EMBO/soda-nel huggingface dataset:
Mutant Lef1 controls Gata6 in sebaceous gland development and cancer (2019)
Figure 2

Figure 2: Gata6 expression is independent from Wnt/β-catenin signaling.

(A) Sections of WT embryonic skin at different HF stages stained for Gata6, Lef1, Sox9 and counterstained with Dapi. Black asterisks indicate overexposed areas of nonspecific Lef1 staining in the suprabasal epidermis. Quantification of the percentage of cells labelled for both Gata6 and Lef1, or Gata6 and Sox9 in stages 4-5 HF are shown (upper right panel). Data are means ± SD and were obtained from 9 HF from 3 mice.

(B) Sections of E18.5 WT and Lef1-/- mouse skin stained for Lef1 and Gata6. Deletion of Lef1 does not impair Gata6 expression.

(C) Sections of WT and K14ΔNβcateninER (K14ΔNβ-catER D2 and D4 strains) adult dorsal skin stained with antibodies against Krt14 and Gata6. Topical treatment with 1 or 6 doses of 4OHT activates β-catenin leading to anagen induction and ectopic hair follicles but not Gata6 expression.

(D) Sections of E18.5 WT and K14Cre/βCat Flox(ex3)/+ mouse skin stained for Sox9 and Gata6. Activation of Wnt/β-catenin signaling during epidermis development does not induce Gata6 expression but results in ectopic expression of the HFSC marker Sox9.

Data information: (A-C) Scale bars: 50 μm. (D) Scale bar: 25 μm.
Mutant Lef1 controls Gata6 in sebaceous gland development and cancer (2019)
Figure 3

Figure 3: Gata6 progenitors contribute to SG morphogenesis.

(A) Bright-field images of whole-mount WT and Gata6 cKO epidermis, showing abnormal SG upon loss of Gata6. Quantification of hypotrophic SG in WT, heterozygous (Het) and Gata6 cKO epidermal whole-mounts. Data are boxplots with indication of means. Box limits are minimum and maximum values. An average of 165 HF per mouse (from 3 to 4 mice per genotype) was analyzed. (*) p-value < 0.05; (**) p-value < 0.005; unpaired Student's t-test.

(B) Schematic representation of plasmid constructs used for in utero lentiviral infection. Whole-mounts or sections of adult infected epidermis with empty vector (EV) or Gata6-ires-GFP (G6OE) lentivirus were stained for GFP and Fasn. In vivo overexpression of Gata6 leads to ectopic Fasn expression in the HF/SG unit. White dotted lines define SG, and yellow dotted lines define a cyst. Note that the cyst is mostly negative for Fasn in agreement with its SD-like phenotype. White arrows indicate GFP-positive infected cells. These cells are stained with Fasn only in G6OE condition. H&E-stained skin from G6OE mice shows a cyst with SG elements in the HF unit (black arrows). Staining for Gata6 (both endogenous and exogenous) shows that Gata6 expression occurs in a limited number of cells (representative image in upper right panel). Bottom left graph shows quantification of the percentage of clones labelled for both Fasn and GFP in the SG, HF and IFE compartments. Data are means ± SD and originate from 4 EV mice and 8 G6OE mice (average of 11 clones per mouse).

(C) Whole-mounts adult infected epidermis with EV or G6OE lentivirus stained with LipidTOX. Ectopic Gata6 cells are not stained with LipidTOX indicating an incomplete sebaceous maturation.

(D) Lineage tracing experiments in Gata6EGFPCreERT2:Rosa26-fl/STOP/fl-tdTomato (Gata6creER ROSA-dTom) mice. A single dose of 4OHT was injected in pregnant females at E16.5. Tail skin from pups was collected at P13. Representative example of whole-mount epidermis showing tdTomato labelled cells counterstained with Dapi (top left panel). Right panels show the different Z-stacks related to this whole-mount. Gata6 progenies are mainly found in the upper SG/JZ. Localization of Gata6 progenies (dTomato+) was quantified in 20-26 pilosebaceous units per mouse (N=4 mice) (bottom left panel). Data are means ± SD. As a control, quantification was also performed in Rosa26-fl/STOP/fl-tdTomato (ROSA-dTom) mice that were treated similarly to Gata6creER ROSA-dTomato mice.

