1. Microinjection of mRNA and DNA
Mary Mullins, UPenn
Why inject mRNA?
Gain-of-function experiments (today): over & ectopic expression
Dominant negative (pseudo loss of function) expts
Mutant (today) or morpholino (tomorrow) rescue
Epistasis: what genes can and cannot rescue your mutant?
Where does your mutant gene lie in a pathway?
Why inject DNA?
Gain-of-function experiments: over & ectopic expression
Dominant negative expts
Promoter analysis
Transgenics--in vivo labelling of subsets of cells or subcellular domains,
promoters, ectopic expression (UAS/Gal4, heat shock, Cre-lox).
Why? A few reasons include:…..
When mRNA and when DNA?
1. mRNA gives reliable widespread expression. HOWEVER, it’s short-lived:8-14h.
2. DNA persists much longer, can be tissue-specific, BUT gives mosaic exp.

2. A BMP Activity Gradient in D-V Pattern Formation
BMP antagonists
BMP
Wild type D V
Dorsalized
mutants V D

3. Dorsalization and Zebrafish Fate Map
Class 4 and 5 dorsalizations
tail V D
yolk

4. A BMP Signaling Pathway
/Lost-a-fin /
Alk8/Laf

5. Microinjection of mRNA
Examine mutant
phenotype

6. + somitabundtc24, a dominant maternal mutation ALL progeny mutant--
class 4 to 5 dorsalizations
somitabundtc24 is an antimorphic mutation
(“dominant negative”) of smad5 (Hild et al., 1999)

7. Tail bud stage after lecture tonight
(cross your fingers)
wild type
rescued somitabun
somitabun (smad5)
chordin overexpression in wild type

8. Circumferential somites in class 4 and 5 mutant embryos
Wild type
Class 5 dorsalization

9. Dorsalized mutant phenotypes, 1 dpf
somitabun/smad5
somitabun/smad5
Inject mutant embryos
with smad5 mRNA to
rescue the phenotype

10. Dorsalized mutant phenotypes, 1 dpf
somitabun/smad5
Complete rescue
Partial
rescue
somitabun/smad5
Inject mutant embryos
with smad5 mRNA to
rescue the phenotype

11. BMP antagonists Wild type BMP D V Ventralized Dorsalized D V V D
Non- and partial-rescued somitabun mutants,
chordin ectopic expression
bmp4 ectopic expression

12. Zebrafish Fate Map
tail V D
yolk
Ventralization

13. Ventralized and Dorsalized Phenotypes at
Bud Stage/ End of Gastrulation
Ventralized
Wild type
Dorsalized
James Dutko

14. Ventralized Phenotypes at 1 dpf
bmp4 mRNA overexpression phenotypes
James Dutko

15. Inject through the chorion
--Advantage:embryos remain protected by chorion after injection, good survival
--Disadvantage: Need a good needle to penetrate the chorion and not break needle
Agar troughs
NOTE orientation of wells relative to needle: straight side to left, needle to right.
Important--very little media in trough, so chorion exposed above media.
The surface tension keeps embryo in well, when pull out needle.
Otherwise when try to withdraw needle, embryo comes with it.
Keep agar plate for tomorrow. Seal with parafilm and put into refrigerator.

16. Ventralized phenotypes
Hammerschmidt and Mullins, 2002
Overexpression phenotypes of bmp4