Additional file 3: of Successful production of genome-edited rats by the rGONAD method

Table S1. Tyr-mediated mutations in F1 (DA male/WKY female) rat. Table S2. Tyr-mediated mutations in F1 (WKY male/DA female) rat. Table S3. Tyr-mediated mutations in F1 offspring. Table S4. Coat-color phenotypes recovered from albino in WKY rat. Table S5. CRISPR/Cas9 target sequence and ssODN used. (PDF 46 kb

Supplementary Figures 1 to 8 and supplementary table S1 from The fission yeast NDR kinase Orb6 and its signalling pathway MOR regulate cytoplasmic microtubule organization during the cell cycle

Microtubule organization and reorganization during the cell cycle is achieved by regulation of the number, distribution and activity of microtubule-organizing centres (MTOCs). In fission yeast, the Mto1/2 complex determines the activity and distribution of cytoplasmic MTOCs. Upon mitosis, cytoplasmic microtubule nucleation ceases; inactivation of the Mto1/2 complex is triggered by Mto2 hyperphosphorylation. However, the protein kinase(s) that phosphorylates Mto2 remains elusive. Here we show that a conserved signalling network, called MOR (morphogenesis Orb6 network) in fission yeast, negatively regulates cytoplasmic MTOCs through Mto2 phosphorylation to ensure proper microtubule organization. Inactivation of Orb6 kinase, the most downstream MOR component, by attenuation of MOR signalling leads to reduced Mto2 phosphorylation, coincident with increased number of both Mto2 puncta and cytoplasmic microtubules. These defects cause the emergence of uncoordinated mitotic cells with cytoplasmic microtubules, resulting in reduced spindle assembly. Thus, the regulation of Mto2 by the MOR is crucial for cytoplasmic microtubule organization and contributes to reorganization of the microtubule cytoskeletons during the cell cycle

Cofilin1 Controls Transcolumnar Plasticity in Dendritic Spines in Adult Barrel Cortex

<div><p>During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure <i>in vitro</i>, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.</p></div

Effects of CFL1 expression rescue on impaired experience-dependent plasticity.

<p>(A) Design of three mutant CFL1s that are resistant to miR-CFL1_1 (resCFL1s). Seven or eight nucleic acids within the target sequence of miR-CFL1_1 were mutated such that amino acid sequence did not change. (B) An <i>in vitro</i> test of CFL1 expression rescue by each resCFL1 in rat CFL1 (WT)- and miR-CFL1_1-expressing HEK293T cells. The mock group expressed only WT CFL1. <i>n</i> = 4 for all groups. resCFL1_1, <i>p</i> = 5.8 × 10^-6; resCFL1_2, <i>p</i> = 2.2 × 10^-5; resCFL1_3, <i>p</i> = 1.3 × 10^-4 versus miR-CFL1_1 group, Tukey-Kramer’s multiple comparison test. (C) An <i>in vitro</i> test of the effect of miR-CFL1_1 on resCFL1_1 expression. <i>n</i> = 3 for both groups. <i>p</i> = 0.30, <i>t</i>-test. (D) A schematic diagram of the bicistronic lentiviral vector that co-expresses resCFL1_1 and mCherry via a P2A peptide. (E) Targeted injection of Lenti-CaMKIIα-ChR2-eYFP-miR-CFL1_1 and Lenti-CaMKIIα-mCherry-P2A-resCFL1 to D2 column identified with intrinsic signal imaging induced focal expression of ChR2-eYFP and mCherry. Scale bar, 500 μm. (F) Fluorescent images of a coronal section infected with the two vectors. Scale bar, 300 μm. (G) Confocal images of an infected area showing co-expression of ChR2-eYFP and mCherry in L2/3 neurons. Scale bar, 20 μm. (H) A representative raster plot (100 trials are shown in horizontal row) and peristimulus time histogram (PSTH) of a putative ChR2+ neuron recorded from L2/3 in D2 column of the rat showed in E. (I) A representative raster plot (50 trials) and PSTH of the same neuron with (H), showing responses to D1 whisker deflections. (J) Comparison of average responses recorded from ChR2+ neurons in D2 L2/3 of deprived rats expressing miR-CFL1_1 and resCFL1 with those recorded from ChR2+ neurons in deprived rats expressing only miR-CFL1_1 and those recorded from WT deprived rats. Data of miR-CFL1_1 deprived and WT deprived groups were the same with those shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002070#pbio.1002070.g003" target="_blank">Fig. 3D</a>. <i>n</i> = 17 units (from three rats) for the miR-CFL1_1+resCFL1 deprived group. *<i>p</i> = 0.0069, Tukey-Kramer’s multiple comparison test.</p

