Fig. 4

A 30% reduction in rps4x disrupts brain formation in zebrafish embryos.
A Schematic shows the workflow for microscopic analysis of morpholino-based inhibition and rescue of rps4x expression. B Schematic of the rps4x coding region in zebrafish. The position of the splice bocking morpholino is denoted (red line, MO), as is the position of the forward and reverse primers (red arrow heads) used for RT-PCR analysis of knockdown. C RT-PCR analysis of rps4x transcript in morpholino-injected embryos 48hpf show increased abundance of intron-containing transcripts is associated with lower levels of normal rps4x transcript. Based on these data 0.3 µM morpholino reduced normal rps4x transcript abundance ~30%. The associated sequencing trace showing retention of intron 4 is shown. D Live confocal images of 48hpf embryos stained with BODIPY-ceramide show morpholino inhibition of rps4x disrupts formation of the mid and hindbrain. The ventral hindbrain (VHb), the neuroepithelial fold (NE), and first ventricular space (V1) are labeled. n = 20–25 embryos per condition from 3 biological matings. E Live images of BODIPY-ceramide stained embryos co-injected with 0.3 M morpholino and either wild type¹³ or p.(Arg221Gln) variant containing rps4x mRNA shows only WT mRNA improves morphant phenotypes. n = 15–20 embryos per condition from 3 biological matings. F Live images of BODIPY-ceramide stained wild type embryos injected with either wild type or p.(Arg221Gln) variant containing rps4x mRNA show the variant containing mRNA disrupts normal brain development. n = 18–20 embryos per condition from 3 biological matings. Percent values indicate number of embryos with the pictured phenotypes. For all images the upper panels represent the dorsal view of the brain and lower panels the ventral view. Scale bar = 10 µM.