning of mesoderm
• rescued by injection of wt transcript (unusual case)
germ line replacement strategy
germ line replacement strategy
• GOAL: propagate mutant germ line through WT host using blastula transplantation
GFP-nos1-3’UTR
marks PGCs with
GFP (unfortunately
not yet at blastula
stg.)
Deadend
morpholinos
eliminate the host
germline cell
autonomously
Cells don’t have to be
transplanted to the
margin
Cells have to be
removed from the
margin
Ciruna et al. PNAS 2002
Today’s schedule:
AM: inject lineage marker into donor embryos
1:00 PM: demo: Michael Granato: blastula transplants on the
dissecting microscope;
1:30PM Cecilia Moens: germ cell transplants
2:00PM- 4:30: Your turn.
4:30PM-6:00: Gastrula Stage Transplants with Cecilia
Tonight after the talks: mount selected chimeras for confocal
time-lapse overnight.
See you in the lab at 1:00pm!
(avoid too much caffeine)
A few details:
Agarose molds for injection and transplants:
Adaptive Science Tools
31 Gifford DriveWorcester, MA 01606-3535
(774) 239-6133
TU-1: six ramps containing one 90-degree and one 45-degree beveled
side for micro-injection
PT-1: transplant mold with 150 divets to accept single embryos
Pipette Holders:
World Precision Instruments MPH3 (specify outer diameter of pipettes
and diameter of optional brass handle)
micromanipulator:
Fine Science Tools MM-33 (needs magnetic stand)
Or World Precision Instruments M3301R
Borosilicate Glass pipettes:
World Precision Instruments (match outer diameter to pipette holder)