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Knockout of Rrp7a in zebrafish causes brain defects resembling primary microcephaly.a Expression of rrp7a in 2 dpf zebrafish analyzed by in situ hybridization. Scale bars, 0.1 mm. b Bright-field images showing the morphology of three dpf rrp7+/+ and rrp7a−/− mutant zebrafish larvae. Scale bars, 0.2 mm. c–f Quantification of morphological differences between rrp7+/+ and rrp7a−/− zebrafish larvae. Trunk length c (P = 0.330), normalized eye diameter d (P = 4.563E-16), dorsal head size e (P = 4.243E-10) and lateral head size f (P = 4.139E-14) were quantified (n = 19, biologically independent animals). Measured lengths and areas are indicated in red lines in b (lower panel). g Haematoxylin and eosin (HE) staining of transverse tissue sections from a 3 dpf rrp7+/+ (upper panels; i–iii) and rrp7a−/− mutant (lower panels; i–iii) zebrafish larvae. Location of sections is indicated by red dotted lines in b (upper panel). Note the reduction in cells in the pallium (p) of rrp7a−/− compared with rrp7+/+ (marked in zoomed box, and quantified at right, n = 3, biologically independent animals). Data are presented as mean ± SD (P = 0.007). h hypothalamus, le lens, mc Meckel´s cartilage, mo medulla oblongata, ot optic tectum, sp subpallum. Scale bars, 0.1 mm. h mRNA rescue of rrp7a mutants. Normalized eye diameter and lateral head size were quantified and plotted as box and whiskers, whiskers min to max. i Left: northern blot (top) of total RNA from pooled zebrafish larvae analyzed with 18S rRNA and 28S rRNA probes, respectively, and qRT-PCR (bottom) displaying relative 18S/28S rRNA ratios with wt set to 1 (data average of n = 5 independent experiments). Data are presented as mean ± SD, no in cross phenotype: P = 0.296, in cross phenotype: P = 1.423E-04. Right: Northern blot analysis of processing intermediates of somatic (late) rRNA in rrp7a+/− crosses and a schematic drawing of somatic rRNA processing intermediates inferred from northern blots. Hybridization against 45S, and mature 18S and 28S rRNA were used as internal molecular markers. Unpaired, two-tailed Student’s t test. (c–g and i) and one-way ANOVA (h, normalized head size: box 1 vs 3 P = 0.035, 2 vs 4 P = 0.050). **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s.: not significant.
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