FIGURE SUMMARY
Title

The knockdown of maternal glucocorticoid receptor mRNA alters embryo development in zebrafish

Authors
Pikulkaew, S., Benato, F., Celeghin, A., Zucal, C., Skobo, T., Colombo, L., and Dalla Valle , L.
Source
Full text @ Dev. Dyn.

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.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Terms:
Stage Range: 2-cell to Prim-5

Phenotypes of embryos or larvae at 1, 3, and 5 dpf after treatment with grATG1MO alone or together with MOp53 as compared to control groups (WT or treated with grmismMO or grsplicMO) 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 grATG2MO are also shown.

A:grATG1MO-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 grmismMO control at 5 dpf. B: Left: alkaline phosphatase staining showing reduced or absent sub-intestinal veins (SIV, arrow) in grATGMO 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 grATG1MO 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 grATG1MO 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 grATG1MO and grATG2MO, grmismMO, and grsplicMO 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, grATG1MO grmismMO and grsplicMO embryos at 10 and 24 hpf.

Alcian blue staining of zebrafish WT and MO-injected embryos at 6 dpf (except grmismMO and grsplicMO that were analyzed at 5 dpf) showing alterations of the cartilaginous structures in grATGMO 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 grATG1MO-injected embryos at 12 and/or 24 hpf. Below: Fold changes in gene expression in grATG 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 grATG1MO-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.

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage Range: Sphere to 5-9 somites

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.

Acknowledgments
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