p53 MO Attenuates Cell Death Induced by MOs against Novel Target Genes with Divergent Effects on Craniofacial Phenotypes (A) Brightfield and fluorescent images of 1 dpf embryos injected with MOs targeted against three novel proteins: SP2054, SP2063 (two different MOs for each target: MO1 and MO2), and SP2035. Brightfield images depict observed anterior morphological defects: anterior-ventral concavity (black arrows) and depressed hindbrain (black arrowhead). Fluorescent images indicate areas of apoptosis visualized by live embryo acridine orange staining. Insets show a magnification of apoptotic foci in the head region. Upon p53 coinjection (+p53 MO), there is a significant attenuation of these phenotypes. These results are quantitated for each target in the right graph panel. (B) Brightfield images of 4.5 dpf embryos stained with Alcian Blue to visualize representative craniofacial phenotypes. Arrowheads indicate Meckel′s cartilage (M), arrows indicate ceratohyal arch (Ch). Stars indicate the five branchial arches (Cb). Upon MO targeting of the three novel genes, two types of craniofacial phenotypes were observed: mispatterning of the Meckel′s cartilage and ceratohyal (mispatterned phenotype), and loss of all branchial arches, ceratohyal and severe hypoplasia of the Meckel′s cartilage (severe loss phenotype). (C) Quantitation of the p53 MO effect on craniofacial phenotypes induced by MO targeting of three novel genes. Targeting of SP2054 with MO1 induced a high level of severe loss phenotype, while SP2054 MO2 showed mainly a mispatterned phenotype. None of these craniofacial phenotypes were affected by p53 MO. SP2035 knockdown induced both types of craniofacial abnormalities and the proportion of these was not affected by p53 co-knockdown. SP2063 MOs MO1 and MO2 induced a craniofacial mispatterning phenotype that was partially rescued by p53 MO, suggesting that this craniofacial phenotype is a secondary effect of off-target neural death.
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