EMBO/soda-nel huggingface dataset:
ALX1-related Frontonasal Dysplasia Results From Defective Neural Crest Development and Migration (2020)
Figure 4

Figure 4: NCC apoptosis, cell cycle and differentiation.

A. Homozygous ALX1165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1165L/165L NCC (black). The data on the left represents the mean percentage of Annexin V positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for ten minutes. Representative experiment for each condition is shown. Data is represented as pooled mean ± SEM of three independent experiments. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.

. *: Significantly different from the basal apoptosis rate: p=3x10-12 between control NCC basal apoptosis and induced apoptosis, and between ALX1165F/165F NCC basal apoptosis and induced apoptosis. **: Significantly different from control NCC (p=0.0004).

B. Expression levels of cyclins CCNA2 (blue) and CCND1 (orange) in NCC at passages 2 and 3 of ALX1165L/165L and ALX1165F/165F NCC. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. *: Significantly different from control NCC at passage 2 (p=0.001 between control and ALX1165F/165F NCC at passage 2 for CCNA2, p=0.0052 for CCND1). **: Significantly different from control NCC at passage 3 (p=0.0494 between control and ALX1165F/165F NCC for CCNA2, p=0.0008 for CCND1).

C. Fluorescence Activated Cell Sorting (FACS) experiments showed that control ALX1165L/165L NCC (green) exhibited increased expression of mesenchymal markers CD90, CD105, and CD73 with culture time (passages 1 through 4), whereas homozygous ALX1165F/165F NCC (blue) showed a consistent expression of the markers expressed at passage 1 throughout. Further, control ALX1165L/165L NCC showed a downregulation of CD57 expression with culture time, while ALX1165F/165F NCC maintained the same level of CD57 across passages. Data are presented as the mean percentage of positive-stained cells across passage numbers. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. *: Significantly different from control NCC. For CD90, p=0.0013 at passage 3 and p=0.0207 at passage 4. For CD105, p=0.0016 at passage 2, p=0.00004 at passage 3 and p=0.0021 at passage 4. For CD73, p=0.0060 at passage 2, p=0.00004 at passage 3 and p=0.0114 at passage 4. For CD57, p=0.0026 at passage 2, p=0.000003 at passage 3 and p=0.000007 at passage 4.
ALX1-related Frontonasal Dysplasia Results From Defective Neural Crest Development and Migration (2020)
Figure 5

Figure 5: ALX1165F/165F NCC show a migration defect and a difference in BMP secretion.

A. Mutant ALX1165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L/165L NCC (black). Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24-hour period. For fluorescent pictures, cells were stained in serum free media containing 3.6µM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 hours using a Keyence BZ-X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200µm. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.*: Significantly different from control NCC (p<0.0001).

B. Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1165F/165F NCC (blue) secrete less BMP2 and more BMP9 compared to control ALX1165L/165L NCC (green). Data are represented as pooled mean ± SEM of three clones from each genotype. Statistical significance was determined with an ANOVA test. A p-value <0.05 was considered significant. *: Significantly different from control NCC (p =0.0424 for BMP2 and p=0.0192 for BMP9).

C. Addition of soluble BMP2 or CV2, a BMP9 antagonist, to the culture medium could partially rescue the migration defect of ALX1165F/165F NCC. At the beginning of the assay, 10, 50, or 100 ng/ml of soluble BMP2, CV2, or a combination of the two at 100 ng/ml each were added to the culture medium, and the cells were monitored over the next 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar: 400µm.
ALX1-related Frontonasal Dysplasia Results From Defective Neural Crest Development and Migration (2020)
Figure 6

Figure 6: alx1 function in Zebrafish.

A. Dissected flatmount wild type and alx1-/- zebrafish larvae craniofacial cartilages after Alcian blue staining, the anterior points to the left of the page in all images. The ventral cartilages appear normal, but the alx1-/- anterior neurocranium (ANC) appears narrow, with the midline element that is derived from the frontonasal NCC being absent. The Meckel's cartilage (arrow, MC) is also diminutive. Scale bar: 200 µm.

B. Zebrafish alx1 mutants (blue) show reduced detectable expression of alx1 but increased expression of alx3, alx4a compared to wild type controls (green). alx4b expression levels are similar between wild type and alx1-/- lines. Data is represented as the mean of all pooled embryos from three different clutches. The RT-qPCR relative expression values were normalized to elfa and 18S expression using the ΔΔCT method. Data from each clutch were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. Statistical significance denoted by *; p<0.0001 between WT zebrafish and alx1-/- at all measured time points; at 10 ss, 24 hpf and 36 hpf for alx3; and at 24 hpf and 36 hpf for alx4a. Refer to Table EV2 for p-values.

