Figure 1.

Generation of Pclo^gt/gt mutant animals. A, Sleeping beauty transposon mutagenesis was used to generate gene trap (gt) piccolo KO rats. The transposon element was inserted into exon 3 of the piccolo genomic sequence and caused a stop in the reading frame. Adapted from Ackermann et al. (2019). B, Pairs of heterozygous (Pclo^wt/gt) males and females produced litters with Mendelian distribution. Pie chart demonstrates the birth rates of homozygous WT (Pclo^wt/wt), homozygous gene trap mutation (Pclo^gt/gt), and heterozygous (Pclo^wt/gt) pups (data collected from 63 litters: Pclo^wt/wt = 195 pups; Pclo^wt/gt = 387 pups; Pclo^gt/gt = 137 pups). C, Western blot analysis of brain lysates prepared from postnatal day 2 (P2) animals to confirm the loss of full-length Piccolo protein from the brain. A band of the Piccolo-corresponding size of 560 kDa is detectable in lysates prepared from Pclo^wt/wt and Pclo^wt/gt animals, as well as bands between 70 and 420 kDa, representing smaller Piccolo isoforms. Nearly all of these are absent in Pclo^gt/gt brain lysates (data are representative of three independent experiments), although smaller bands between 100 and 70 kDA are still present in Pclo^gt/gt brain lysates. D, Image of postnatal day 1 (P1) littermates. E, Quantification of the body length of P0–P2 Pclo^wt/wt and Pclo^gt/gt pups (Pclo^wt/wt = 5.5 ± 0.077 cm, n = 23; Pclo^gt/gt = 5.15 ± 0.070 cm, n = 27; 6 independent litters; unpaired t test, t₍₄₈₎ = 3.394, **p = 0.0014). F, Quantification of the body weight of P0–P2 Pclo^wt/wt and Pclo^gt/gt pups (Pclo^wt/wt = 8.09 ± 0.203 g, n = 39; Pclo^gt/gt = 7.31 ± 0.166 g, n = 35; 12 independent litters; unpaired t test, t₍₇₂₎ = 2.937, **p = 0.0044). G, Image of brains dissected from P1 Pclo^wt/wt and Pclo^gt/gt pups. H, Quantification of the brain weight of P0–P2 Pclo^wt/wt and Pclo^gt/gt pups (Pclo^wt/wt = 0.293 ± 0.00533 g, n = 7; Pclo^gt/gt = 0.289 ± 0.00758 g, n = 3; 3 independent litters; unpaired t test, t₍₈₎ = 0.423, p = 0.683). I, Nissl-stained sagittal sections from P2 rat brains show no overt differences between Pclo^gt/gt and Pclo^wt/wt pups. Animals used in this experiment are as follows: wt1; ko1. J, Image of 4% PFA-perfused brains from Pclo^wt/wt and Pclo^gt/gt animals at 3 months of age. K, Quantification of the brain weight showing that Pclo^gt/gt brains are significantly lighter than Pclo^wt/wt brains (Pclo^wt/wt = 2.098 ± 0.074 g, Pclo^gt/gt = 1.435 ± 0.021 g; n = 6, Mann–Whitney U test, U = 0, **p = 0.0022). L, Nissl-stained sagittal sections from 3-month-old rat brains reveal microcephaly in Pclo^gt/gt compared with Pclo^wt/wt. Ventricles are larger, and cerebellum, pons, cerebrum, and subcortical regions are smaller. Animals used in this experiment are as follows: wt11; ko13. Scale bars: G, J, 1 cm; I, 1 mm; L, 0.5 cm. Error bars indicate SEM.