Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A-C) Immunofluorescence on cross-sections of 54 hpf atp1b1a sibling (A), atp1b1a mutant (B), and atp1b1a mutant injected with st14a MO (C) showing pAKT (red) in basal cells in atp1b1a-/- but not in atp1b1a-/- , st14a MO (N = 3, n = 41–46). (D-F) Immunofluorescence on whole mounts, lateral views on trunk region for pRPS6 (red) and tp63 (green) in 54 hpf atp1b1a sibling (D), atp1b1a mutant (E), and atp1b1a mutant injected with st14a MO (F) (N = 3, n = 27–32). (G-I) Confocal images of the tail region of live 54 hpf embryos transgenic for NFkB-RE:eGFP injected with control MO (G), injected with atp1b1a MO (H) or co-injected with atp1b1a and st14a MO (I). Intensity of the GFP signal is color-coded. (J-L) Immunofluorescence of BrdU incorporation (red) of 56 hpf wild-type sibling (J), atp1b1a mutant (K), and atp1b1a mutant injected with st14a MO (L). (M-O) Whole mount in situ hybridization of mmp9 in 58 hpf wild-type sibling (M), atp1b1a mutant (N), or atp1b1a mutant injected with st14a MO (O) (N = 2, n = 18–24). (P) Quantification of GFP signal obtained from the fin fold region of embryos as shown in (G-I) (n = 3–5). (Q) Quantification of BrdU-positive cells in the fin fold area (n = 3–5). (R) RT-qPCR showing relative quantities of mmp9 transcript of 58 hpf atp1b1a-/- mutants in comparison to their atp1b1a+/+ and atp1b1a+/- siblings, either containing (bars 1+2) or lacking ( st14a-/- ; bars 3+4) functional Matriptase-1 (cDNA obtained from 15 pooled embryos each, N = 3). Significances in (P-R) were determined via a one-way ANOVA and Tukey’s post hoc test; ns, not significantly different (p>0.05); *,**,***,**** significantly different with p<0.05, p<0.01, p<0.001, p<0.0001, respectively. Scale bars: 10 μm (A,D,G,J,M).

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Immunofluorescence, Mutagenesis, Injection, Transgenic Assay, BrdU Incorporation Assay, In Situ Hybridization, Quantitative RT-PCR, Functional Assay

Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A-B) RT-qPCR showing no significant change of st14a and hai1a transcript levels in atp1b1a mutants compared to their siblings (n = 3, cDNA obtained from pools of 15 embryos each at 56 hpf, significances were determined via Student’s t-test; ns, non-significant difference; p>0.05). (C-E) Reporter assay for Matriptase activity towards Par2 cleavage showing that zebrafish Matriptase1a cleaves AP-Par2b more efficiently at lower osmolalities. HEK293 cells were transfected with empty pcDNA3, pcDNA3+AP-Par2b, and pcDNA3+St14a. After 24 hrs in regular / isotonic medium (tonicity of 320 mOsm), cells were exposed to media of 320 mOsm, 270 mOsm, 230 mOsm, and 150 mOsm for 15 and 45 minutes, respectively. (C) At 15 min, absolute luminescence values of AP released into the supernatant progressively increase with increasing hypotonicity / lower tonicity. (D,E) Ratios of luminescence between isotonic and hypotonic media indicate an up to 3.79-fold increase (in 150 mOsm medium) after 15 min (D) and a 2.51-fold increase after 45 min (E). Ratios were determined from values as shown in (C), deducting baseline luminescence (bar 1 in C) from the luminenscences of co-transfected samples obtained at different osmolarities (bars 3–6 in C), normalized against the value at 320 mOsm (isotonic); n = 5, significances were determined via a one-way ANOVA and Tukey’s post hoc test; columns with same superscript letter (a,b,c) are not significantly different (p>0.05). (F) Immunoblot analysis for processed / activated Matriptase-1 (ST14) showing that culturing of MCF-10A cells for 24 hours in hypotonic medium (150 mOsm) leads to a 3.78-fold increase in active endogenous Matriptase-1 compared to cells cultured in isotonic medium (320 Osm). Bar diagram displays mean value of proteins normalized to loading control GAPDH (n = 3); ns, not significantly different (p>0.05); **, ***, significantly different with p<0.01, p<0.001, respectively. (G-H) Scribble knockout (SCRIB KO) in human MCF-10A cells does not affect protein levels of full length ST14 or HAI1. Representative western blots show unaltered amounts of endogenous full-length ST14 (G) and HAI1 (H) proteins in SCRIB-KO cells compared to knockout cells with re-introduced Scribble (SCRIB KO + SCRIB), cultured in media of 320 mOsm, 230 mOsm, or 150 mOsm for 1 hr. Bar diagrams display mean value of proteins normalized to loading control GAPDH (n = 2); all differences are not statistically significant (p>0.05). (I) Loss of Scribble increases active ST14 in media of different osmolalities. Representative western blots showing active ST14 and GAPDH of SCRIB-KO and SCRIB-KO + SCRIB cells cultured in media of 320 mOsm, 230 mOsm, and 150 mOsm for 24 hrs, with highest numbers in cells lacking the epithelial polarity protein Scribble protein and exposed to hypotonic stress. Bar diagram displays mean value of proteins normalized to loading control GAPDH (n = 3). Significances were determined via a one-way ANOVA and Tukey’s post hoc test; columns with same superscript letter (a,b,c,d) are not significantly different (p>0.05).