Schematic illustration of the circuit-specific cofilin action on barrel map plasticity.

<p>Cross-sectional view of the D1 and D2 barrel columns. L2/3 neurons in the D2 column exhibit a CFL1-dependent neuronal response increase to horizontal transcolumnar inputs from the spared D1 column. By contrast, the response decrease to ascending intracolumnar inputs from L4 is CFL1 independent. In the distal portion of the supragranular layer of the D2 column, spine densities of dendrites receiving transcolumnar inputs from the D1 column increase in a CFL1-dependent manner during sensory deprivation.</p

Effects of CFL1 KD on spine density during sensory deprivation.

<p>(A, B) Lentiviral vectors employed for co-expressing eGFP and miRNAs (A) and for expressing tdTomato (B). (C) The D1 and D2 barrel columns were identified via intrinsic signal optical imaging. (D) Virus injection was targeted to L2/3 of the D1 and D2 columns. (E) A representative parasagittal section showing expression of tdTomato (D1) and eGFP (D2). Scale bar, 300 μm. (F) Magnified view of an eGFP-expressing region in the parasagittal section shown in (E). Scale bar, 100 μm. (G) Confocal images of a rectangle region shown in (F). Scale bar, 20 μm. (H) A representative dendritic branch in the D2 column is shown that made a putative synaptic connection with a tdTomato+ D1 axonal bouton. Dendritic spines were counted that were localized at a distance less than 15 μm from an identified putative synaptic connection. A magnified view of the putative synaptic connection is shown in the inset. Scale bar, 10 μm. (I) Representative images of the dendritic branches within the distal portion in the D2 column. Scale bar, 10 μm. (J) Spine densities measured in the distal portion. <i>n</i> = 18, 17, 19, and 19 branch segments for miR-Neg non-deprived (ND), miR-Neg deprived (D), miR-CFL1_1 ND, and miR-CFL1_1 D groups, respectively. WT ND, <i>p</i> = 1.1 × 10^-4; miR-CFL1_1 ND, <i>p</i> = 1.8 × 10^-5; miR-CFL1_1 D, <i>p</i> = 0.0027 versus WT D group, Tukey-Kramer’s multiple comparison test. (K) Cumulative frequency histogram of spine density. WT ND, <i>p</i> = 0.037; miR-CFL1_1 ND, <i>p</i> = 3.6 × 10^-4; miR-CFL1_1 D, <i>p</i> = 3.6 × 10^-4 versus WT D group, Kolmogorov-Smirnov test with Bonferroni’s correction. (L−N) Same as (I−K) but of dendritic branches measured within the proximal portion. <i>n</i> = 14, 18, 16, and 10 branch segments for miR-Neg ND, miR-Neg D, miR-CFL1_1 ND, and miR-CFL1_1 D groups, respectively. WT ND, <i>p</i> = 0.86; miR-CFL1_1 ND, <i>p</i> = 0.97; miR-CFL1_1 D, <i>p</i> = 0.93 versus WT D group, Tukey-Kramer’s multiple comparison test.</p

Efficiency and specificity of CFL1 KD through miR-CFL1.