C. Dissected flatmount of zebrafish embryos injected with Alx1DN, after Alcian blue staining. The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium (ANC) and a profound hypoplasia of the Meckel's and ventral cartilages. In the most severely affected zebrafish, a nearly abrogated ANC was observed. Scale bar: 200 µm.

D. Lineage tracing experiments in control and Alx1DN mutant embryos revealed aberrant migration of anterior cranial NCC when alx1 is disrupted. In the control animal, the anterior cranial NCC always migrate to contribute to the median portion of the ANC. In contrast, the anterior cranial NCC labeled in the Alx1DN animals fail to migrate to the median ANC, where the ANC structure is narrower and the labeled cranial NCC are found in an anterior and lateral ectopic location (white asterisks). Scale bar: 250µm.

Expanded View (EV) Figure Legend
A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord (2021)
Figure 1

Figure 1. Microclusters of spinal cord neural progenitor cells have positively correlated HES5 levels.

(a) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo (left panel); Draq5 live nuclear stain with nuclear segmentation overlay (right panel); scale bar 30 μm.

(b) Venus::HES5 nuclear signal corresponding to tissue in (a) obtained by applying nuclear segmentation onto Venus channel.

(c) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity (Materials and Methods) corresponding to segmented image in (b). Black line surrounding representative microclusters.

(d) Dimensions of microclusters in cell numbers with high and similar levels of HES5 in apical-basal axis (left panel) and dorso-ventral axis (right panel) at E9.5 (10 microclusters, 3 slices, 3 exps), E10.5 (10 microclusters, 9 slices, 3 exps) and E11.5 (10 microclusters, 3 slices, 3 exps). NS - no significant difference in one-way ANOVA p=0.46 (A-B), p=0.38 (D-V). Bars show mean ±SD.

(e) Pearson correlation coefficient observed in segmented E10.5 homozygous Venus::HES5 spinal cord ex-vivo slices showing correlation between mean nuclear Venus::HES5 intensity in any cell compared to up to 8 nearest neighbours (see Materials and Methods); dots indicate average per slice; bars indicate mean and standard deviation of 5 slices from 3 experiments (dataset is different from (d).

(f) Pearson correlation coefficient of mean nuclear Venus::HES5 intensity in relationship to distance; red dots indicate average Venus::HES5 correlation per slice of 12 slices from 3 experiments with corresponding red line indicating polynomial fit (order 2); gray dots with black line indicate correlations and polynomial fit from 5 randomizations of intensities analysed in the same way (see Materials and Methods).

(g) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo. Scale bar 30 μm, D- dorsal, V- ventral.

(h) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity of (g); centre of intensity shown with *.

(i) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity in (h) (only 1 side of ventricle) after radial gradient removal (see Materials and Methods).

(j) Pearsons correlation coefficient of mean nuclear Venus::HES5 intensity with distance after subtraction of radial gradient in Venus::HES5 intensity; red dots represent average in each of 12 slices from 3 experiments.
A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord (2021)
Figure 2

Figure 2. HES5 microclusters are spatially periodic along dorsal-ventral axis of spinal cord.

(a) 20x snapshot of an ex-vivo slice culture of E10.5 spinal cord from Venus::HES5 heterozygous knock-in mouse, transverse section; delineated region (blue) correspond to data shown in (b-c). D- dorsal, V- ventral.

(b) Spatial profile of Venus::HES5 intensity averaged over 2.5hrs with 0 distance representing the ventral end of kymograph; black line represents the trend in Venus::HES5 data across the domain produced using an polynomial order 6 (see Materials and Methods).

(c) Detrended spatial profile of Venus::HES5 corresponding to data shown in (b).

(d) Lomb-Scargle Periodogram analysis of detrended Venus::HES5 data in (c); horizontal line indicates Lomb-Scargle significance level p=0.0001; red arrowhead indicate significant peaks.

(e) Auto-correlation analysis of detrended Venus::HES5 spatial profile in (c) with multiple peaks indicating spatial periodicity; significant peaks (red arrowhead) lie outside gray area indicating 95% significance based on bootstrap approach (see Materials and Methods) and non-significant peaks (black arrowhead).

(f) Peak to peak distance in auto-correlation from detrended Venus::HES5 signal collected in apical regions of spinal cord between E9.5-E11.5; bars indicate mean and SD of individual slices from 3 independent experiments; Kruskal-Wallis test not significant, p=0.44.

(g) Representative example of auto-correlation from detrended Draq5 nuclear signal with peak to peak distances indicative of inter-nuclear distance in live tissue; gray area denotes 95% confidence area for Draq5.

(h) Peak to peak distance in auto-correlation of detrended Draq5 spatial profile in apical regions of spinal cord between E9.5-E11.5; bars indicate mean and SD of individual slices from 3 independent experiments; Kruskal-Wallis test not significant, p=0.3.

