Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 4. (A) Glands inhibited with rFGFR2b were rescued with FGF7 and FGF10, which increased the number of end buds. Glands were treated with 1.6 µg/ml of rFGFR2b, and increasing doses of FGFs were added. Glands cultured with rFGFR2b were rescued only by FGF7 (500 ng/ml) and FGF10 (1000 ng/ml), and not by FGF1 (500 ng/ml) and FGF3 (1000 ng/ml), by other ligands for FGFR2b, or by FGF2 (200 ng/ml) or BMP7 (200 ng/ml). (B) A combination of neutralizing antibodies to FGF1, FGF7 and FGF10 was required to significantly inhibit branching morphogenesis (ANOVA, **P<0.01). Individual antibodies to FGFs (25 µg/ml), or combinations of either two or three antibodies or similar concentrations of either mouse or goat IgGs, were incubated with E12 SMGs for 48 hours. Individual anti-FGF antibodies or combinations of two anti-FGF antibodies (not shown) did not significantly inhibit branching. The number of buds was expressed as a ratio of the number of buds at 48 hours/number of buds at 2 hours (T48/T2).

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Cell Culture, Incubation

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 6. FGF-induced morphogenesis is a result of localized proliferation. (A) Individual FGFs, FGF1 (200 ng/ml), FGF2 (100 ng/ml), FGF4 (100 ng/ml), FGF7 (100 ng/ml) and FGF10 (500 ng/ml), stimulate proliferation of epithelial buds by 8 hours, before any morphogenesis has occurred. BMP4 (100 ng/ml) and BMP7 (100 ng/ml) did not stimulate epithelial proliferation above control levels. Scale bar: 50 µm. BrdU was detected with a Cy3-labeled antibody, the nuclei were stained with SYBR-green, and fluorescence was quantitated using MetaMorph software and expressed as the ratio of BrdU:SYBR-green pixels. At least five glands/condition were used for quantitation, and the experiments were repeated twice with similar results. (B) FGFs induce localized proliferation resulting in distinct morphologies by 44 hours. FGF1 and FGF10 stimulate proliferation at the tips of the ducts. FGF2 and FGF7 stimulate proliferation over the entire buds, and FGF7 also induces duct proliferation. BrdU was detected with a Cy3-labeled antibody, and nuclei were stained with SYBR-green. Scale bar: 100 µm.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Control, Labeling, Staining, SYBR Green Assay, Software, Quantitation Assay

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 5. (A) Individual FGFs and BMPs have distinct morphological effects on isolated epithelium cultured in growth factor-reduced Matrigel for 44 hours. FGF1-, FGF4- and FGF10-treated epithelium form duct-like structures, whereas FGF2 and FGF7 promote bud formation. Epithelium treated with FGF8, BMP4 or BMP7 alone do not grow. Numbers indicate concentrations in ng/ml. (B) Combinations of FGFs resulted in phenotypes with more complex morphology than any growth factor alone. FGF4/FGF1 resulted in larger glands with longer ducts. FGF10/FGF2 and FGF7/FGF1 both resulted in elongated stalks and enlarged buds. Combinations of FGF10 or FGF7 with either BMP4 (shown) or BMP7 (similar result, not shown) did not grow, suggesting BMPs antagonize FGF7 and FGF10. FGF2/FGF7/FGF10 resulted in multiple stalks and buds. FGF1/FGF2/FGF10 resulted in the most complex morphology with elongated ducts, multiple branch points and enlarged terminal epithelial buds. Scale bars: 200 µm.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Isolation, Cell Culture

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 7. FGFR1 and FGFR2 are expressed throughout the epithelium. (A) FGFR1 staining (red) is increased around the edge of the bud with FGF7 treatment, and near the tip of the duct with FGF10 treatment. E- cadherin (green) defines the cell junctions of the epithelium, and SYBR-green stains the nucleus (pseudocolored blue). (B) FGFR2 staining (red) is localized throughout the epithelium in a punctate pattern and shows less cell membrane localization with FGF7 than with FGF10 treatment. E-cadherin (green) defines the cell junctions of the epithelium and SYBR- green stains the nucleus (pseudocolored blue). Scale bars: 20 µm.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Staining, SYBR Green Assay, Membrane