Data information: (A-C) Scale bars: 50 μm. (D) Scale bar: 25 μm.
Mutant Lef1 controls Gata6 in sebaceous gland development and cancer (2019)
Figure 4

Figure 4: Effect of Gata6 loss in K14ΔNLef1 sebaceous tumors.

(A) Sections of mouse skin tumors were stained with antibodies to Gata6 and Krt14. A papilloma tumor found in WT mice treated with DMBA and TPA (left panel), is compared with sebaceous tumors found in K14ΔNLef1 mice (right panels).

(B) Tumor burden and tumor incidence in DMBA-treated K14ΔNLef1 (n=11) and K14ΔNLef1:cKO (n=8) mice. (***) p-value < 0.001; Wilcoxon matched-pairs signed rank test.

(C) Representative H&E-stained tumors of K14ΔNLef1 and K14ΔNLef1:cKO mice with quantification of each tumor type relative to the total number of tumors in each group (middle panel) and as the average absolute number of tumors per mice (right panel). A total of 268 tumors were analyzed. 143 tumors were found in 7 K14ΔNLef1 mice (103 sebaceous tumors and 40 papillomas), while 125 were found in 5 K14ΔNLef1:cKO mice (55 sebaceous tumors and 70 papillomas). (**) p-value < 0.005, Student's t-test.

(D) Representative images of mouse skin tumor sections labelled with antibodies to Plet1 and Atp6v1c2.

Data information: (A, D) Scale bars: 100 μm. (C) Scale bar: 1 mm. (B, C) Data are means ± SEM.
Mutant Lef1 controls Gata6 in sebaceous gland development and cancer (2019)
Figure 5

Figure 5: Gata6 controls DNA Mismatch Repair response during sebaceous tumorigenesis.

(A) Heat-map highlighting differentially expressed genes (DEG) between WT and Gata6 cKO keratinocytes from (Wang et al, 2017). Gata6 direct transcriptional targets from Chip-Seq data (Donati et al, 2017) are shown in dark grey.

(B) KEGG pathway analysis performed on down-regulated genes upon Gata6 loss.

(C) MSI analysis performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice (5 mice in each group). Representative microsatellite profiles of K14ΔNLef1 and K14ΔNLef1:cKO tumors. Peak heights were normalized to the highest peak in each microsatellite profile to obtain the relative frequency of each allele for 5 different markers. "0" indicates the position of the highest peak in bp. (*) p-value < 0.05; (**) p-value < 0.001; paired t-test.

(D) Representative immunohistochemistry stainings against Mlh1 performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice. 4 mice were included in each group. A technical control is displayed (without primary antibody incubation). Semi-quantitative analysis of Mlh1 staining and statistical analysis (χ2 test) were performed.

(E) Representative immunohistochemistry stainings against Msh2 and semi-quantitative analysis performed as in (D).

Data information: (D and E) Scale bar: 250 μm.
Mutant Lef1 controls Gata6 in sebaceous gland development and cancer (2019)
Figure 6

Figure 6: Gata6 expression is conserved in human normal skin and found in tumors harboring a sebaceous differentiation.

(A) Normal human scalp skin sections stained with Gata6 and PanKeratin antibodies. Endogenous Gata6 expression is found in the JZ, upper SG and SD. As in mouse skin, Gata6 is not expressed in sweat glands (SwG).

(B) Representative sections of human skin tumors stained with Gata6 and PanKeratin antibodies. Quantification of the percentage of Gata6-positive tumors within each histopathological group (middle left panel) and quantification of the percentage of Gata6-positive cells within each positive sample (middle right panel). Note that the least positive sample showed about 3% of Gata6-positive cells. The analysis was performed on 73 different tumor samples. SSC: squamous cell carcinoma, SCAP: syringocystadenoma papilliferum, EMPD: extramammary Paget's disease, nod./superf./scler. BCC: nodular/ superficial/ sclerodermiform basal cell carcinoma, Steatocystoma M.: steatocystoma multiplex, FSCH: folliculosebaceous cystic hamartoma. Heat-map displaying the -log(p-value) of χ2 test computed between the different tumor types (bottom left panel).

(C) Gata6 expression (measured in Transcripts Per Million, TPM) in human SCC (n=6), BCC (n=6) and sebaceous carcinoma (n=14) samples from (North et al, 2018). Data are means ± SD. (**) p-value ≤ 0.005, Student's t-test.

