EMBO/soda-nel huggingface dataset:
PTENε suppresses tumor metastasis through regulation of filopodia formation (2021)
Figure 7

Figure 7. PTENε suppresses the aggressiveness of cancer cells.

A. Immunofluorescence staining of F-actin in PTENε or PTENε Y210L overexpressed Hela PTEN-/- cells and control cells. PTENε-GFP, PTENε Y210L-GFP, or mock construct was introduced into HeLa PTEN-/- cells respectively. The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm (left panel). White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane.

B. Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t-test. ****, p<0.001.

C. Representative images of transwell migration and invasion assays in Hela PTEN-/- cells and H4 cells stably transfected with PTENε, PTENε Y210L, or an empty control (magnification, 200x). The scale bars represent 50 μm.

D. Statistical analysis of transwell migration and invasion assays in Hela PTEN-/- cells and H4 cells stably transfected with PTENε, PTENε Y210L or an empty control (magnification, 100x; five random per well). Data are presented as the mean ± SEM based on three independent experiments and were analyzed with the unpaired t-test. *, p<0.01. ****, p<0.001.

E. Adenine Base Editing mediated endogenous PTENε knockout in Hela cells. Immunoblotting analyses were used to verify the level of PTENε loss in Hela cells.

F. Representative images of transwell migration and invasion assays in Hela PTENε+/- cells at 200× magnification (left panel, the scale bars represent 50 μm). (right panel) Migrated or invaded cells were counted at five random fields at 100× magnification per well to assess cell migration and invasion ability. Data are presented as the mean ± SEM based on three independent experiments and were analyzed with the unpaired t-test. ***, p<0.001; ****, p<0.0001.

G. Left panel: immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells. Middle panel: representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model (n=4). H & E, hematoxylin-eosin staining. The scale bars represent 600 mm (upper panel) and 500μm (lower panel) separately. Right panel: the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules (data are presented as mean ± SD based on three independent replicates). The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group. ****, p<0.0001 in the unpaired t-test.

Expanded View Figure legends
MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis (2020)
Figure 3

Figure 3. Crossovers are positively associated with SNP density at the local scale.

A. SNPs/kb in physical windows of increasing size (kb) around crossover midpoints (red), or for matched randomly chosen positions (grey). Printed above the plot for each window are circles coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population (Col×Ws, Col×Bur (Lawrence et al, 2019), Col×Ct, Col×Ler (Serra et al, 2018b; Rowan et al, 2019) and Col×CLC) is printed above each plot, and the number of positions analysed is printed inset.

B. As for A, but analyzing the indicated Col/Ler transition and transversion polymorphisms around Col×Ler crossovers (Rowan et al, 2019) (red), or the same number of random positions (grey).

C. As for A, but analysing Col/Ler SNPs/kb around crossovers from wild type, HEI10-OE, recq4a recq4b or HEI10-OE recq4a recq4b (Serra et al, 2018b). All populations were generated from Col×Ler hybrids. Printed beneath are the estimated average numbers of Class I and Class II crossovers per meiosis for each genotype, measured via genotyping-by-sequencing (Serra et al, 2018b; Ziolkowski et al, 2017). To the right are plots showing the significance of SNPs/kb differences between crossover sets, across the windows tested (Bonferroni adjusted t-tests).
MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis (2020)
Figure 4

Figure 4. Remodelling of the crossover landscape in msh2.

A. Histograms of observed crossovers per F2 individual in the indicated wild type and msh2 populations, from Col×Ct, Col×Ler and Col×CLC crosses (blue). The Poisson expectation is plotted in red. Data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b F2 populations are also shown for comparison (Serra et al, 2018b; Ziolkowski et al, 2017). Mean values are indicated by black dotted lines.

B. Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes, with mean values shown by horizontal dashed lines. Data are shown for wild type (red) and msh2 (blue) crossovers generated from Col×Ct, Col×Ler and Col×CLC hybrids. SNPs per 200 kb are shaded in colour (blue=Col/Ct, green=Col/Ler, pink=Col/Cvi). The positions of telomeres (TEL) and centromeres (CEN) are labeled. The number of crossovers analysed is printed inset.