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 8. FGF7- and FGF10-mediated morphogenesis is ERK1/2 dependent. FGF7-mediated morphogenesis is also PI3K dependent. FGF7- (200 ng/ml) and FGF10- (1500 ng/ml) mediated morphogenesis is inhibited by the FGFR inhibitor SU5402 (SU, 2.5 µM), the MEK1/2 inhibitor UO126 (UO, 10 µM), and by rFGFR2b (R2b, 5 µg/ml), but not by the broad PKC inhibitor GO6983 (GO, 1.5 µM), or by rFGFR1b (R1b, 10 µg/ml). FGF7-mediated morphogenesis is also inhibited by the PI3K inhibitor LY294002 (LY, 10 µM). Star indicates an inhibited phenotype. The inhibitors were added to the media for 44 hours, and the number of buds (for FGF7) and the length of ducts (for FGF10) were measured using MetaMorph software and expressed as a ratio at 44 hours/time 0. *, P<0.5; **, P<0.01. See Movie 2 in supplementary material, which shows FGF7- and FGF10-treated epithelium cultured for 36 hours.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Software, Cell Culture

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 9. FGF-mediated morphogenesis is MMP-dependent. (A, part a,b) MMP2 is localized in the mesenchyme and in the epithelium at the periphery of the growing buds, while MMP9 is localized only in the mesenchyme. Perlecan, produced by the mesenchyme, stains the basement membrane. LSCM sections of E13 cultured glands. Scale bar: 100 µm. (A, part c,d) MMP2 is localized at the periphery of the epithelium and in residual mesenchyme cells (dotted outline) in both (c) FGF7- and (d) FGF10-treated epithelium. (A, part e,f) MMP9 is localized only in the residual mesenchyme cells (dotted outline) in both FGF7- and FGF10-treated epithelium. α6 integrin and peanut lectin (PNA) are epithelial cell markers. Scale bars: 20 µm. (B) The relative levels of gene expression of MMPs, FGFs and FGFRs were compared by real-time PCR in salivary epithelium treated with either FGF7 (200 ng/ml) or FGF10 (1500 ng/ml) for 44 hours. MMP2 gene expression was increased ~4-fold, FGF1 expression was increased ~2.5-fold and FGFR1b expression was increased ~2-fold in FGF7-treated epithelial rudiments, when compared with FGF10-treated epithelial rudiments. (C) FGF7 increases MMP2 production and activation after 44 hours. The culture media from epithelium treated with FGF7 or FGF10, as well as media from a dish with laminin-1 alone, was assayed by gelatin zymography after 24 and 44 hours of FGF treatment. (D) FGF7- and FGF10-mediated morphogenesis is inhibited by GM6001 (5 µM), a broad MMP inhibitor, and by a neutralizing antibody to FGF1 (25 µg/ml). Neither a DMSO carrier control (shown) nor an IgG control (not shown) inhibited morphogenesis.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Produced, Membrane, Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Activation Assay, Zymography, Control

Journal: Development (Cambridge, England)

Article Title: FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

doi: 10.1242/dev.01690

Figure Lengend Snippet: Fig. 10. Working model of how FGF7 and FGF10 signaling through FGFR2b regulates morphogenesis. The model summarizes our findings, and the dotted lines show other potential mechanisms: MMP2 may regulate FGFR1 cleavage; FGF1 expression may stimulate both FGFR1 and FGFR2; cofactors or co-receptors may specify the localization of FGF binding and, therefore, where proliferation occurs.

Article Snippet: Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7 (MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody previously used for neutralization of FGF10 activity (sc7375-L) (Harada et al., 2002), and a goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the culture medium at concentrations of 10, 25 and 50 μg/ml.