Data information: (A and B) Scale bars: 100 μm.
Mutant Lef1 controls Gata6 in sebaceous gland development and cancer (2019)
Figure 7

Figure 7: Gata6 is mutated or downregulated in human sebaceous carcinomas with a high mutational burden.

(A) Differentiation status (poor, moderate or well-differentiated), circumscription (not infiltrative, focally infiltrative or infiltrative), log10 total number of mutations and Gata6 mutations in 32 human sebaceous carcinomas (SebC) separated in 3 subgroups: pauci-mutational, MSI-related and UV-related as published in (North et al, 2018). For Gata6 mutations, the most deleterious mutation is displayed. n.a.: not assessed.

(B) Schematic representation of Gata6 protein displaying the different protein domains: PEST, GATA-type transcription activator, Zinc-finger (ZnF), Nuclear Localization Sequence (NLS) and the different Gata6 missense mutations and deletions found in human SebC. Heat-map displaying the predicted effect (deleterious or neutral) of W8C, S33C, G61R and S511F point mutations as assessed by PredictSNP, Polyphen-1, Polyphen-2, SIFT, SNAP and SNAP2 algorithms (right panel). Percentages indicate the level of confidence of the predictions.

(C) Gata6 expression (measured in Transcripts Per Million, TPM) in human pauci-mutational (pauci-mut) SebC (n=4), MSI-related SebC (n=4), UV-related SebC (n=5) samples. Data are means ± SD. (*) p-value ≤ 0.05, Student's t-test.

(D-F) Graphs showing the number of mutations per megabase (Mutations/Mb) (D), the number of Somatic Single-Nucleotide Variants (SSNV) (E) and indel (F) of Gata6mut (n=3) or Gata6wt (n=6) MSI-related SebC (left panels) and of Gata6mut (n=3) or Gata6wt (n=7) UV-related SebC (right panels). Data are means ± SD.

(G) Heat-map displaying the mean expression level of MMR genes (measured in TPM) in Gata6wt (n=10) and Gata6mut (n=3) SebC.

Expanded View Figure legends
The conserved C2 phospholipid-binding Domain in Delta contributes to robust Notch signalling (2021)
Figure 1

Figure 1. Structure and binding properties of Drosophila ligands

(A,B) Left panels. The structures of the N-terminal regions from Drosophila Delta (A) and Serrate (B) are shown in a cartoon representation (rainbow coloured from blue at N-terminus to red at C-terminus). These have been overlaid on their mammalian equivalents DLL-4 (A) and Jagged-1 (B) in the context of their complexes (PDB entries 4xlw and 5uk5 respectively) with Notch-1 (cartoon representation, coloured grey). The structure of isolated Drosophila Notch is also depicted in each panel (cartoon, rainbow coloured) superposed on the respective copy of mammalian Notch-1 (cartoon, grey) for each complex. The overlays demonstrate the high degree of conservation in domain structures and arrangements between the Drosophila and mammalian homologues. Right panels. A close-up view of the C2 domains of each ligand overlaid with their mammalian equivalent. These demonstrate conservation of overall fold but large differences in the apical loops, particularly in the β1-2 and β5-6 loops.

(C) Isolated structure of N-terminal Delta, with residues deleted in ∆β1-2 highlighted as red Van Der Waals spheres.

(D,E) Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes (D) Binding is reduced in Delta∆β1-2, but not to the Serrate equivalent using liposomes composed of PC:PS:PE-fluoroscein (80:15:5). Notch binding to Drosophila Delta NE3 (E) WT and ∆β1-2 (Delta∆β1-2) variants both bind to Notch, unlike variant with F204A substitution in DSL domain. Comparisons were performed with a two-tailed unpaired t-test. Values are shown as scattered data points with the dark lines representing the means. ns, no significant difference, **P < 0.01; ***P < 0.0001.
The conserved C2 phospholipid-binding Domain in Delta contributes to robust Notch signalling (2021)
Figure 2

Figure 2. Dl β1-2 loop mutant generated by genome editing.

(A) Two gRNAs flanking the Dl Exon 2 were used to replace the exon with a modified version where 5 amino-acids in the β1-2 loop were removed. Red lettering highlights the genomic sequence of the β1-2 loop.

