Journal: Development (Cambridge, England)

Article Title: Hemidesmosomes and Notch signaling regulate epidermal differentiation via delamination

doi: 10.1242/dev.205210

Figure Lengend Snippet: Mosaic loss of Itgb4 alters embryonic basement membrane organization without overt blistering. (A) Schematic of the LUGGIGE technique. (B,C) Single-plane confocal images of E17.5 mosaic epidermis showing integrin-β4 reduction beneath Itgb4 4124 RFP + knockdown cells (B), and associated quantification from n ≥6 embryos per condition; independent images from each litter are represented by a unique shape (triangle, circle or square) (C). (D) Spinous thickness determined by quantifying cytokeratin 10 (K10) immunofluorescence in E16.5-E17.5 control (WT), Itgb4 4124 and Itgb4 2326 epidermis; each dot in represents average thickness per animal. (E-H) Transmission electron micrographs (E,G) and quantification (F,H) of hemidesmosomes (HDs) in E17.5 control and highly transduced Itgb4 4124 nape skin. Total HD density is quantified in H and maturity in J; yellow arrowheads indicate mature and magenta arrowheads immature HDs. (I-L) Single-plane confocal images of E17.5 Itgb4 4124 knockdown epidermis showing integrin-α6 (I) and laminin-β3 (K) with intensity quantification (J,L). Basement membrane is indicated with cyan dashed line; blue arrowheads (B,I) represent areas of non-transduced (RFP − ) basal cells. Scale bars: 25 µm (B,I,K); 0.5 µm (E); 0.25 µm (G). ns, not significant; * P <0.05, ** P <0.01, **** P <0.0001 (unpaired t -test or Mann-Whitney test).

Article Snippet: The following TaqMan Probes were used: Ppib (Mm00478295_m1) and Itgb4 (Mm01266840_m1).

Techniques: Membrane, Knockdown, Immunofluorescence, Control, Transmission Assay, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Hemidesmosomes and Notch signaling regulate epidermal differentiation via delamination

doi: 10.1242/dev.205210

Figure Lengend Snippet: Itgb4 loss leads to decreased basal occupancy but does not impact division orientation or polarity. (A) Confocal images of Scramble control and Itgb4 knockdown epidermis showing RFP + relative to K10; differentiation index (SB:B transduction ratio) is shown for each image. (B,C) Plots of suprabasal versus basal transduction for scramble controls (B) and Itgb4 4124 and Itgb4 2326 (C) epidermis; each dot indicates one field of view (FOV) with solid circles <50% basal transduction and empty circles >50%; data collected from n ≥4 biological replicates per condition. (D) Quantification of differentiation indices (SB:B ratio) for FOVs with <50% basal transduction; each shape represents a unique litter; whiskers represent min/max; box represents 25th-75th percentile with center line as median. (E) WT and Itgb4 4124 RFP + telophase cells (survivin, green) in planar, oblique and perpendicular orientations. (F) Cumulative frequency plot of division angles for WT (black) and Itgb4 4124 RFP + (orange) and RFP − (gray) cells; no significant differences between groups by Kolmogorov–Smirnov cumulative frequency test. (G) Bar graph of orientation types, binned by 30° increments. (H) Schematic of ex vivo live imaging with en face and PS-Multiview settings. (I) xz projections of dividing cells at pre-anaphase (t=−5 min), anaphase (t=0 min) and post-anaphase (t=60 min) time points. (J) Line graphs of division orientation angle per cell between anaphase and 60 min post-anaphase in Itgb4 4124 RFP − (gray) and RFP + cells (orange) with bar graph depicting outcomes of initial oblique cells at right. (K) PS-Multiview timelapse of a WT cell (pink) undergoing delamination. Basement membrane is indicated with cyan dashed lines. Scale bars: 10 μm (E,I,K); 25 μm (A). ns, not significant; * P <0.05, **** P <0.0001 [Mann-Whitney test (D), Kolmogorov-Smirnov test (F), Chi-squared test (G,J)].

Article Snippet: The following TaqMan Probes were used: Ppib (Mm00478295_m1) and Itgb4 (Mm01266840_m1).

