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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?

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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?

  • Determining cell autonomy or non-autonomy

  • Identifying later functions for genes with essential early functions

  • 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

Wt >wt

gbx- >wt

r3

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:

aggregation of blastomeres from pre-implantation embryos

Cre-mediated tissue-specific recombination (“tissue-autonomy”)

In the zebrafish:

Transplantation of cells between wild-type and mutant (or morphant) embryos.

Cell-type specific expression of a dominant-negative protein

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



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.


donors

hosts

6

‘deep’

‘shallow’

25

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

  • 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


Donors:

H2A-GfP (ubiquitous, nuclear GFP); RhD

Isl1-GfP (motoneurons, cytoplasmatic GFP); RhD

mnx1-GfP (motoneurons, cytoplasmatic GFP) ;RhD

Brn3:GFP (RGC, cytoplasmatic GFP;

RhD

Ath5:GFP (RGC, cytoplasmatic GFP;

RhD

Hosts: Wildtype


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!


• 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 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



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)


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