Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.
Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.
Thummel
Ryan
R
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA. thummel.1@nd.edu
Bai
Shan
S
Sarras
Michael P
MP
Song
Peizhen
P
McDermott
Jeffrey
J
Brewer
Jeffrey
J
Perry
Martin
M
Zhang
Xiaoming
X
Hyde
David R
DR
Godwin
Alan R
AR
D000818:Animals; D018274:Electroporation; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics* / Q000378:metabolism*; D012038:Regeneration / Q000502:physiology*; D015027:Zebrafish / Q000033:anatomy & histology* / Q000502:physiology*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism*
D018398:Homeodomain Proteins; D017468:Receptors, Fibroblast Growth Factor; D029961:Zebrafish Proteins; C460082:msx1b protein, zebrafish