Techniques: Expressing, Binding Assay

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure 1. The RP1 protein is located in the outer segment portion of the photoreceptor axoneme. Frozen sections (A–C) or isolated photoreceptor outer segments (D) from adult C57BL/6 mouse retinas were double immunostained with antibodies to Rp1andacetylated-tubulin(A,D),Rp1andRom1(B),orRp1andRPGR(C).Thecoimmunostainedsampleswereviewedwith aZeissLSM510Metaconfocalmicroscope.ForA–C,separatedimagesforthetwolabelsarepresentedincolumns1and2.Merged imagesareshownincolumns3and4,withhigher-magnificationviewsincolumn4.Representativequantitativeanalysesofthe immunostaining patterns are shown in column 5. For these analyses, the intensity of the different fluorescent labels was evalu- ated in each pixel along a line drawn through the axoneme using the LSM 510 Meta analysis software (lines A4, B3, C4). The separated signal intensities for each dye were plotted as a function of distance. A, D, The Rp1 protein colocalizes with the acetylated-tubulin-labeledaxonemeintheoutersegmentbutnotintheconnectingcilium,asindicatedbythegreensignalthat extends above the outer segment into the connecting cilium (A4, D3, arrowheads). B, Rp1 is located in the proximal portion of outersegments,asindicatedbythecolocalizationoftheRp1andRom1signals.C,TheRp1signaldoesnotoverlapwiththeRPGR signalintheconnectingcilium,butratherthereisagapbetweenthetwosignals(C4,arrow).E,Diagramofrodphotoreceptorcell. The region of the connecting cilium and the base of the outer segment are enlarged on the right to show the axoneme in the connectingciliumandoutersegment.Thisophthalmicorientation,withtheoutersegmentsofthephotoreceptorspointingdown, is used in all the figures. IS, Inner segment; OS, outer segment; ac-tu, acetylated -tubulin.

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: Isolation, Immunostaining, Software

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure 2. Immunoelectron microscopy of RP1 in the axoneme. Ultrathin sections of mouse retina were probed with anti-C-Rp1 antibodies, followed by gold-conjugated secondary anti- bodies.A,Lower-magnificationimageshowingthattheRp1labelingislocatedalongthelength of the axoneme (arrowheads), in both the connecting cilium (CC) and outer segment (OS). B, Higher-magnificationimageshowingthatRp1isconcentratedintheoutersegmentportionof the axoneme, next to the newly formed discs (arrow).

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: Immuno-Electron Microscopy

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure 3. Rp1 coassembles with microtubules. A, Microtubules were assembled from cyto- solic extracts of mouse retina by repeated cycles of polymerization–depolymerization. The complete cytosol (total), the supernatant of the first spin, and the final microtubule pellet (MT pellet) were analyzed by Western blot analysis using two different anti-Rp1 antibodies, as indicated. The 240 kDa RP1 protein was detected in the microtubule pellet by both antibodies. B, Diagram of the full-length RP1 cDNA and four cDNA fragments used for COS-7 transfection experiments.TheseconstructscontainaC-terminalV5epitopetagtofacilitateidentificationof the recombinant RP1 proteins. The RP1 codons included are indicated within the body of each construct. The DCX domains are indicated in black; the V5 epitope tag is indicated in white. C, The ability of recombinant N1-RP1 and N2-RP1 proteins produced in COS-7 cells to bind to microtubuleswastestedusingacosedimentationassay.Thestartingmaterial(total),superna- tant, and microtubule pellet (MT Pellet) were analyzed by Western blot analysis using anti-V5 antibodiestodetecttherecombinantRP1proteins.Thesizesofthedetectedproteinsareshown ontheright.TheN1-RP1protein(80kDa),whichcontainstheDCXdomains,co-sedimentswith themicrotubulepellets.TheN2-RP1protein(53kDa),whichlackstheDCXdomains,wasfound in the supernatant.

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: Western Blot, Transfection, Recombinant, Construct, Produced

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure4. TheDCXdomainsinRP1areactive.TheRP1cDNAconstructsindicatedontheleftweretransfectedintoCOS-7cells, andthecellswerecoimmunostainedwithanti-V5antibodiestodetecttherecombinantRP1proteins(A1–D1,red)andantibodies to -tubulin to detect the microtubule cytoskeleton (A2–D2, green). Merged images are shown in column 3. Higher- magnificationviewsfromtheimagesincolumn3areshownincolumn4.A,B,Thefull-lengthandN1-RP1proteinscolocalizewith cytoplasmic microtubules. C, Treatment of cells transfected with the N1-RP1 construct with 20 M nocodazole for 2 hr before immunostainingeliminatestheRP1andmicrotubulenetworks.D,TheN2-RP1proteindoesnotcolocalizewithmicrotubulesbut rather is diffuse in the cytoplasm.