(B-C) Adult wings from Dl∆β1-2 flies. No defects are detected in wings from Dl∆β1-2/+ (B), Homozygous Dl∆β1-2/ Dl∆β1-2 have extra vein tissue near L5 and uneven L2 veins (arrowheads; C).

(D) Microchaetae are arranged in rows on the thorax of control (yw) flies; these become disordered and more dense in Dl∆β1-2/Dl∆β1-2. White rectangle indicates area scored for E

(E) Number of microchaetes per central area (white rectangle in D) in the indicated genotypes.

Data information: ***P<0.0001 (Unpaired t-test). Each dot represents an individual fly, and light or dark shading indicates individuals from independent genetic crosses.

On the violin plot, dashed line represents the median and the dotted lines show the quartiles. Scale bars correspond to 200 μm (B-C) and 500 μm (D).
The conserved C2 phospholipid-binding Domain in Delta contributes to robust Notch signalling (2021)
Figure 3

Figure 3. Dl∆β1-2 enhances vein thickening from loss of function Dl alleles and suppresses Notch phenotype.

(A-C) Representative images of adult females wings in combinations of Dl∆β1-2 with loss of function Delta alleles. In combinations with Dlrev10 (A), Dl∆Exon2 (B), or Df(3R)DlFx3 (C), vein thickening is strongly enhanced (right panels) compared to heterozygous mutants alone (left panels). Vertical square brackets indicate the regions used for vein thickness quantification.

(D) Quantification of wing-vein thickness in females of the indicated genotypes.

(E) Representative images of adult female wings demonstrate that Dl∆β1-2 rescues the wing notching phenotype, caused by a Notch loss of function allele (N55e11). Horizontal square bracket indicates the L5 vein "delta" at the intersection with the wing margin analyzed in EV2E.

(F) Quantification of wing notching in females of the indicated genotypes, Dl∆β1-2 rescues notching in a similar manner to Dlrev10.

Data information: ***, P<0.0001(Unpaired t-test). Light, dark shading indicates data points from independent genetic crosses. On the violin plots, dashed lines represent the median and the dotted lines show the quartiles. Scale bars A-E correspond to 200 μm.
The conserved C2 phospholipid-binding Domain in Delta contributes to robust Notch signalling (2021)
Figure 4

Figure 4. Dl∆β1-2 has compromised Notch response in photoreceptor fate decisions.

(A) Schematic representation of Notch reporter E(spl)mδ0.5 expression during photoreceptor differentiation. Expression is initiated in R3 and R4 of the 5-cell pre-cluster and becomes restricted to R4 as Notch activity resolves. Light orange indicates photoreceptors with R3 in light green and R4 in dark green. MF marks the morphogenetic furrow, boxed region indicates the region shown in B.

(B) Equatorial region of eye imaginal discs where E(spl)mδ0.5 expression (green) becomes restricted to a single photoreceptor in each cluster (magenta), as detected in control and Dl∆β1-2/+ discs (top panels). In Df(3R)DlFx3 /+ and Dl∆β1-2/Df(3R)DlFx3 discs (bottom panels) E(spl)mδ0.5 expression is reduced (Df(3R)DlFx3 /+) or absent from several clusters (Dl∆β1-2/Df(3R)DlFx3) indicative of reduced Notch signalling. Scale bars correspond to 10 μm.

(C) Proportion of photoreceptors clusters that fail to express the E(spl)mδ0.5 reporter in the indicated genotypes. ns, no significant difference, *** P<0.0001 (one-way ANOVA). On the violin plot, dashed line represents the median and the dotted lines show the quartiles.
The conserved C2 phospholipid-binding Domain in Delta contributes to robust Notch signalling (2021)
Figure 5

Figure 5. Dl∆β1-2 exhibits normal sub-cellular localisation.

(A) Apical view of wing imaginal disc with homozygous Dl∆β1-2 clones (GFP negative) stained for Dl (red) and Cadherin (blue). (A') Z-projection of apical layers spanning a Dl∆β1-2 clone (GFP negative) located at the DV boundary. No change in apical localisation of Dl (red) and Cadherin (blue) is detected. Panels on the right show the Dl (grayscale) apical localisation on the wing imaginal disc.

(B) Uptake assay at t=0. After exposure to extracellular anti-Dl antibody, Dl protein (red) is detected at similar levels apical to Cadherin (blue) in wild-type (GFP) and Dl∆β1-2 mutant tissue (GFP negative). (B') Cross-sectional view of B, Dl protein (red) is present apically relative to Cadherin (Blue) which marks adherens junctions. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.