C. Data as for B, but analyzing crossovers in wild type (red) and msh2 (blue), or SNPs along proportionally scaled chromosome arms orientated from telomeres (TEL) to centromeres (CEN). Crossover data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b Col×Ler F2 populations are also shown (Serra et al, 2018b; Ziolkowski et al, 2017).
MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis (2020)
Figure 5

Figure 5. Crossover association with polymorphism is reduced in msh2 at the local scale.

A. Histograms showing the mean distance (megabases, Mb) of observed double crossovers (DCO, red vertical line), compared to 2,000 matched sets of randomly generated distances (RAN, black). The mean distance of the random sets is shown in by the vertical grey line.

B. SNPs/kb values were calculated in physical windows of increasing size (kb) around crossover midpoints in wild type (red) and msh2 (blue). The same values were calculated for matched randomly chosen positions (grey). Printed above the plot for each window are circles that are coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population analysed (Col×Ct, Col×Ler, Col×CLC) and its genome average SNP frequency (SNPs/kb) is printed above the plots and the number of crossover positions analysed is printed inset. Beneath are plots showing the significance of SNPs/kb differences around crossovers between the indicated genotypes.
MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis (2020)
Figure 6

Figure 6. MSH2 accumulates on meiotic chromatin during early prophase I.

A. Representative DAPI-stained spreads of pachytene, diakinesis, metaphase-I, dyad and tetrad meiotic stages, for wild type and msh2, in Col×Ler or Col×CLC hybrid backgrounds. B. Male meiocytes immunostained for MSH2 (red) and ASY1 (green), and stained for DAPI (blue) in wild type (Col) or msh2.

C. As for B, but immunostaining wild type male meiocytes for MSH2 (red), MSH4 (green) and staining chromatin with DAPI (blue).

Data information: All scale bars indicate 10 μm.
MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis (2020)
Figure 7

Figure 7. Crossover remodelling via juxtaposition of heterozygous and homozygous regions is MSH2 dependent.

A. Crossover frequency (black) and SNP density (purple) per 200 kb measured from the Col×Ct F2 population. The position of the centromere (CEN, vertical dashed line) and 420 FTL transgenes (red and green vertical lines) used to measure crossover frequency are indicated.

B. Plots of chromosome 3 showing positions of Col/Col (black) or Col/Ct (purple) homozygous or heterozygous genotypes at the indicated marker positions (circles). The positions of the 420 FTL transgenes are indicated (red and green vertical lines).

C. 420 crossover frequency (cM) in the HOM-HOM, HET-HET, HET-HOM, HOM-HET genotypes shown in B, in either wild type or msh2. Values for replicate individuals are plotted, in addition to the mean (red).

D. As for C, but comparing genotypes in wild type or HEI10-OE backgrounds. Values for replicate individuals are plotted, in addition to the mean (red).
The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse (2021)
Figure 1

Figure 1. FOXM1 is expressed in the human embryonic cerebral cortex and induces formation of gyrus-like structure.

(A) The expression pattern of FOXM1 in fetal human cortex at GW15. VZ, ventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone. Right panels show higher magnification images. Arrows show that FOXM1+cells were colocalized with SOX2+ cells. Scale bars, 100μm (left); 50μm (right).

(B) Staining for NESTIN and FOXM1 in human neural progenitor cells cultured in vitro. Down panels show higher magnification images. Scale bars, 50μm(up); 10μm(down).

(C) Staining for SOX2 and FOXM1 in human neural progenitor cells cultured in vitro. Down panels show higher magnification images. Dashed shapes show that FOXM1+cells were colocalized with SOX2+ cells in human neural progenitor. Scale bars, 50μm(up); 10μm(down).

(D) Staining for Pax6 in E15.5 mice after IUE at E13.5. Arrowheads indicate Pax6+ cell in enlarged SVZ. Right panels show higher magnification images. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars,50μm(left); 20μm (right).

(E) Graph shows the percentage of Pax6+ cells among GFP+ cells (n = 5 each group; two-tailed

unpaired t-test, control vs. hFOXM1, P=0.0032; mFoxm1vs. hFOXM1, P=0.0121).

(F) Graph shows the percentage of Pax6+ cells among GFP+ cells in enlarged SVZ (n = 5 each group; two-tailed unpaired t-test, control vs. hFOXM1, P<0.0001; mFoxm1vs. hFOXM1, P<0.000 1).