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: Transfection, Construct

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure 5. N1-RP1 stabilizes microtubules. Cells transfected with either the N1-RP1 (A) or N2-RP1 (B) cDNA constructs were treated with 0, 2.5, 5, or 10 M nocodazole for 2 hr, as indicated.Thecellswerethencoimmunostainedwithanti-V5(red)andanti--tubulin(green) antibodies to assess the stability of the microtubule network in transfected cells. Nuclei were counterstained with DAPI (blue). Cells transfected with N1-RP1 retained intact cytoplasmic microtubule networks up to 5 M nocodazole (A1–A3). N2-RP1 did not colocalize with micro- tubules, and treatment of N2-RP1-transfected cells with all concentrations of nocodazole caused loss of microtubule networks (B1–B4). After treatment with 10 M nocodazole, the cytoplasmic microtubules of the N1-RP1-transfected cells were also partially disrupted, al- though some polymerized microtubules remained in transfected cells, especially in the micro- tubule organizing centers. Even the microtubule organizing centers have been lost in the sur- rounding untransfected cells and N2-RP1-transfected cells subjected to 10 M nocodazole (A4–B4).

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: Transfection, Construct

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure6. N1-RP1stimulatesmicrotubulepolymerization.N1-RP1andN2-RP1recombinant proteins were incubated with purified tubulin at 37°C, and the extent of polymerization into microtubules was measured by absorbance at 340 nm over 60 min at 1 min intervals. The N1-RP1 protein was almost as efficient as the positive control Taxol at promoting microtubule assembly. In contrast, the N2-RP1 protein was much less active. Tubulin alone served as a negative control.

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: Incubation, Purification, Positive Control, Negative Control

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure 7. Rp1 proteins with the DCX domains stabilize the photoreceptor axoneme. A, The wild-type and targeted Rp1 loci are depicted. The DCX domains contained in exons 2 and 3 are showninyellow.Theneomycinselectioncassettesareshowninblue.The10aminoacidmyctag oftheRp1-mycalleleisshowninred.B,Photoreceptoraxonemeswereisolatedfromtheretinas of mice with the genotypes indicated and coimmunostained with antibodies to Rp1 (red) and -tubulin (green). The stained axonemes were then viewed by confocal DIC and fluorescence microscopy.AxonemesfromRp1 /andRp1myc/mycmicewerewellpreservedandmeasured 11–12 m from the basal body (arrows) to the distal end. In contrast, axonemes from the Rp2–3/2–3miceweremuchshorter,measuring3m.TheRp1signalintheRp2–3/2–3

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: Staining, Fluorescence, Microscopy

Journal: Journal of Neuroscience

Article Title: The Retinitis Pigmentosa 1 Protein Is a Photoreceptor Microtubule-Associated Protein

doi: 10.1523/jneurosci.1335-04.2004

Figure Lengend Snippet: Figure 8. The DCX domains in the Rp1 protein are active in vivo. A, Frozen sections of retina from mice with the genotypes indicated were stained with antibodies to acetylated -tubulin and viewedbyconfocalmicroscopy.Individualaxonemeswereidentifiedbyviewingtheconfocalimagesathighmagnification.Theaxonemesweretraced(exampletracesinred),andthecordlengths of50axonemesfromeachtypeofmousewerethenmeasuredusingtheLSM510Metasoftware.ONL,Outernuclearlayer;IS,innersegment;CC,connectingcilium;AXN,axoneme;OS,outersegment; RPE, retinal pigment epithelium. B, The mean lengths SD of the 50 axonemes from each genotype of mouse are indicated. The length of the axonemes in the Rp2–3/2–3 mice is significantly shorter than those of the wild-type and Rp1-myc mice (*p 0.01). C, Frozen sections of the retinas from 2-week-old double mutant Rp1myc/2–3 mice were coimmunostained with pairs of antibodiestodetecttheRp1-2–3protein(anti-C-Rp1,red),theRp1-mycprotein(anti-myc,green),andtheconnectingcilium(anti-RPGR,blue).Themergedimagesandquantitativeanalysesare shown for each antibody pair. The analysis profiles start proximally and proceed distally into the outer segment. The Rp1-myc protein is localized correctly to the outer segment portion of the axoneme (C1, C3). In contrast, the Rp1-2–3 protein is mislocalized into the connecting cilium and inner segment (C1, C2). D, Diagram of the junction between the inner and outer segments of a rod photoreceptor cell, showing the location of the wild-type Rp1 and Rp1-myc proteins (green) and the Rp1-2–3 deletion protein (red).

