Anti-zebrafish Gnrh3 Gap polyclonal antibody is specific to zebrafish Gnrh3.

(A) The immunostaining of Gnrh3-ir soma and fibers (white arrow; Aa) in the brain with anti-zebrafish Gnrh3 Gap was eliminated when the antibody was substituted with pre-immune serum (Ab). (B) Immunostaining with anti-Gnrh3 Gap (green; Ba) on brain sections of gnrh3:tdTomato (red; Bb) adults shows co-localization between Gnrh3-ir soma and gnrh3:tdTomato-labeled soma (Bc), indicating the specificity of the antibody to Gnrh3. White arrows indicate Gnrh3-tdTomato-expressing soma that were positively stained by anti-Gnrh3 Gap. (C) Immunostaining with (Ca-Cc) anti-zebrafish Gnrh3 Gap or (Cd) pre-immune serum in COS7 cells transfected with (Ca) control pcDNA3.1 plasmid, (Cb) gnrh2-pcDNA3.1 plasmid, or (Cc,d) gnrh3-pcDNA3.1 plasmid. The cells that express zebrafish Gnrh3 and are immunostained with anti-zebrafish Gnrh3 Gap (red) are indicated by white arrows (Cc). Scale bars = 50 µm.

gnrh3 mRNA but not Gnrh3 protein detectable in gnrh3^-/- fish.

(A) Immunohistochemistry on adult coronal brain sections (Aa) using anti-zebrafish Gnrh3 Gap (green) demonstrates the presence of Gnrh3 signal in the form of somas (white arrows) and fibers in the gnrh3^+/+ pre-optic area of the brain (Ab). However, no Gnrh3 signal was found in the gnrh3^-/- pre-optic area (Ac) or in any other region of the brain. (B) In situ hybridization on adult sagittal brain sections (Bd) using gnrh3 DIG-labeled riboprobes. The anti-sense gnrh3 riboprobe demonstrated mRNA (red) in the ventral telencephalon and pre-optic area of both gnrh3^+/+ (Ba) and gnrh3^-/- (Bb) fish. The sense gnrh3 riboprobe demonstrated no gnrh3 mRNA signal in any brain regions in the gnrh3^+/+ fish (Bc). Scale bars = 50 µm.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

gnrh3^-/- juveniles exhibit normal Gnrh3 neuronal migration.

In both gnrh3^+/+ gnrh3:tdTomato (B) and gnrh3^-/- gnrh3:tdTomato (C) fish, Gnrh3-tdTomato-expressing soma located in the olfactory region and ventral telencephalon project fibers that extend posteriorly toward the hypothalamus and pituitary stalk (pituitaries not shown; A). Z-stack images were taken at 20x magnification on a Leica Microsystems DMi8 confocal microscope with a resolution of 1024 x 1024 and a z-step size of 0.10. All images were analyzed and assembled with Image J and Adobe Photoshop. Scale bars = 100 µm. OB = olfactory bulb. T = telencephalon. OT = optic tectum. ON = optic nerves. POA = pre-optic area. P = pituitary.

Adult gonadal morphology and gametogenesis do not differ between gnrh3^+/+ and gnrh3^-/- fish for both males and females.

(A-D) Gross gonadal morphology of adult male (A,C) and female (B,D) fish of both genotypes: gnrh3^+/+ (A,B) and gnrh3^-/- (C,D). (E-H) Gonadal histology with hematoxylin and eosin staining, demonstrating no differences between gnrh3^+/+ and gnrh3^-/- gametogenesis for both males and females. Male gnrh3^-/- testes contain spermatozoa in the lumens of testicular cysts (G), similar to gnrh3^+/+ testes (E). Female gnrh3^-/- ovaries contain all stages of vitellogenesis (H), including late-vitellogenic oocytes, which is similar to gnrh3^+/+ ovaries (F). White asterisks = mature spermatozoa in lumens of spermatocysts. Black stars = mature, late vitellogenic oocytes. Scale bars = 125 µm (testes) and 250 µm (ovaries).

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Gnrh3 protein not detectable by another primary antibody in gnrh3^-/- adult brains.