(i) Schematic of multiple non-overlapping regions of interest identified as Apical, Intermediate and Basal in the spinal cord tissue; width of regions in the apical-to-basal direction was 15μm.

(j) Peak to peak distance in auto-correlation of detrended Venus::HES5 spatial profile corresponding to Apical, Intermediate and Basal regions of spinal cord at E10.5; dataset is different from (h); markers indicate average distance per experiment with a minimum of 3 z-stacks per experiment and 2 repeats (left and right of ventricle) analysed per z-stack; bars indicate mean and SD of 6 independent experiments; Kruskal-Wallis test not significant, p=0.115; distances correspond to 4-5 cells considering the inter-nuclear distance in DV quantified in (h).
A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord (2021)
Figure 4

Figure 4. Notch inhibition increases HES5 pattern persistence.

(a) Start:Finish Venus::HES5 intensity ratio in E10.5 Venus::HES5 heterozygous spinal cord slices treated with control (DMSO) and Notch inhibitor DBZ (2 μM) observed over 16h; bars indicate mean and standard deviation of DMS0 (n=3 experiments) and DBZ (n=4 experiments); 2-tailed t-test ****p=0.0001.

(b) Representative spatiotemporal plots of the detrended Venus::HES5 pattern along ventral-dorsal direction in DMSO control (left panel) and DBZ conditions (right panel) obtained by averaging kymographs data in the same region over 2 hr time intervals.

(c) Schematic indicating the correspondence between Venus::HES5 spatial oscillator represented as detrended level and phase angle characteristics; the spatial oscillator traverses repeated cycles including start (HES5 low-orange arrowhead), middle (HES5-teal arrowhead) and end (HES5 low-red arrowhead) which in phase space corresponds to phase angles 0, π and 2π respectively.

(d) Phase maps corresponding to DMSO (left panel) and DBZ (right panel) detrended Venus::HES5 data shown in (b).

(e) Phase synchronisation measure (see Materials and Methods) of the detrended Venus::HES5 spatial oscillator measured over time in E10.5 Venus::HES5 spinal cord periodic slices treated in DMSO vs DBZ conditions up to 10h; dots indicate DMSO (21 kymographs, n=3 experiments) and DBZ (19 kymographs, n=4 experiments); bars indicate mean and SD; 2 tailed Mann-Whitney test with significance ****p<0.0001.

(f) Spatial peak: trough fold change in Venus::HES5 intensity profile in the D-V axis measured at 2h and 10h in DMSO and DBZ treated E10.5 Venus::HES5 spinal cord slices; dots indicate average over 3 z-slices from DMSO (n=3) and DBZ ( n=4) experiments; lines indicate median per condition; 1 tailed unpaired t test with significance *p<0.05.

(g) Percentage of ex-vivo slices with significant spatial period detected after 10-12hrs of DMSO and DBZ conditions; significant spatial period defined as multiple significant peaks in auto-correlation detected above the 95% confidence bounds; dots indicate % per experiment; bars denote median and inter-quartile range of DMS0 (n=3) and DBZ (n=4) experiments; 1 tailed t test with significance **p=0.0062.

(h) Peak:to:peak distance in auto-correlation plots of detrended Venus::HES5 spatial profile in DMSO and DBZ treated E10.5 Venus::HES5 spinal cord slices; gray dots represent significant mean peak-to-peak distance of DMSO (100) and DBZ (105) auto-correlation functions collected from 3 z-stacks per slice and 2 repeats (left and right of ventricle) with multiple timepoints; bars indicate median per experiments from DMSO (n=3) and DBZ (n=4) experiments; error bars indicate SD; 2 tailed Mann-Whitney test ****p<0.0001.
A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord (2021)
Figure 7

Figure 7. Type of HES5 spatial pattern and coupling strength correlates with rate of differentiation in motorneuron and ventral interneuron domains.

(a) Transverse cryosection of E10.5 Venus::HES5 spinal cord. Venus::HES5 endogenous signal, OLIG2 - motorneuron progenitor marker, NGN2 - early marker of neuronal commitment; scale bar 20 μm.

(b) Relative nuclear intensities of Venus::HES5 and NGN2 in motorneuron and interneuron progenitors; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments; Kruskal-Wallis with Dunn's multiple comparison test adjusted p-values **p=0.0032, ***p<0.001.

(c) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity within motorneuron (MN) and interneuron (IN) domains, corresponding to segmented image in (a).

(d) Dimension of microclusters in DV axis for MN and IN domains; microclusters counted contained cells with high and similar levels of HES5 (Materials and Methods); bars show mean ±SD; data consists of 34 microclusters measured from 5 sections and 3 independent experiments; 2-tailed Mann-Whitney test. **** p<0.0001.