Article Snippet: The primary antibodies used were chicken polyclonal anti-C -Rp1 (Liu et al., 2002), monoclonal anti-myc (Cell Signaling Technology, Beverly, MA), anti-acetylated -tubulin (clone 6 –11B-1; Sigma, St. Louis, MO), antiRom1 (Rom 1D5; a gift from Dr. Robert Molday, University of British Columbia, Vancouver, Canada), rabbit polyclonal anti-RPGR, and antiRPGR interacting protein (RPGRIP) (gifts from Dr. Tiansen Li, Harvard University, Cambridge, MA).

Techniques: In Vivo, Staining, Mutagenesis

Journal: Nature genetics

Article Title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions

doi: 10.1038/s41588-018-0274-x

Figure Lengend Snippet: a, Schematic of DNMT3A protein and domains. Position of microcephalic dwarfism (MD) mutations (red) and Tatton-Brown-Rahman syndrome (TBRS) overgrowth (grey) mutations (Tatton-Brown et al. 2014) (MTase, DNA methyltransferase domain) b, The heterozygous de novo c.988T>C mutation results in substitution of a Tryptophan residue (patient 1 and 2). The heterozygous de novo c.997G>A mutation results in substitution of an Aspartic acid residue (patient 3). Both residues are conserved in vertebrates c , and replaced with a physiochemically dissimilar residue: Arginine (p.W330R) and Asparagine (p.D333N) respectively. Sequence alignments, Clustal Omega. d, The W330R and D333N mutations cause extreme growth failure and microcephaly (red diamonds, n=3 independent patients), in direct contrast to DNMT3A overgrowth patients (grey circles, n=13 and n=12 patients, respectively for height and OFC). Height and head circumference (OFC) plotted as z-scores (s.d. for population mean adjusted for age and sex). Dashed lines at −2 and +2 s.d indicate 95% confidence interval for general population. Horizontal bars, mean values for respective patient groups. TBRS morphometric data reproduced from Tatton-Brown et al. 2014 11 .

Article Snippet: Immunoblotting was performed using antibodies to Dnmt3a (Novus Biologicals NB120–13888; 1:500), EZH2 (Cell Signaling #5246S; 1:1000) and Actin (Sigma A2066; 1:5000).

Techniques: Mutagenesis, Residue, Sequencing

Journal: Nature genetics

Article Title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions

doi: 10.1038/s41588-018-0274-x

Figure Lengend Snippet: a, Murine Dnmt3a W326R protein, containing the orthologous substitution to W330R, is stably expressed, in contrast to corresponding overgrowth PWWP mutations (W293del, I306N). Immunoblotting of cell lysates from CRISPR/Cas9 genome-edited mouse embryonic stem cells (mESC). Multiple independent cell lines, with genotypes as indicated. Representative of n=3 (WT, W326R lines) and n=2 (W293del, I306N) independent experiments. Immunoblots are cropped. b, Structural modelling of the PWWP domain predicts the W330R mutation to disrupt interaction with H3K36me3. The highlighted amino acids (blue) form a cage that binds trimethylated lysine 36 (purple). The amino acids altered in MD patients (tryptophan at codon 330 and aspartate at codon 333) are labelled in red. Backbones of PWWP and histone H3 N-terminal tail depicted in grey and pink respectively. c,d, Recombinant PWWP WT but not PWWP W330R protein binds H3K36me3 peptide. ( c ) Schematic of streptavidin pull-down of biotinylated histone peptides. ( d ) Coomassie stained gel of eluted protein from histone peptide pull-downs (cropped). Input, 9% of total protein. Histone peptide H3 (aa 21–44). H3K36me0 corresponding unmodified peptide. Representative of n=3 expts. e, PWWP W330R does not bind H3K36me2, H3K36me3 or other histone-tail modifications. MODified™ Histone Peptide Array representing 384 distinct or combinatorial histone modifications probed with recombinant PWWP proteins as indicated. Below, magnified insets of row L7–11 (histone 3 aa26–45) and K1–3 (histone 3 aa16–35) demonstrates that PWWP WT binds to H3K36me2 (L9) and H3K36me3 (L10), but PWWP W330R does not. Representative of n = 2 independent expts; see also .

