Spatio-temporal expression of gr mRNA during zebrafish development evidenced by whole-mount in situ hybridization performed at the indicated stages. All embryos are lateral views with the animal pole up (1- and 2-cell embryos and at 6, 9, and 10 hpf) and head up pointing to the left (15 and 24 hpf). Note that expression levels of gr mRNA are lost at 9 and 10 hpf and again detected at 15 hpf.

Phenotypes of embryos or larvae at 1, 3, and 5 dpf after treatment with gr^ATG1MO alone or together with MOp53 as compared to control groups (WT or treated with gr^mismMO or gr^splicMO) or after rescuing with t-gr2 mRNA. Fish in Class II are more severely affected than in Class I. Animals are presented as lateral view, anterior to the left. Bar = 500 µm. The effects of gr^ATG2MO are also shown.

A:gr^ATG1MO-injected embryos failed to inflate the swim bladder (red arrowhead) and displayed a voluminous yolk sac and yolk extension (small arrow), pericardial oedema (asterisk), disrupted myoseptal melanophores (red arrow), and less developed trunk fin (black arrowhead) compared with gr^mismMO control at 5 dpf. B: Left: alkaline phosphatase staining showing reduced or absent sub-intestinal veins (SIV, arrow) in gr^ATGMO embryos as compared to well-organized SIV in WT embryos at 3 dpf; results were the same using the Tg(fli1a:EGFP)y1 zebrafish transgenic line (right). C: Histology of zebrafish gr^ATG1MO and WT embryos at 6 (sagital sections) and 5 (trasversal sections) dpf. Ov, otic vesicle; Li, liver; In, intestine; Yo, yolk sac; Sb, swim bladder. D: Top: expression of mnx1 in gr^ATG1MO and WT embryos as detected by WISH. Lateral view shows mnx1 expression in the swim bladder epithelium (sb) at 48 hpf and 5 dpf. Other domains of expression are rhombomeres 5 and 6 (r-5,6), neural tube (nt), and endocrine pancreas (p). Bottom: Effects of gr^ATG1MO and gr^ATG2MO, gr^mismMO, and gr^splicMO on swim bladder inflation in larvae at 5 dpf. Results show the percentage average (means ± SD) of individual larvae (n = 20; in triplicate) that developed the gas bladder. Means with asterisks indicate statistically significant differences from controls (P < 0.05). E: TUNEL analysis to detect apoptotic nuclei in WT, gr^ATG1MO gr^mismMO and gr^splicMO embryos at 10 and 24 hpf.

Alcian blue staining of zebrafish WT and MO-injected embryos at 6 dpf (except gr^mismMO and gr^splicMO that were analyzed at 5 dpf) showing alterations of the cartilaginous structures in gr^ATGMO morphants. Malformations are of greater severity in class II than class I embryos. Right, middle, and left images: lateral, dorsal, and ventral views, respectively. M, Meckel′s cartilage; pq, palatoquadrate; ch, ceratohyal; bb, basibranchial; e, ethmoid plate; hb, hypobranchial.

WMISH showing expression of the developmental markers, six3a and shha, in WT and gr^ATG1MO-injected embryos at 12 and/or 24 hpf. Below: Fold changes in gene expression in gr^ATG MO-injected embryos compared to WT (set at 1). Values represent the mean ± S.E. (n = 3). ***P < 0.001, differences in expression levels are significantly different.

Fold changes in gene expression of caspase 8, grp1, igf2α, mcm6 in gr^ATG1MO-injected embryos compared to WT (set at 1). Values represent the mean ± S.E. (n = 3). Asterisks indicate that expression levels are significantly different: *P < 0.05; **P < 0.01; ***P < 0.001. The inset shows the RT-PCR results as visualized by agarose gel.

Supp. Fig. S1. WMISH showing expression of the developmental markers, chd, egr2b, emx1, myod1, and pax2a, in WT and grATG1 MO-injected embryos at different developmental stages.