Zebrafish embryos carrying a loss-of-function mutation in the trh gene cannot hatch. (A) Annotated illustration of the zebrafish trh gene and mutation strategy. Guide RNA was designed to target the translation start site and generated a 4-bp deletion immediately downstream of the start codon. (B) Hatching percentage in clutches resulting from crosses of different genotypes. Bottom images show representative pictures of the clutches at 3 dpf. n = 300 eggs per cross. (C) Brightfield images of trh+/+ (wild-type), trh+/−, and trh−/− mutants at 3 dpf. White arrowheads mark the hatching gland. The insets show magnified views of the hatching glands.

Exogenous Trh rescues the hatching phenotype of trh−/− zebrafish embryos. (A) Intracerebroventricular injection of intact trh−/− embryos. Brightfield images of a saline-injected embryo (top) and an embryo injected with Trh (bottom). Images on the right are magnified views of the boxed area in the corresponding left images that show HGCs (circled). The time elapsed from injection is shown at the top. (B) Quantification of hatching gland integrity in saline-injected (n = 8) and Trh-injected (n = 7) embryos. niGV, normalized inverted gray values. (C) Intramuscular injection of decapitated trh−/− embryos. Brightfield images of a saline-injected embryo (top) and an embryo injected with Trh (bottom). Images on the right are magnified views of the boxed area in the corresponding left images that show HGCs (circled). The time elapsed from injection is shown at the top. (D) Quantification of hatching gland integrity in saline-injected (n = 4) and Trh-injected (n = 5) decapitated embryos. (E) Confocal images showing colocalization of Trh receptors (green puncta) on HGCs in prehatching embryos. HGCs are outlined. (F) Effect of F0 deletion of Trh receptors on hatching success.

Trh neurons form a transient circuit on the primary head sinus. (A) Immunolabeling of the Trh circuit (green) in prehatching 2-dpf embryos (top) and posthatching 3.5-dpf embryos (bottom). The red arrowheads mark the location of hindbrain Trh projections in prehatching embryos. Illustrations on the right show schematic representation of the Trh circuit before and after hatching. E, eye; HB hindbrain; OV, otic vesicle; P pituitary. (B) Lateral view of a 2-dpf prehatching embryo with labeled vasculature (green) and Trh (magenta). The bottom image is an enlargement of the boxed area in the top picture. The images were generated by surface rendering of a confocal image using Imaris software. The illustration on the top depicts the vasculature structure and names of the main blood vessels. (C) Ventral views of a prehatching embryo. Details are the same as in (B), except that the top image is a raw confocal image. DC, duct of Cuvier; PHBC, primary hindbrain channel; PHS, primary head sinus; PMBC, primary midbrain channel.

Trh activates the hatching gland of medaka (O. latipes). (A) Schematic illustrations of the differences in hatching glands (arrowheads) between zebrafish and medaka. The zebrafish hatching gland is located on the yolk sac, whereas medaka HGCs line the palate and the gill area. (B) Immunolabeling of the Trh circuit in prehatching 7-dpf medaka embryos (left) and posthatching 8-dpf medaka embryos (right). Red arrowheads mark the location of hindbrain Trh projections in prehatching embryos. (C) Intracerebroventricular injection of medaka embryos. Brightfield images of a saline-injected embryo (top) and an embryo injected with Trh (bottom). Images on the right are magnified views of the boxed area in the corresponding left images that show HGCs (circled). The time elapsed from injection is shown at the top. (D) Quantification of hatching gland integrity in saline-injected (n = 5) and Trh-injected (n = 5) embryos. (E) Histochemistry [hematoxylin and eosin (H&E) staining] of the gill area of a noninjected, unhatched embryo (top left), unhatched embryos injected with saline (bottom left) or Trh (bottom right), and a naturally hatched embryo (top right). Pink-stained HGCs are marked with black arrowheads.