Article Snippet: Immunoblotting was performed using antibodies to Dnmt3a (Novus Biologicals NB120–13888; 1:500), EZH2 (Cell Signaling #5246S; 1:1000) and Actin (Sigma A2066; 1:5000).

Techniques: Stable Transfection, Western Blot, CRISPR, Mutagenesis, Recombinant, Staining, Modification, Peptide Microarray

Journal: Nature genetics

Article Title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions

doi: 10.1038/s41588-018-0274-x

Figure Lengend Snippet: a, DNA methylation in DNMT3A W330R/+ patient fibroblasts significantly differs from controls. Unsupervised Ward clustering based on Pearson correlations of all probes from Illumina EPIC arrays for n=2 independent patients and 2 independent controls. Pvclust , approximately-unbiased p -values using 1000 bootstraps. b,c, A methylation signature is evident in DNMT3A W330R patient cells across tissues, comprising 1140 sites of increased methylation. ( b ) Heat map of differentially methylated regions (DMRs) hypermethylated in patient fibroblasts and peripheral blood leukocytes (PBLs). P1, P2, patients ( DNMT3A W330R/+ ); C1-C4 healthy controls; O1, O2, TBRS overgrowth patients. ( c ) Quantification of DNA methylation for DMRs (n=1140 DMRs) depicted in panel ( b ). Box, 25 th -75 th percentile; whiskers, full data range; centre line, median; Δ%mCpG, percent change of methylation relative to mean of control. p value, two-sided, paired Wilcoxon rank sum tests for mean of control probes vs mean patient probes. d, Gene ontology analysis of genes associated with hypermethylated DMRs. Top ten significant hits shown. Color indicates Benjamini-Hochberg adjusted FDR significance level, genes associated with DMR probes (n=907 genes) versus genes associated with all probes on the array (n=18159), two-sided Fisher’s exact test. e, Exemplars of DNA binding factors and morphogens associated with DMRs. f, Representative genome browser views of hypermethylated DMRs demonstrating increased DNA methylation at key developmental genes in microcephalic dwarfism patient samples. All tracks scaled 0–100% mCpG, DNA methylation. CGI, CpG islands.

Article Snippet: Immunoblotting was performed using antibodies to Dnmt3a (Novus Biologicals NB120–13888; 1:500), EZH2 (Cell Signaling #5246S; 1:1000) and Actin (Sigma A2066; 1:5000).

