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.

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

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

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

Microinjection of mRNA Examine mutant phenotype

+ 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)

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

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 embryos with smad5 mRNA to rescue the phenotype

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

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

Zebrafish Fate Map tail V D yolk Ventralization

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

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 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.

Ventralized phenotypes Hammerschmidt and Mullins, 2002 Overexpression phenotypes of bmp4