(C) Uptake assay after 30 minutes, internalised anti-Dl (red) enters the endocytic route and in the cross sectional view (C') can similarly be detected as puncta along the cell axis in wild-type (GFP) and homozygous Dl∆β1-2 (GFP negative) tissue. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.

Scale bars: A, 50 μm; A' B and C,10 μm.
Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids (2021)
Figure 2

Figure 2. Mitochondrial PS decarboxylation by Psd1p prefers di-unsaturated PS molecular species.

(A, B) PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (A) percentage of the lipid-incorporated 13C15N-label recovered in PE, and (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.

(C) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.

Data information: Exponentially growing cells were pulsed for 20 min with 13C315N-serine at 30oC prior to lipid extraction and lipid analysis by shotgun lipidomics. Molecular species representing at least 1% of both labeled PS and labeled PE are shown in panel B (as mol% of total). Compared to *PS and *PE, the amount of 13C215N -PC synthesized was negligible (cf. (Elbaz-Alon et al, 2014)), and therefore not included in the analysis. Data are presented as the mean of 3 biological replicates, with the individual values indicated. Underlying data for this figure can be found in Dataset EV1.
Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids (2021)
Figure 4

Figure 4. Loss of mitochondrial lipid biosynthetic enzymes enhances the accumulation of saturated acyl chains under conditions of SCT1 overexpression.

(A) The molecular species-selectivity of PS-to-PE conversion is recapitulated in wild type BY4741. WT cells overexpressing SCT1 vs. control were pulsed for 20 min with 2H3-serine prior to lipid extraction and ESI-MS/MS analysis of 2H3-PS (*PS) and 2H3-PE (*PE). Data is presented as mean of 4 biological replicates with the individual values indicated. Underlying data for this figure can be found in Dataset EV2.

(B) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test.

(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.

(D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).
Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids (2021)
Figure 5

Figure 5. ERMES and vCLAMP mutants retain preferential decarboxylation of di-unsaturated PS, and accumulate saturated acyl chains upon overexpressing SCT1.

(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.

(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.

(C, D) PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed as (C) percentage of the lipid-incorporated 13C15N-label recovered in PE, and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.

(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.

Data information: For experimental details see the legend to Figure 2. Data in C, D, E are presented as the mean of 3 or 2 (vps39∆) biological replicates, with the individual values indicated. Underlying data for this figure can be found in Dataset EV3.
Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids (2021)
Figure 6

Figure 6. Effect of exogenous ethanolamine and choline on the accumulation of saturated acyl chains in ERMES mutants and vps39∆.

SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.
CPAP promotes timely cilium disassembly to maintain neural progenitor pool (2016)
Figure 1

Fig. 1.

Seckel cells display overly long cilia and reduced proliferation

a, Seckel cells contain normal centrosome numbers and mitotic spindle poles. Interphase centrosomes of Seckel cells have no CPAP immunoreactivity but recruit similar levels of centrosomal proteins (ii and iii).

b, Seckel cells display overly long cilia. Arl13b labels cilium. Scale bar 1 μm. Quantification of cilia length, *** p<0.001; t-test; Scatter plot showing mean with ±SD (iii). Cells displaying normal (0-5 μm), long (5-10 μm) and extra long (>10 μm) cilia. *** p<0.001; Ordinary Two-way ANOVA (iv). Average cilia length of WT and Seckel cells (v). n = 3 independent experiments, 228 cells for WT and 297 cells for Seckel. Error bars are s. e. m. *** p<0.001, t-test.

c, Electron microscopy (EM) of Seckel and WT fibroblasts. In contrast to WT, Seckel basal body has disorganized microtubule of doublet and triplets (red arrows and red circles in schematics).

d, Longitudinal serial-sectioning EM of WT (top) Seckel fibroblasts (bottom). In contrast to WT basal bodies that have structurally normal distal and sub-distal appendages (yellow arrows and red triangles of DA and SDA in schematics), Seckel basal body displays either lack or misplaced appendages. n > 15 basal bodies.

e, Seckel cells show reduced EdU incorporation after 72 hrs. Error bars are s. e. m. ** p<0.01, ttest; n = 4 independent experiments, 727 cells for WT and 844 cells for Seckel.