Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb

Abstract

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. Developmental Dynamics 235:336 -346, 2006.

FGF signaling regulates rod photoreceptor cell maintenance and regeneration in zebrafish

“…To test whether Fgf signaling is required for either Müller glia or progenitor cell proliferation, adult Tg(hsp70:dn-fgfr1) fish and their wild-type siblings were exposed to constant bright light and heat-shocked daily. Eyes were harvested at 2 and 4 dpl and immunolabeled for PCNA, a widely-used marker for cell proliferation in retinal regeneration and other systems (Fimbel et al, 2007; Kassen et al, 2007; Kassen et al, 2010; Leung et al, 2005; Mahler and Driever, 2007; Thummel et al, 2006; Thummel et al, 2010; Thummel et al, 2008a; Thummel et al, 2008b; Vihtelic and Hyde, 2000; Vihtelic et al, 2006), and Rhodopsin, which labels rod photoreceptor outer segments (ROS). At 2 dpl, transgenic animals were indistinguishable from wild-type siblings (Fig.…”

“…More recent studies have also implicated a role for Fgf signaling in tissue regeneration in adult zebrafish. Fgf signaling has been shown to be necessary for proper regeneration of the adult zebrafish caudal fin (Lee et al, 2005; Poss et al, 2000; Thummel et al, 2006; Whitehead et al, 2005) and heart (Lepilina et al, 2006). …”

Specific NuRD components are required for fin regeneration in zebrafish

“…Fins were photographed immediately after injection and at 24 hours post-injection with a stereomicroscope (MZ16) and camera (DFC480) (both Leica). The percentage of regeneration was calculated as previously described [18,26]. Areas of the dorsal and the ventral half of regenerating fins were measured with Image J 1.43 q software.…”

Fgf Signaling is Required for Photoreceptor Maintenance in the Adult Zebrafish Retina

“…After injury, MGC are able to give rise to multipotent progenitor cells, which proliferate and substitute all types of neurons to reconstitute the previous tissue architecture [17], [18], [19]. Investigations in zebrafish fin, heart and brain revealed a crucial role for Fgf signaling during homeostasis and regeneration [20], [21], [22], [23], [24]. Furthermore, inhibiting Fgf signaling from prenatal stages onwards causes degeneration of rod cells in the mouse retina.…”