Immunohistochemistry staining results for Dnajc9 and Col17a1 protein in zebrafish cornea. Both Dnajc9 (green) and Col17a1 (red) proteins are present in the dual cell layers of the developing zebrafish cornea (3 days after fertilization [d.p.f.]). In the adult zebrafish cornea (13 months after fertilization [m.p.f.]), Dnajc9 and Col17a1 expression is restricted to the external surface membrane of the superficial epithelial cells. 4′,6-Diamidino-2-phenylindole staining (blue) indicates cell nuclei. cn = Cornea; epi = epithelium; ln = lens; rt = retina; st = stroma.

Photomicrographs obtained after whole-mount in situ hybridization was performed using antisense probes to the Col17a1a, Col17a1b, and Dnajc9 genes on embryos 50 hours after fertilization and visualized with alkaline phosphatase–mediated staining. Col17a1a is observed in a punctate pattern on the epithelium, including over the developing cornea. Col17a1b transcripts are evident in the neuromast cells. Diffuse staining for Dnajc9 transcript was observed in the head region and retinal proliferative zone of the zebrafish embryos (lateral and dorsal views shown).

Photographs of zebrafish injected with 0.25 pmol Col17a1 TB morpholino (MO), 0.5 pmol Dnajc9 SB (disease exon) MO, or 0.5 pmol control MO obtained 3 days after fertilization (dpf). Col17a1a TB morphants present with morphologic changes in the shape of the tail tip and occasional heart edema (arrow). Although most Dnajc9 SB (disease exon) morphants appear unaffected, several demonstrated spine curvature (arrow).

Dnajc9 protein is up-regulated in response to stress. Immunohistochemistry on cryosections from 2 dpf zebrafish embryos revealed increased expression of Dnajc9 throughout the head, including the epithelium and cornea (cn), after 24 h exposure to 1.5% ethanol (EtOH) in E3 media, compared to untreated controls. Dnajc9 expression is maintained after 1 hour of recovery in E3 media. Lens (ln), cornea (cn) and retina (rt).