(G­) Staining for upper neuron marker Cux1 in P3 mice after IUE at E13.5. Arrowhead and asterisk indicate deformation of the cortical surface at regions containing electroporated cells. Right panels show higher magnification images. Arrows show GFP+ Cux1+ double-positive cells. Scale bars,100μm(left); 20μm (right).

Data information: At least three biological replicates are shown. Data are represented as means ± S.E.M. Two-tailed unpaired student's t test, *P < 0.05. **P < 0.01. ***P < 0.001.
The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse (2021)
Figure 2

Figure 2. Cortical expansion and folding are induced by human FOXM1 expression in mice.

(A) Representative images of whole brains from wild-type (WT) and hFOXM1f/+-NesCre (hFOXM1-cKI) mice pups at P0. Scale bar,100μm.

(B) Quantification of cortical width and length at P0 (WT n=7; hFOXM1-cKI n=7; two-tailed

unpaired t-test, P= 0.0361for width, P= 0.2866 for length).

(C) Quantification of brain/body weight at P0 (WT n=9; hFOXM1-cKI n=9; two-tailed unpaired t-test, P= 0.0004).

(D) Quantification of cortex thickness at P0 (WT n=7; hFOXM1-cKI n=11; two-tailed unpaired t-test, P= 0.0002).

(E) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice folding structure. Asterisks indicate gyrus and arrows indicate sulcus structures. A1, A2, B1and B2 (DAPI) are high-magnification images. Scale bars, 200μm(left); 100μm(right).

(F) Quantification of GI of WT and hFOXM1-cKI mice pups at P0. The quantification of the GI was analyzed in pre-selected hFOXM1-cKI mice with folding. (n = 6 each group; two-tailed

unpaired t-test, P= 0.0010).

(G）Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice. White dash lines represent the positive area. Scale bar,100μm.

(H) Graph shows the number of Satb2+cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0086).

(I) Graph shows the number of Ctip2 +cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0025).

(J) Staining for DAPI in P3 WT and hFOXM1-cKI mice. Asterisks indicate gyrus and arrows indicate sulcus structures. Scale bars, 500μm .

(K) Staining for Cux1 in P0 WT and hFOXM1-cKI mice. Scale bar,100μm.

(I) Graph shows the number of Cux1+cells in 4×104µm2 at P0 (WT n=3; hFOXM1-cKI n=3; two-tailed unpaired t-test, P= 0.0451).

Data information: At least three biological replicates are shown. Data are represented as means ± S.E.M. Unpaired two-tailed Student's t test. *P < 0.05. **P < 0.01. ***P < 0.001.
The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse (2021)
Figure 3

Figure 3. Cortical basal progenitor cells are increased in FOXM1 conditional knockin mice.

(A) Staining for Pax6 and p-Vim in E15 WT and hFOXM1-cKI mice. The right A and B are high-magnification images. Arrows indicate double positive of Pax6+ p-Vim+ cells with cells with a basal process (arrowheads). Scale bars, 50μm (left); 5μm (right).

(B) Graph shows the percentage of Pax6+ cells in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0002).

(C) Graph shows the number of Pax6+ p-Vim+ cells in 4x10^4 μm2 in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0003).

(D) Staining for Sox2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.

(E) Graph shows the number of Sox2+ cells in 4x104 μm2 in enlarged SVZ (WT n=3; hFOXM1-cKI n=4; two-tailed unpaired t-test, P=0.0292).

(F) Staining for Tbr2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.

(G) Graph shows the number of Tbr2+ cells in 4x104 μm2 in enlarged SVZ (WT n=6; hFOXM1-cKI n=9; two-tailed unpaired t-test, P=0.0003).

(H) Staining for HOPX in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.

(I) Graph shows the number of HOPX+ cells in 4×104 μm2 in enlarged SVZ (n = 4 each group; two-tailed unpaired t-test, P= 0.0044).

(J) Staining for Ki67 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.

(K) Graph shows the percentage of Ki67+ cells in enlarged SVZ (n = 4 biological replicates; two-tailed unpaired t-test, P=0.0038).