(e) Peak to peak distance in auto-correlation plots of de-trended Venus::HES5 spatial profile in MN and IN domains; this is a measure of spatial period in Venus::HES5 profile along dorsal-ventral axis of spinal cord; grey data points represent mean peak to peak distance of at least 3 slices with left and right ventricle analysed separately in 6 experiments; black dots show median per experiment and line shows overall median; 2-tailed Mann-Whitney test p-values ****p<0.00001.

(f) Cell-cell concentration differences in HES5 between neighbours, normalised to mean concentration of HES5 in that domain; grey data points represent normalised concentration difference between a pair of neighbours, bars shows mean and SD; 2 independent experiments; 2-tailed Mann-Whitney test with p-values ***p = 0.003, ****p<0.00001.
A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord (2021)
Figure 8

Figure 8. NGN2 expression is spatially periodic and positively correlates with the HES5 pattern.

(a) Detrended spatial profile of NGN2::mScarlet-I intensity from transverse slice of E10.5 spinal cord from heterozygous knock-in mouse in ventral-dorsal direction;red indicates motorneuron (MN) domain, blue interneuron domain (IN).

(b) Auto-correlation analysis of detrended NGN2::mScarlet-I intensity spatial profiles from motorneuron and interneuron domains; multiple peaks indicating spatial periodicity; significant peaks (red triangle) lie outside black dotted lines indicating 95% significance based on bootstrap approach (see Materials and Methods) and non-significant peaks (black triangle).

(c) Ratio of NGN2:HES5 spatial period in the same tissue; grey dots show ratio for single image from 4 experiments; line shows overall median and error bars 95% confidence limits.

(d) Peak to peak distance in auto-correlation plots of de-trended NGN2::mScarlet-I spatial profile in motorneuron (MN) and interneuron (IN) domains as a measure of spatial period in NGN2 expression along dorsal-ventral axis of spinal cord; Grey data points represent mean peak to peak distance in a single slice, n=33, left and right ventricle analysed separately in 4 experiments; black line shows overall mean, error bars show SD; 2-tailed Mann-Whitney test with exact p-value *** p=0.0003.

(e) Detrended spatial profile of Venus::HES5 (black) and NGN2::mScarlet-I (red) intensity from the same transverse slice of E10.5 spinal cord in ventral-dorsal direction. (f) Example cross-correlation function of Venus::HES5 with NGN2::mScarlet-I (blue), Venus::HES5 with brightfield signal (black), and NGN2::mScarlet-I with brightfield signal (red) from the same transverse slice of E10.5 spinal cord; markers indicate the presence of two types of co-ordination namely in-phase (red arrowhead) and out-of-phase (black arrowhead).

(g) Phase shift showing absolute lag distance corresponding to in-phase peak in Venus::HES5 vs NGN2::mScarlet-I cross-correlation function of spatial intensity profiles from the same slice. 34 individual data points from 6 slices, 2 experiments; red line indicates average inter-nuclear distance in D-V; bars show mean±SD; 2-tailed Mann-Whitney test not significant, p=0.32.

(h) Pseudo-color look-up tables applied to mean nuclear Venus::HES5 and NGN2 staining intensity in motorneuron (MN) and interneuron (IN) domains. Venus::HES5 microcluster and single NGN2 high cell (red arrow) in IN domain; Alternating high-low expression of Venus::HES5 in MN, red arrows show high cells.

(i) Graphical summary: Through a combination of experimental and computational work we characterised the HES5 dynamic expression in the mouse E10.5 ventral spinal cord. We found evidence that progenitors located in two domains (motorneuron, MN and interneuron, IN) give rise to distinct spatiotemporal characteristics that are indicative of differences in coupling strength and can explain increased differentiation rates observed in MN.

Expanded View Figure Legends
In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer (2019)
Figure 1

Figure 1

In vivo installation by base editing of oncogenic mutations in a model of triple negative breast cancer.

(A) Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgAkt1E17K.

(B) EditR (Kluesner et al., 2018) was used to calculate the frequency (%) of C-to-T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.

(C) Overview of the intraductal injections performed in WapCre;Brca1F/F;Trp53F/F;Col1a1invCAG-BE3/+ (WB1P-BE3) females with high-titer lentiviruses encoding Myc cDNA and either a non-targeting (NT) sgRNA (Lenti-sgNT-Myc) or the sgRNA targeting Akt1 (Lenti-sgAkt1E17K-Myc).

(D) Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc (n=12) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (58 days after injection vs 72 days after injection, P < 0.01 by Mantel-Cox test).

(E) Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in 3 independent tumors from WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc. Arrowheads highlight cytosines of the protospacer that show base editing.

(F) EditR was used to calculate the average frequency (%) of C-to-T conversion at C7 of the protospacer in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgAkt1E17K-Myc. Data are plotted as mean + standard deviation (n=7).