<p>(A) CFL1 mRNA KD efficiency of two miRNAs (miR-CFL1_1 and miR-CFL1_2) targeted against different sequences within the CFL1 gene, assessed by using CFL1-overexpressing HEK 293T-cells. CFL1 mRNA levels were normalized to those of the miR-Neg group. <i>n</i> = 3 for all groups; miR-CFL1_1, <i>p</i> = 2.7 × 10^-8; miR-CFL1_2, <i>p</i> = 2.8 × 10^-8 versus miR-Neg, Dunnett’s multiple comparison test. (B) KD efficiency of miR-CFL1_1 and miR-CFL1_2 for endogenous CFL1 protein, assessed by using PC-12 cells. “WT” (wild type) indicates PC-12 cells that were not infected with lentivirus. (C) Two neighboring coronal sections obtained from a miR-CFL1_1-expressing rat are shown, one stained with an antibody against CFL1 (left) and the other stained with an antibody against NeuN (right). The eYFP fluorescence image (middle) was obtained from the NeuN-stained section. Scale bar, 300 μm. (D) Magnified view of the rectangular region indicated in (C). Scale bar, 150 μm. (E, F) Same as (C and D), but of two neighboring coronal sections derived from a miR-Neg virus-injected rat. (G, H) Confocal images of eYFP+ or eYFP− region in a coronal section obtained from a miR-CFL1_1-expressing rat stained with antibodies against NeuN (blue) and CFL1 (red). Scale bar, 50 μm. (I) Percentage of CFL1+ cells in NeuN+ cells measured in miR-CFL1_1- or miR-Neg-expressing rats. <i>n</i> = 3 for all groups. *<i>p</i> = 0.0003, t-test with Bonferroni’s correction. (J) Effects of miR-CFL1 on mRNA expression of genes related to CFL1, assessed in PC-12 cells. <i>n</i> = 3 for all groups. miR-CFL1_1 of CFL1, <i>p</i> = 2.6 × 10^-7; miR-CFL1_2 of CFL1, <i>p</i> = 5.2 × 10^-7 versus miR-Neg, Dunnett’s multiple comparison test. (K) Effects of miR-CFL1 on expression of ADF protein in PC-12 cells. (L) Three successive coronal sections obtained from a miR-CFL1_1-expressing rat are shown, one stained with an antibody against ADF (middle) and another stained with an antibody against CFL1 (right). Scale bar, 300 μm.</p

Additional file 1: of i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases

Figure S1. Fetuses recovered from the i-GONAD procedure to edit the Tyr gene. Figure S2. Germline transmission of Tyr-gene-corrected allele. Figure S3. Generation of reporter knock-in mice at the Tis21 locus using the GONAD method. Figure S4. Generation of indel mutation in the Tyr locus of various mouse strains using i-GONAD. Figure S5. Generation of indel mutation in the Kit locus of C3H/HeSlc and C57BL/6NCrSlc mouse strains using i-GONAD. Figure S6. Knock-in of ssODN into Cdkn1a and Cdkn2a loci in the C57BL/6NCrl mouse strain using i-GONAD. Figure S7. Restoration of Tyr mutation of albino Jcl:MCH(ICR) mice by ssODN-based knock-in using i-GONAD with AsCpf1. Figure S8. i-GONAD-used females retain reproductive capability. Table S1. Generation of Foxe3 knock-out mice using conventional GONAD and i-GONAD approaches. Table S2. Correction of Tyr mutation by ssODN knock-in using the i-GONAD method. Table S3. Correction of Tyr mutation by zygote microinjection of CRISPR/Cas9 components. Table S4. Restoration of agouti gene expression by elimination of retrotransposon sequence using the i-GONAD method. Table S5. Generation of reporter gene knock-in mice using i-GONAD with ssDNA as donors. Table S6. Editing of the Hprt locus using i-GONAD with AsCpf1. Table S7. Correction of Tyr mutation using the i-GONAD with AsCpf1. Table S8. CRISPR target sequences and the types of gRNA used. Table S9. Sequences of the oligonucleotides used in this study. (PDF 7440 kb