(L) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1-cKI mice. White arrows represent BrdU and EdU double-positive cells. Scale bars, 50μm(left);20μm(right).

(M) Graph shows the percentage of BrdU and EdU double-positive cells in E15 WT and hFOXM1-cKI mice (n = 6 each group; two-tailed unpaired t-test, P=0.0413).

Data information: At least three biological replicates are shown. Data are represented as means ± S.E.M. Unpaired two-tailed Student's t test. *P < 0.05. ***P < 0.001.
The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse (2021)
Figure 4

Figure 4. Human FOXM1 unique sequence promotes the generation of basal progenitor cells.

(A) Structure of human FOXM1gene, mouse Foxm1 gene, mutant of human gene FOXM1 (hFOXM1▲exon9) and mutant mouse gene Foxm1 (mFoxm1+hexon9).

(B) Cell immunostaining for HOPX in control, hFOXM1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm.

(C) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=3; hFOXM1-FL n=6; hFLΔexon9 n=5; one-way ANOVA, F (2, 12) = 29.13, control vs. hFOXM1, P=0.0002;

control vs. hFOXM1Δexon9, P=0.9080).

(D) Cell immunostaining for HOPX in control, mFoxm1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm.

(E) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=4; mFoxm1-FL n=6; mFL+h exon9 n=4; one-way ANOVA, F (2, 11) = 8.163, control vs. mFoxm1, P= 0.4747,

control vs. mFoxm1+h exon9, P= 0.0066).

(F) Western blot analysis for basal RG marker (HOPX) in control, hFOXM1 and mutants in mice primary neural progenitors 3 days after lentiviral infection. β-actin was detected as loading control.

(G) Statistics of relative intensity of HOPX (n = 3 biological replicates, one-way ANOVA, F (2, 6) = 5.084, control vs. hFOXM1 P=0.0435, control vs. hFOXM1Δexon9 P= 0.3991 for HOPX).

(H) Western blot analysis for HOPX in control, mFoxm1 and mutants in mice primary neural progenitors 3 days after lentiviral infection. β-actin was detected as loading control.

(I) Statistics of relative intensity of HOPX (n = 3 biological replicates; one-way ANOVA

F (2, 6) = 8.111, control vs. mFoxm1, P= 0.6024, control vs. mFoxm1+hexon9, P=0.0192 for HOPX).

(J) Staining for Tbr2 in E15.5 mice after IUE at E13.5. Down panels show higher magnification images. White arrows represent GFP and TBR2 double-positive cells. Scale bars, 50μm.

(K) Graph shows the percentage of GFP+Tbr2+/GFP+ in enlarged SVZ (n = 3 each group; one-way ANOVA, F (3,8) =8.787, P=0.0065).

Data information: At least three biological replicates are shown. Data are represented as means ± S.E.M. one-way ANOVA was used for statistical analysis. n.s., no significant, *P < 0.05. **P < 0.01. ***P < 0.001.
The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse (2021)
Figure 5

Figure 5. FOXM1 regulates proliferation of neural precursor cells through Lin28a.

(A) Gene ontology analysis for up-regulated genes based on RNA-seq data in E15 hFOXM1-cKI mice.

(B) Gene ontology analysis for down-regulated genes based on RNA-seq data in E15 hFOXM1-cKI mice.

(C) Volcano plots shows the gene expression states. The yellow dots are the up-regulated genes, and the blue dots are the down-regulated genes. The Lin28a gene (red dot) is significantly up-regulated.

(D) Western blot analysis for Lin28a in cortex of E15 WT and hFOXM1-cKI mice. β-actin was detected as loading control.

(E) CHIP analysis for hFOXM1,mFoxm1 and hFOXM1▲exon9 binding to the region of the Lin28a promoter in the N2A cells. (n=3 biological repeats).

(F) Staining for Sox2 in E15.5 mice after IUE at E13.5. Arrowheads indicate Sox2+ cell in enlarged SVZ. Scale bar, 50μm.

(G) Graph shows the percentage of Sox2+ cells among GFP+ cells in enlarged SVZ (n = 3 each group; two-tailed unpaired t-test, P=0.0154).

(H) Graph shows the percentage of Sox2+ cells among GFP+ cells (n = 3 each group; two-tailed unpaired t-test, P=0.0018).