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Quantitative RT-PCR, Reporter Assay, Activity Assay, Transfection, Western Blot, Cell Culture, Knock-Out

Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A-C) Loss of st14a in basal cells is neither necessary nor sufficient to normalize mmp9 expression in basal cells of atp1b1a morphants. (A) Schematic of experimental set up in which ventral ectodermal cells either from atp1b1a , st14a double morphant or atp1b1a morphant donors were homotopically transplanted at 6 hpf into the same region of atp1b1a or atp1b1a , st14a double morphant hosts, respectively ( atp1b1a MO, st14a MO> atp1b1a MO or atp1b1a MO> atp1b1a MO, st14a MO). Donor cells express Tg(Ola . Actb : Hsa . hras-egfp)- encoded membrane-tagged eGFP. Whole mount in situ hybridization for mmp9 (blue) and immunohistochemistry for eGFP (brown) of chimeric embryos at 58 hpf, showing that in atp1b1a MO, st14a MO> atp1b1a MO experiments, atp1b1a MO, st14a MO basal cells express mmp9 (B), and are thus not rescued although they lack functional Matriptase-1; whereas in atp1b1a MO> atp1b1a MO, st14a MO experiments, atp1b1a MO basal cells lack mmp9 expression (C), and are thus rescued although they contain Matriptase-1 (n = 6–8). (D-N) Re-introduction of st14a into peridermal cells of atp1b1a , st14a double morphants abrogates the rescue of basal cells, reflected by re-gained epidermal aggregate formation and enhanced mmp9 expression. (D) Immunofluorescence for eGFP-Matriptase1a (green), p63 as a nuclear marker for basal cells (red) and ZO1/Tjp1 as a marker for tight junctions in peridermal cells (red) on transverse section through the epidermis of a 48 hpf embryo transgenic for peri : Gal4 zc1044a and UAS : gfp-st14a fr58Tg , counterstained with DAPI (blue). Transgene-encoded Matriptase-1 is restricted to peridermal cells and localized at their basolateral membranes. (E-H’) Brightfield images of representative live 56 hpf embryos transgenic for peri : Gal4 zc1044a or peri : Gal4 zc1044a ; UAS : eGFP-st14a controls (E,F) and injected with atp1b1a and st14a morpholinos ( atp1b1a MO, st14a MO) (G,H); lateral views of entire embryos (E-H), and magnified views of tail region of same embryos (E’-H’). In contrast to atp1b1a MO, st14a MO embryos without transgenic re-introduction of st14a (G,G’), the atp1b1a MO, st14a MO embryos transgenic for peri : Gal4 zc1044a ; UAS : eGFP-st14a displays epidermal aggregates (H,H’), comparable to global atp1b1a single mutants (compare with L’). (I-L’) Representative whole mount mmp9 in situ hybridizations, revealing re-gained strong mmp9 expression in basal cells of 72 hpf atp1b1a MO, st14a MO embryo transgenic for peri : Gal4 zc1044a ; UAS : eGFP-st14a (L,L’, the latter counterstained for the basal cell marker p63), but not in the atp1b1a MO, st14a MO embryos lacking transgene-driven peridermal st14a re-expression (K). (M) Quantification of epidermal phenotype of 56 hpf embryos with respective genotypes, as shown in (E-H) (n = 20–46). (N) Quantification of mmp9 in situ signal of 72 hpf embryos with respective genotypes, as shown in (I-L) (n = 12–38). Scale bars: 20 μm (B,L’), 10 μm (D), 500 μm (E), 100 μm (E‘,I).

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Functional Assay, Immunofluorescence, Marker, Transgenic Assay, Injection, In Situ

Journal: PLOS Genetics

Article Title: Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects

doi: 10.1371/journal.pgen.1010873

Figure Lengend Snippet: (A) Model of Matriptase activity restriction in the wild-type zebrafish epidermis. Hai1a is tightly associated with Matriptase, thereby inhibiting its activity, both in peridermal and basal cells. In addition, Matriptase trans-layer signaling from the periderm to the basal layer is restrained by confined levels of Matriptase at the basal side of peridermal cells. (B) Upon loss of Hai1a, Matriptase 1 is no longer inhibited (red star), leading to the cleavage of adjacent Par2b (orange asterisk), which in turn activates the EGFR-PLD pathway, to induce hyperproliferation and expression of mmp9 (a marker for EMT). Mild hypotonicity (small blue star), most likely due to compromised epidermal integrity, further enhances Matriptase activity levels. Note that additional pathways downstream of Par2b have been described, which lead to additional pre-neoplastic events, like sterile inflammation; however, they do not include PI3K . (C) More extreme hypotonicity in the pericellular space (due to loss of ATP1b1a or Pax2a; large blue star) causes (moderate) Matriptase activation even in the presence of Hai1a. This, however, only has subtle effects on epidermal cells ( , ), unless occurring in conjunction with the loss of epithelial polarity (due to loss of ATP1b1a or Lgl2), allowing Matriptase to shift towards the basal side of peridermal cells, thereby getting into physical contact and to cleave / activate Par2b and other, not yet identified targets (indicated by?) on underlying basal keratinocytes in trans. These targets activate a PI3K-pAKT-mTORC1-NFkB pathway in basal cells resulting in pre-neoplastic events like hyperproliferation, EMT and, in contrast to hai1a mutants, strong invasiveness of basal cells.

Article Snippet: The following antibodies were used: mouse anti-GAPDH (Millipore, MAB374, 1:10000), goat anti-Hai1 (R&D Systems, AF1048-SP, 1 ug/ml), rabbit anti-Matriptase/MT-SP1 (Sigma Aldrich, IM1014-50UG, 1:1000), mouse anti-Scribble ([ ], clone 7C6.D10, 1:100), sheep anti-mouse HRP (Amersham, NA931V, 1:4000), donkey anti-rabbit HRP (Amersham, NA9340V, 1:4000), and rabbit anti-goat HRP (Invitrogen, 81–1620, 1:4000).

Techniques: Activity Assay, Expressing, Marker, Activation Assay