Genetic Mosaic/Chimeric Analysis in the Zebrafish

1. 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

2. 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

3. 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

4. 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

5. 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

6. 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

7. Neural fates can be mapped at shield stage Woo and Fraser, 1996

9. Blastula Chimera Analysis Instructors: Jim Fadool Andres Collazo Michael Granato

10. 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.

11. 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

12. 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

13. 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!

15. • 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

16. germ line replacement strategy

17. 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

19. 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.

20. See you in the lab at 1:00pm! (avoid too much caffeine)

21. 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)