(I) LIN28A-shRNA1 rescues the neuronal progenitor cell increase in hFOXM1-cKI mice. Brain slice staining for Sox2 two days after IUE at E13. Scale bar, 50μm.

(J) Graph shows the percentage of Sox2+ cells among GFP+ cells (n=3 each group; one-way

ANOVA, F (2, 6) = 11.32, Control-GFP(WT) vs. Control-GFP (cKIf/+), P= 0.0105; control-GFP (WT) vs. LIN28Ash1-GFP (cKIf/+), P= 0.7712).

Data information: At least three biological replicates are shown. Data are represented as means ± S.E.M. Student's t test: *P<0.05, **P<0.01.
The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse (2021)
Figure 6

Figure 6. LIN28A is downstream target of hFOXM1 and rescues the phenotype hFOXM1 knockout in human progenitor cell.

(A) Schematic showing the method used to generate FOXM1+/+ and FOXM1-/- human NPCs.

(B) Representative cases of FOXM1+/+ and FOXM1-/- NPCs clones. Scale bar, 20μm.

(C) Graph shows the number of cells per clone in FOXM1+/+ and FOXM1-/- human NPCs (FOXM1+/+ n=12; FOXM1-/- n=13; at least three biological replicates. two-tailed unpaired t-test, P= 0.0014).

(D) Overexpression of FOXM1 or LIN28A rescues the human neural progenitor cell reduction in FOXM1-/- cell lines. Western blot analysis for SOX2, PAX6 and HOPX. β-actin was detected as loading control.

(E) Statistics of relative intensity of SOX2, PAX6 and HOPX (PAX6: n=3 biological replicates; one-way ANOVA, F (2,6) = 8.716, FOXM1-/-(Control) vs. FOXM1-/-(hFOXM1), P= 0.0173, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.0476. SOX2: n=5 biological replicates; one-way ANOVA, F (2, 12) = 14.72, FOXM1+/+(Control) vs. FOXM1-/-(Control), P= 0.0015, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P=0.9906. HOPX: n=4 biological replicates, one-way ANOVA F (2, 9) = 25.28, FOXM1+/+(Control) vs. FOXM1-/- (Control), P= 0.0018, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.2211).

(F) The model for the role of hFOXM1 in cerebral cortex. Human FOXM1 expression causes neural precursor cells increase through up-regulating the level of LIN28A. The increased neural precursor cells subsequently increase in the number of new born neurons and induce cortical extension and folding in mice.

Data information: At least three biological replicates are shown. Data are represented as means ± S.E.M, unpaired student's t test, one-way ANOVA. n.s., no significant, *P < 0.05, **P < 0.01, ***P < 0.001.

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The E3 ligase Highwire promotes synaptic transmission by targeting the NAD synthesizing enzyme Nmnat (2019)
Figure 1

Figure 1. Excess presynaptic dNmnat is sufficient to depress evoked release without altering NMJ growth. (A) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), dNmnat (DVGlut-Gal4 > UAS-dNmnat), or Wnd (DVGlut-Gal4 > UAS-Wnd). (B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 13 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=36, F=12.66, p<0.0001. UAS-RFP vs UAS-dNmnat p < 0.0001 (****), UAS-RFP vs UAS-Wnd p = 0.0093 (**)]. (C) Quantification of mean (±SEM) mEPSP amplitudes, in which 75 consecutive spontaneous events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=11.66, p = 0.0001. UAS-RFP vs UAS-dNmnat p=0.994 (NS), UAS-RFP vs UAS-Wnd p = 0.0003 (***)]. (D) Quantification of quantal content, which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude, and then averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=9.286, p = 0.0006. UAS-RFP vs UAS-dNmnat p=0.0015 (**), UAS-RFP vs UAS-Wnd p = 0.0003 (***)]. (E) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in the electrophysiology experiments in A-D. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). (F) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [UAS-RFP n = 23, UAS-dNmnat n = 21, UAS-Wnd n= 20 NMJs. 1-way ANOVA w/Tukey's multiple comparisons, DF=63, F=68.31, p < 0.0001. UAS-RFP vs UAS-dNmnat p=0.9818 (NS), UAS-RFP vs UAS-Wnd p < 0.0001(****)].