Knockdown of ASS1 causes morphological brain alterations with neuronal cell loss and reduced brain size. At the 1-cell stage, wildtype or tg(elavl3:eGFP) transgenic zebrafish larvae were injected with either MO targeting the zebrafish homologue of ASS1 or mock or combined injection of ASS1-targeting MO together with the human ASS1 mRNA was performed. Subsequently, zebrafish larvae were imaged using a binocular (fluorescence) microscope (MZ16F, Leica) at 1 or 3 dpf, respectively (A-C). Lateral view with anterior to the left (A, B) or top view with anterior to the left (C). Relative brain size of indicated cohorts in comparison to the mock-injected control (D). Alternatively, injected zebrafish larvae were subjected to quantification of L-citrulline concentration by HPLC (E). MO-injected wildtype zebrafish larvae exhibited reduced brain size and disorganized mes- and diencephalic structures (arrow head) with dissolved midbrain-hindbrain-boundary (asterisk), most prominent at 1 dpf (A, B). For comparative reasons, brain structures have also been marked in the wildtype and mRNA-treated cohort (A). Consistently, tg(elval3:eGFP) transgenic larvae injected with ASS1-targeting MO showed reduced brain size as indicated by eGFP-expressing neurons, predominantly involving the optic tectum (C), corresponding to approximately 80% of the mock-injected transgenic control (D). Intriguingly, coinjection of MO together with human ASS1 mRNA (450 pg/μL) rescued the morphological phenotype and restored brain size (A-D). Moreover, zebrafish larvae coinjected with MO and human ASS1 mRNA exhibited normalized L-citrulline concentration (E). Data are expressed as mean +/− SD of relative brain size (percentage of normal) in comparison to negative control (D) or μmol per g protein (E; whole body lysates; N = 5 with 50 larvae per group and experiment; ANOVA, *P < 0.05).
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