Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Abstract

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.

Agrin function associated with ocular development is a target of ethanol exposure in embryonic zebrafish

“…The LG2 agrin MO, which we have shown previously to produce mild to severe phenotypes depending on the concentration of MO injected into one-cell embryos (Kim et al, 2007), was employed for these studies. We have shown previously that all agrin MO-induced defects are specific and not p53-mediated off-target defects (Liu et al, 2008). MOs were solubilized in water at a concentration of 0.1-1.0 mM before injection into one to two-cell stage embryos.…”

“…Whole-mount in situ hybridization was performed as previously described, with probe hybridization at 65°C (Kim et al, 2007; Liu et al, 2008). Digoxygenin-labeled riboprobes were transcribed from cDNAs encoding agrin , Pax6a , Mbx1 , or Shh .…”

“…Studies from our laboratory have also shown that agrin binds Fgfs to modulate optic nerve growth, as well as eye development (Kim et al, 2003; Liu et al, 2008). Likewise, our recent studies show that retinal Pax6a and ath5 gene expression is perturbed following agrin knockdown in zebrafish embryos, and is accompanied by perturbed optic stalk development (Liu et al, 2008). Since Pax6 and ath5 gene expression can be regulated by Shh signaling (Ericson et al, 1997; Kay et al, 2005; Miyake et al, 2005; Neumann and Nuesslin-Volhard 2000), these data suggest that agrin function may be required for proper Shh signaling during eye development.…”

“…Altering agrin expression in both transgenic mice (Fuerst et al, 2007) and zebrafish morphant embryos (Kim et al, 2007; Liu et al, 2008) induces many morphological phenotypes related to ocular development, which include microphthalmia and optic nerve hypoplasia. Studies from our laboratory have also shown that agrin binds Fgfs to modulate optic nerve growth, as well as eye development (Kim et al, 2003; Liu et al, 2008). Likewise, our recent studies show that retinal Pax6a and ath5 gene expression is perturbed following agrin knockdown in zebrafish embryos, and is accompanied by perturbed optic stalk development (Liu et al, 2008).…”

Zebrafish-Encoded 3-O-Sulfotransferase-3 Isoform Mediates Herpes Simplex Virus Type 1 Entry and Spread

“…[10][11][12][13][14] Enzymes that modify HS proteoglycans play essential roles during zebrafish (ZF) embryo development. [15][16][17][18] Recently, a combinatorial expression pattern of 3-OST gene family was shown in the ZF model. 19 In addition, adult ZF was presented as a new model system for HSV-1 infection of the nervous system.…”