Skip to main content
. 2013 Jul 11;93(1):173–180. doi: 10.1016/j.ajhg.2013.05.021

Figure 3.

Figure 3

Morphology of Embryos Injected with the WT or Mutant RIT1 mRNA

In vitro transcription of each mRNA was performed with the mMESSAGE mMACHINE kit (Applied Biosystems) according to the manufacturer’s instructions. Synthesized mRNAs were purified with G-50 Micro Columns (GE Healthcare) and subsequently adjusted to a 300 ng/μl concentration for microinjection. Approximately 1 nl (300 pg) of RNA in water with 0.2% phenol red was injected into the cytoplasm of 1-cell-stage zebrafish embryos. Injected embryos were incubated at 28°C until observation.

(A) At 11 hpf, the shapes of the embryos injected with the WT sense or antisense mRNA were round, a normal morphology as observed in the uninjected embryos. In contrast, embryos expressing mutations (c.236A>T [p.Gln79Leu], c.242A>G [p.Glu81Gly], and c.284G>C [p.Gly95Ala]) are oval and compressed along the dorsal-ventral axis, indicative of a gastrulation defect. Note that cells have a hump in the head region at the anterior end of the body axis, the earliest manifestation of a craniofacial defect.

(B) Lateral views at 48 hpf are shown. Embryos expressing mutations (c.236A>T [p.Gln79Leu], c.242A>G [p.Glu81Gly], and c.284G>C [p.Gly95Ala]) formed swollen yolk sacs equally along the anterior posterior axis but did not show narrowing in the caudal half, which was clearly visible in the uninjected embryos and in those injected with the WT sense or antisense mRNA. In the craniofacial area, misshapen head and jaw structures and small eyes with hypoplasia on the ventral side were observed (middle panel); these phenotypes are consistent with the gastrulation defect. Shapes of the hearts (highlighted by red dotted lines) are shown in the right panel at a higher magnification. Normal looping of the heart tube and correct formation of two distinct chambers are observed in embryos injected with the WT sense or antisense mRNA. When mutations (c.236A>T [p.Gln79Leu], c.242A>G [p.Glu81Gly], and c.284G>C [p.Gly95Ala]) were expressed, looping was incomplete, resulting in stretched straight heart tubes. Constrictions at the atrial-ventricular canal are obscure, and the heart chambers are hypoplastic. Abbreviations are as follows: A, atrium; and V, ventricle.