Elsevier

Experimental Eye Research

Volume 93, Issue 5, November 2011, Pages 726-734
Experimental Eye Research

FGF signaling regulates rod photoreceptor cell maintenance and regeneration in zebrafish

https://doi.org/10.1016/j.exer.2011.09.003Get rights and content

Abstract

Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors in the light-damaged retina, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors. These data suggest that rod photoreceptor homeostasis and regeneration is Fgf-dependent and that rod and cone photoreceptors in adult zebrafish are regulated by different signaling pathways.

Highlights

fgfr1 was found to be expressed in the adult zebrafish retina. ► FGF-2 induced rod precursor proliferation and photoreceptor neuroprotection. ► Fgf signaling is not required for retinal progenitor amplification or migration. ► Fgf signaling is required for the regeneration of rod, but not cone, photoreceptors.

Introduction

Zebrafish, like other teleost fish, have the ability to regenerate multiple tissues and organs as adults, including appendages (i.e., fins) (Geraudie and Singer, 1992, Johnson and Weston, 1995, Morgan, 1901), cardiac tissue (Poss et al., 2002), spinal cord (Becker et al., 1997), and retina (Bernardos et al., 2007, Cameron, 2000, Vihtelic and Hyde, 2000). For example, following photoreceptor cell ablation by constant intense light treatment, Müller glia reenter the cell cycle to produce neuronal progenitors that continue to proliferate and migrate to the photoreceptor layer, where they ultimately differentiate into new rod and cone photoreceptors (Vihtelic and Hyde, 2000). Although a few proteins were recently shown to be required at various stages in the retinal regeneration process (Craig et al., 2010, Fausett et al., 2008, Qin et al., 2009, Thummel et al., 2010, Thummel et al., 2008b), relatively little is known about the signaling pathways that mediate the regenerative response in the retina.

One candidate for regulating adult retinal regeneration is the fibroblast growth factor (Fgf) signaling pathway. Fgfs are a family of secreted small polypeptides that bind to specific transmembrane receptor tyrosine kinases (Fgfrs). Ligand binding induces receptor dimerization and activation. Depending on the cellular context, activated receptors stimulate downstream signaling pathways that lead to cell proliferation, differentiation, migration, or survival (Turner and Grose, 2010). Fgf signaling has been implicated in many biological processes such as induction and patterning events during embryonic development (Crossley et al., 1996, Martin, 1998, Ohuchi et al., 1997, Peters and Balling, 1999, Reifers et al., 1998, Vogel et al., 1996, Zhu et al., 1996), tissue maintenance (Campochiaro et al., 1996, Stone et al., 1999), wound healing (Ortega et al., 1998), and cancer pathogenesis (Turner and Grose, 2010).

Fgf signaling was first shown to be important in regenerating the amphibian limb. Components of the Fgf signaling pathway were necessary for normal regeneration of newt limbs (Boilly et al., 1991, Poulin et al., 1993, Zenjari et al., 1997), and fgf8 expression was associated with successful hindlimb regeneration in Xenopus tadpoles (Christen and Slack, 1997). Blocking Fgf signaling by application of specific Fgfr inhibitors to Xenopus tadpoles suppressed premetamorphic hindlimb regeneration (D’Jamoos et al., 1998), whereas implanting Fgf2-soaked beads rescued the regeneration of de-innervated axolotl limbs (Mullen et al., 1996). 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).

To our knowledge, this is the first work to describe the role of Fgf signaling in adult zebrafish retinal regeneration. Here we show that intravitreal injections of FGF-2 induce rod precursor cell proliferation and neuroprotection during intense light damage to photoreceptors. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line (Lee et al., 2005), we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors following light damage, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors.

Section snippets

Zebrafish

Four zebrafish (Danio rerio) lines were used in this study: wild-type AB, albino, Tg(hsp70:dn-fgfr1) (Lee et al., 2005), and Tg(gfap:GFP)mi2002 (Bernardos and Raymond, 2006). Fish were maintained according to standard rearing protocols. All procedures using animals are in compliance with the ARVO statement for the use of animals in vision research and have been approved by the appropriate university committee on use and care of animals. Heat-shock induction of the hsp70:dn-fgfr1 transgene was

Endogenous fgfr1 is expressed in photoreceptors and inner nuclear layer nuclei in the adult zebrafish retina

Four Fgf receptors (Fgfr1–4) mediate Fgf signaling via homo- and hetero-dimerization (Shi et al., 1993). The expression of all four fgfr subtypes was examined in the adult zebrafish retina by in situ hybridization. While probes for fgfr2, fgfr3, and fgfr4 showed no signal above background (data not shown), fgfr1 was detected in both rod and cone photoreceptors and in the inner nuclear layer (INL), but not in the ganglion cell layer (GCL) (Fig. 1A, B). This expression pattern did not change

Discussion

Here we provide evidence that rod photoreceptor homeostasis and regeneration in adult zebrafish is Fgf-dependent but cone photoreceptors do not have the same dependence. First, we show that intravitreal injections of FGF-2 induced rod precursor proliferation and neuroprotection during intense light damage to photoreceptors. Additionally, using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors.

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    Funding sources: This work was funded by the Center for Zebrafish Research at the University of Notre Dame (DRH), National Institutes of Health grants R21EY019401 (RT), R01EY018417 (RT and DRH), P30EY04068 (RT), GM074057 (KDP), R01EY004318 (PAR and ZQ), and start-up funds to RT, including an unrestricted grant from Research to Prevent Blindness to the Wayne State University, Department of Ophthalmology.

    1

    Current address: Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.

    2

    Current address: Department of Biology, University of Missouri-St. Louis, 8011 Natural Bridge Road, St. Louis, MO 63121, USA.

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