Techniques: Control, Knockdown, Transduction, Ex Vivo, Imaging, Membrane, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Hemidesmosomes and Notch signaling regulate epidermal differentiation via delamination

doi: 10.1242/dev.205210

Figure Lengend Snippet: Loss of integrin-β4 or its ligand laminin-332 increases delamination. (A-C) Clonal lineage tracing, with cartoons of clone types (A), representative image of Itgb4 4124 RFP + delamination clone (C), and distribution of Itgb4 4124 RFP − versus RFP + clones (B). (D) Confocal images of K10 + /K14 + basal cells in WT, Itgb4 4124 , Itgb4 2326 and Lama3 1436 epidermis; asterisks indicate dual-positive cells. (E) Quantification of dual-positive frequency; each dot represents the total percentage for an animal, per genotype. (F,G) Representative images of laminin-β3 intensity in control and Lama3 1436 epidermis (F), with associated quantification (G). (H) Confocal images of control and Lama3 1436 epidermis showing RFP + cells relative to K10; asterisks indicate basal-resident RFP + cells. (I) SB:B ratio (differentiation index) quantification; whiskers represent min/max; box represents 25th-75th percentile with center line as median. In G,I, each point represents a FOV; shapes indicate unique litters; n >3 biological replicates/condition. Basement membrane is indicated with cyan dashed lines. Scale bars: 25 µm (D,F,H); 10 µm i(C). ns, not significant; * P <0.05, ** P <0.01, **** P <0.0001 [Chi-squared test, unpaired t -test (E,G) or Mann-Whitney test (I)].

Article Snippet: The following TaqMan Probes were used: Ppib (Mm00478295_m1) and Itgb4 (Mm01266840_m1).

Techniques: Clone Assay, Control, Membrane, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Hemidesmosomes and Notch signaling regulate epidermal differentiation via delamination

doi: 10.1242/dev.205210

Figure Lengend Snippet: Notch signaling regulates integrin-β4 levels and delamination. (A-D) Confocal images of E17 Cre-negative control versus Rosa NICD epidermis (A) and E16 WT littermate versus Rbpj cKO epidermis (C), with single channel images of integrin-β4 on the right, and associated quantification of fluorescence intensity (B,D); n =3 except for WT control in Rbpj cohort ( n =2). (E-G) Images of Notch reporter (NR) transgenic (E) and LUGGIGE NR-transduced epidermis (F), and associated quantification (G). NR shown in green; RFP marks cells transduced with reporter. (H) Quantification of nuclear YAP in Itgb4 4124 epidermis. (I,K) Confocal images of E17 Cre-negative control versus Rosa NICD epidermis (I) and E16 WT littermate versus Rbpj cKO epidermis (K); asterisks indicate dual-positive cells. (J,L) Quantification of dual-positive cells in WT versus mutant. Each dot represents a biological replicate in G,H,J,L, or FOV in B,D, where shapes designate litters. Basement membrane is indicated with cyan dashed line. Scale bars: 25 μm (A,C,E,F,I,K); 10 µm (F, insets). ns, not significant; * P <0.05; **** P <0.0001 ( t -test).

Article Snippet: The following TaqMan Probes were used: Ppib (Mm00478295_m1) and Itgb4 (Mm01266840_m1).

Techniques: Negative Control, Fluorescence, Control, Transgenic Assay, Transduction, Mutagenesis, Membrane

Journal: MedComm

Article Title: Hemidesmosome Mutations Contribute to the Onset and Severity of Acquired Autoimmune Bullous Diseases

doi: 10.1002/mco2.70627

Figure Lengend Snippet: Pathogenic variants in hemidesmosome assembly‐related genes in pemphigoid disease spectrum decrease skin‐related protein expression. (A) Pattern diagram showing the location and pattern of pathogenic antibody deposition for different PD subtypes. (B) Representative immunohistochemistry images of BP, LigA, DH, and EBA mutation‐carrying and control patient samples stained for ITGA6 , LAMC2 , EPPK1 , and ITGB4 , n = 10, 14 for mutations and controls. Scale bar: 150 µm. (C) Quantitative analysis of IHC results for BP samples. (D) Quantitative analysis of IHC results for LigA samples, n = 11, 11 for mutations and controls. p‐ values are calculated using a two‐tailed Student's t ‐test. * p ≤ 0.05, ** p ≤ 0.01. Each sample is represented as one dot.

Article Snippet: Antibodies for IHC experiments, including LAMC2 (#ab210959, Abcam), EPPK1 (#ab247172, Abcam), ITGA6 (#ab181551, Abcam), LAMB3 (#ab97765, Abcam), JUP (#ab2309, Abcam), ITGB4 (#ab14803, CST), LAMA3 (#ab242197, Abcam), DST (#ab244440, Abcam), and PLEC (#ab32528, Abcam), were used according to the recommended dilution and incubation times.

Techniques: Expressing, Immunohistochemistry, Mutagenesis, Control, Staining, Two Tailed Test