Cellular expression of midkine-a and midkine-b during retinal development and photoreceptor regeneration in zebrafish

Abstract

In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine-b (mdkb). Midkine is a highly conserved heparin-binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration.

Figure 1. Graphical representation of differentially-expressed genes

Panel A illustrates functional gene categories and the number of genes in each category showing increased (+) or decreased (−) expression at 72 hrs after light onset. Genes within the left-most five categories resulted from the analysis of functional gene ontologies, whereas right-most three categories, cell cycle, photoreceptor, growth factors, were selected by hand from the list of differentially-expressed genes. Panel B illustrates the QRT-PCR results for five genes, rhodopsin, red opsin, PCNA, mdka and mdkb. Values above or below each bar represent the fold change in expression for each gene at 72hrs after light onset.

Figure 2. The cellular expression of mdka and mdkb during retinal development

Panels a-e are in situ hybridizations that illustrate the retinal expression of mdka at 30-120 hpf, respectively. The white arrowheads identify mdka-expressing cells in the circumferential marginal zone and retina. The white arrows in panels c-e identify the columnar cells within the inner nuclear layer that express mdka. The yellow arrowhead in panel d identifies the optic nerve. The yellow arrowheads in panel e identify presumptive horizontal cells. Panels f-j are in situ hybridizations that illustrate the expression of mdkb at 30-120 hpf, respectively. The arrow in panel g identifies the ventronasal patch. The arrows in panels g-j demarcate regions of transient mdkb expression. The arrowheads in panels h and i identify the circumferential marginal zone, which does not express mdkb. Note the prominent expression of mdkb within the gcl and inl in panels h-j. onl, outer nuclear layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer. Scale bar equals 50μm.

Figure 3. mdka is expressed by retinal progenitors and mdkb is expressed by differentiated cells

Panel a is an in situ hybridization that illustrates the expression of mdka at 72 hpf. Panel b is the same section as in panel a, but immunostained with antibodies against proliferating cell nuclear layer. Panel c is the digital overlay of panels a and b. Panel d is an in situ hybridization showing the expression of mdkb at 72 hpf. Panel e is the same section as in panel d, but immunostained with antibodies against proliferating cell nuclear antigen. Panel f is the digital overlay of panels d and e. In each panel, the white arrows identify the circumferential marginal zone in, whereas the arrowheads identify cells expressing mdka (panels a and c) and mdkb (panels d and f) within central retina. onl, outer nuclear layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer. Scale bar equals 50μm.

Figure 4. mdka is expressed in Müller glia

Panels a and d illustrate in situ hybridizations of mdka expression in Tg(gfap:GFP)^Mi2001 fish at 120 hpf. Panels b and e illustrate Müller glia immunostained with antibodies against green fluorescent protein. Panel c is the digital overlay of panels a and b; panel f is the digital overlay of panels d and e. In each panel, the three arrows in identify the same three Müller glia. In panel d, the asterisks identify mdka expression in presumptive horizontal cells. Panel g is an in situ hybridizations showing the expression of mdka at 72 hpf. Panel h is the same section as in panel g, but immunostained with antibodies against Prox1. Panel i is the digital overlay of panels g and h. Note that at 72hpf, horizontal cells synthesize Prox1, but do not yet express mdka. Panel j is an in situ hybridization showing the expression of mdka at 120hpf. Panel k is the same section as in panel j, but immunostained with antibodies against Prox1. Panel l is the digital overlay of panels j and k. Note the co-localization of mdka mRNA and Prox1 protein. Panel m is an in situ hybridization showing the expression of mdka in the adult retina. Panel n is the same section as in panel j, but immunostained with antibodies against Prox1. Panel o is the digital overlay of panels d and e. Arrows in j-o identify horizontal cells that express mdka and are immunostained for Prox1. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer; DAPI, nuclear stain 4,6-diamidino-2-phenylindole dihydrochloride. Scale bar equals 50μm; onl, outer nuclear layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer; ipl: inner plexiform layer. Scale bar equals 50μm.

Figure 5. In the light-lesioned retina, mdka and mdkb are expressed by horizontal cells and injury-induced photoreceptor progenitors

Panel a is an in situ hybridization showing the expression of mdka in a control retina. The white arrow identifies mdka-expressing horizontal cells. Panel b is an in situ hybridization showing mdka expression in a retina following 72 hrs of light exposure. Panel c is the same section as in panel b, immunostained with antibodies against PCNA. Panel d is the digital overlay of panels b and c. Arrowheads and arrows in panels b-d identify double-labeled cells in the ONL and INL, respectively. Panel e illustrates an in situ hybridization showing the expression of mdkb in a control retina. Panel f is an in situ hybridization showing mdkb expression in a retina following 72 hrs of light exposure. The arrowhead and arrow identify mdkb-expressing cells in the inner and outer nuclear layers, respectively. Panel g is the same section as in panel f, immunostained with antibodies against PCNA. Panel h illustrates the digital overlay of panels f and g. In panels b-d and f-h, arrowheads and arrows identify double-labeled cells in the ONL and INL, respectively. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; PCNA, Proliferating Cellular Nuclear Antigen; DAPI: nuclear stain 4,6-diamidino-2-phenylindole, dihydrochloride. Scale bar equals 50μm.