Generation of the DHPS deficiency zebrafish model. (A) The first three exons and introns of the dhps gene and selected targeting sites of two antisense morpholino oligomers and PCR primers are shown, as well as the predicted amplicon lengths. (B) RT-PCR analysis confirmed the predicted lengths of the dhps mRNA amplicons from Wt, Ctrl MO and dhps E2I2 MO larvae with two biological repeats. While the length of the PCR amplicon covering the first three exons of dhps mRNA is 375 bp (b) in Wt, Ctrl MO, and dhps E2I2 MO larvae, in the latter two, additional bands appear (a and c). Appearance of the 172-bp amplicon (c), indicating deletion of exon 2 from the dhps mRNA, and reduction in amount of the 375-bp amplicon (b), together confirm partial knockdown of dhps. Appearance of an approximately 450-bp amplicon (a) in dhps E2I2 MO larvae may be due to partial activation of a cryptic splice site induced by antisense blockage of the E2I2 splice site. (C) Analysis of protein expression in dhps knockdown zebrafish. Zebrafish embryos (2 dpf) with knockdown by dhps E2I2 MO or dhps AUG MO were analyzed by Western blot for expression of DHPS, eIF5A^TOTAL, eIF5A^HYP, and total protein (as visualized by REVERT™). (D) Densitometric data for the relative expression of DHPS, eIF5A^TOTAL, and eIF5A^HYP, normalized on the basis of total protein as quantified by REVERT™ staining

Phenotypic analysis of dhps knockdown zebrafish at five days post-fertilization (5 dpf). (A) Lateral view photographs of representative Wt, Ctrl MO, dhps E2I2 MO, and dhps AUG MO larvae, scale bar: 0.5 mm. (B) Chi-square analysis revealed significant differences between knockdown and control groups with regard to the percentages of larvae exhibiting normal development, mild and moderate dysmorphology (p<0.0001)

Partial rescue of dhps knockdown larvae by expression of dhps mRNA at 1 day post-fertilization (1 dpf). (A) Map of pIVT construct with zebrafish dhps cDNA. (B) mRNA overexpression partially shifted the more severe phenotypic categories of dhps knockdown to less severe categories. Chi-square analysis showed significant differences between control, knockdown and rescue groups with regard to the percentages of embryos exhibiting normal development, mild and moderate dysmorphology (P<0.0001). (C) Definition of observed phenotypic categories and sample size of each group

LFP recording of dhps knockdown larvae. (A) Snapshot of LFP signal recorded from 4-dpf Wt, Ctrl MO and dhps E2I2 MO larvae. (B) One-way ANOVA revealed that the frequency of ictal-like events in dhps knockdown larvae was significantly increased compared to control larvae (p ≤ 0.02). Each recording was performed over a period of 20 min. (C) Each pie chart shows percentage of larvae with more than 3 ictal-like events during 20 min of recording. While in Ctrl MO larvae less than 10% of larvae had more than 3 ictal-like events, in dhps E2I2 MO-injected larvae, more than 43% of larvae had more than 3 ictal-like events

GABAergic neuronal dendritic arborization. (A) Larval head with location of optic tectum (blue) and neuropil (light yellow) where that GABAergic neurons project their arbors. (B) Confocal microscopy revealed that dhps knockdown larvae have less complex dendritic arborization in the GABAergic neurons. (C) GABAergic neuronal arbors were quantified in zebrafish larvae at three different ages (3, 5, and 7 dpf). At all three developmental ages, via one way ANOVA there was a significant reduction in the number of neuronal arbors in the dhps knockdown larvae (dhps E2I2 MO) versus both wild-type control larvae (Wt) and control MO-injected larvae (Crtl MO) (see also Table 1 below)