Microinjection of mRNA and DNAWhy inject mRNA?1. Gain-of-function experiments (today): over & ectopic expression2. Dominant negative (pseudo loss of function) expts3. Mutant (today) or morpholino (tomorrow) rescue 4. Epistasis: what genes can and cannot rescue your mutant?

Where does your mutant gene lie in a pathway?

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.

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).

Mary Mullins, UPenn

D

D

V

V

Wild type

Dorsalized mutants

BMP antagonists

A BMP Activity Gradient in D-V Pattern Formation

Dorsalization and Zebrafish Fate Map

tail

DV

yolk

Class 4 and 5 dorsalizations

A BMP Signaling Pathway

/Lost-a-fin

/Alk8/Laf

Microinjection of mRNA

Examine mutant phenotype

+

ALL progeny mutant-- class 4 to 5 dorsalizations

somitabundtc24, a dominant maternal mutation

somitabundtc24 is an antimorphic mutation (“dominant negative”) of smad5 (Hild et al., 1999)

• wild type• rescued somitabun • somitabun (smad5)

• chordin overexpression in wild type

Tail bud stage after lecture tonight(cross your fingers)

Circumferential somites in class 4 and 5 mutant embryos

Wild type Class 5 dorsalization

Dorsalized mutant phenotypes, 1 dpf

somitabun/smad5

somitabun/smad5

Inject mutant embryoswith smad5 mRNA torescue the phenotype

Dorsalized mutant phenotypes, 1 dpf

somitabun/smad5

somitabun/smad5

Complete rescue

Partialrescue Inject mutant embryos

with smad5 mRNA torescue the phenotype

DV

DV

Wild type

BMP antagonists

DV

Non- and partial-rescued somitabun mutants,chordin ectopic expression

bmp4 ectopic expression

Zebrafish Fate Map

tail

DV

yolk

Ventralization

James Dutko

Ventralized and Dorsalized Phenotypes at Bud Stage/ End of Gastrulation

 

Wild typeVentralized Dorsalized

Ventralized Phenotypes at 1 dpf

bmp4 mRNA overexpression phenotypes James Dutko

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

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.

Agar troughs

Keep agar plate for tomorrow. Seal with parafilm and put into refrigerator.

NOTE orientation of wells relative to needle: straight side to left, needle to right.

Ventralized phenotypes

Hammerschmidt and Mullins, 2002Overexpression phenotypes of bmp4