Techniques: DNA Methylation Assay, Methylation, Control, Binding Assay

Journal: Nature genetics

Article Title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions

doi: 10.1038/s41588-018-0274-x

Figure Lengend Snippet: a, Hypermethylated DMRs in DNMT3A W330R patient cells are significantly enriched at poised promoters and polycomb-repressed regions. Plotted, enrichment of chromatin state categories as identified in normal human lung fibroblasts (NHLF) by ChromHMM in patient hypermethylated DMRs. P-values for each enriched category, two-sided Fisher’s exact test hyper-DMR probes (n=10871 probes) vs all probes (n=403348). (ChromHMM: software annotating Chromatin state by a Hidden Markov Model) . b-d, H3K27me3 sites in control dermal fibroblasts correlate with hypermethylated DMRs in patient cells. ( b ) Heat map of normalised H3K27me3 ChIP-seq reads in control fibroblasts (mean of C1, C2) centred on DMRs, ranked by DMR mean H3K27me3 levels. Scale indicates normalised read counts. Window size, 250 bp. ( c,d ) Quantification of H3K27me3 enrichment at hypermethylated DMRs. ( c ) Percentage of Infinium array probes overlapping H3K27me3 peaks in control fibroblasts (red, mean of C1 and C2). All, all probes on the array (n=403348 probes). Hyper-DMRs, probes within hypermethylated DMRs (n=10871). p-value, two-sided Fisher’s exact test. ( d ) Venn diagram displaying overlap of hypermethylated DMRs (n=1140) with H3K27me3 peaks (n=3815) in controls. p value, two-sided Fisher’s exact test. ( e) Genes associated with hypermethylated DMRs (n=907 genes), significantly overlap genes associated with DMVs (n=1,358). Two-sided Fisher’s exact test, genes associated with hyper-DMRs vs all genes represented on array. ( f ) Increased methylation is distributed across H3K27me3 regions, but excluded from H3K4me3 peaks. Representative IGV genome browser views. For all tracks: DNA methylation (magenta, scale 0–100%), H3K27me3 (green, scale 0–4 scaled read counts per 10 7 reads), H3K4me3 (yellow, scale 0–8 scaled read counts per 10 7 reads) in control (C1, C2) and patient (P1, P2) dermal fibroblasts. DNA methylation data for SOX1 and FOXA1 are shown again for comparison with H3K27me3 and H3K4me3. g, Polycomb-marked DNA methylation valleys (DMVs) are hypermethylated in DNMT3A W330R/+ patients. Shown, heat maps of n=1,152 DMVs of normalised H3K27me3/K4me3 read counts for control (C1,C2 mean) and patient (P1,P2 mean) fibroblasts, centred on DMVs and ranked by mean H3K27me3 levels in controls. Δ%mCpG, percent change of DMV methylation relative to mean of controls. Window size, 500bp. h, i, Quantification of data shown in panel g . ( h ) Polycomb-marked DMVs exhibit increased methylation in patient cells, while non-polycomb associated regions do not. Y-axis indicates mean difference between patients and controls: 0, no change; >0 increased in patients; <0 decreased in patients. ( i ) Polycomb-marked DMVs with increased methylation in patient cells, exhibit lower levels of H3K4me3 in controls (C1, C2 mean). Box, 25 th -75 th percentile; (h) whiskers, full data range; (i) whiskers, 1.5x interquartile range; centre line, median. Polycomb marked DMV definition, see methods. (p-values in h,i, two-sided Wilcoxon rank sum tests, polycomb positive (+) (n=524) versus negative (−) (n=628) DMVs).

Article Snippet: Immunoblotting was performed using antibodies to Dnmt3a (Novus Biologicals NB120–13888; 1:500), EZH2 (Cell Signaling #5246S; 1:1000) and Actin (Sigma A2066; 1:5000).

Techniques: Software, Control, ChIP-sequencing, Methylation, DNA Methylation Assay, Comparison

Journal: Nature genetics

Article Title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions

doi: 10.1038/s41588-018-0274-x

Figure Lengend Snippet: a-e, DNA methylation at DMRs occurs during cellular differentiation to embryoid bodies (EBs) and neural progenitor cells (NPCs) in CRISPR/Cas9-edited Dnmt3a W326R mESCs. Bisulfite sequencing of the Hoxc13 locus of ( a ) LIF/serum maintained mESCs, ( b ) after 9 days differentiation to EBs and ( c ) after 9 days neural induction to NPCs. For EBs and NPC differentiation, representative of n=2 independent experiments each. Blocks, independent cell lines; open and closed circles, unmethylated and methylated CpGs, respectively; dots, undetermined methylation status; columns CpG sites; rows individual sequences. Total percentage methylation calculated per sample. ( d ) Genome browser view of RRBS DNA methylation profiles after 9 days neural differentiation. Tracks, independent wild type (dark grey), and Dnmt3a W326R (blue) cell lines. Neural precursor cell H3K27me3 data (magenta) from published ChIP-seq dataset . DNA methylation (scale 0–80%, all tracks). ( e ) Hypermethylated DMRs are enriched for H3K27me3 peaks in wildtype NPCs. Percentage of CpGs observed in RRBS overlapping with H3K27me3 peaks. H3K27me3 data from wild type NPC ChIP-seq dataset . All, all CpGs observed (n=1178718 CpGs). Hyper-DMRs, CpGs within hypermethylated DMRs (3117). P-value, two-sided Fisher’s exact test. f, Hypermethylated gene loci in Dnmt3a W326R NPCs substantially overlap those in patient cells. Venn diagram of orthologous genes (human n=781; mouse n=207) associated with respective DMRs. P-value, two-sided Fisher’s exact test. ( g ) Reduced expression for genes associated with hyper-DMRs is evident during NPC differentiation. RNA-seq data for NPC differentiation experiment from panel d . (n=3 wild-type clones, n=3 Dnmt3a W326R/W326R clones). log2 CPM ratios of Dnmt3a W326R/W326R versus wildtype at 9 day NPC differentiation plotted. Box, 25 th -75 th percentile; whiskers, 1.5x interquartile range from box; centre line, median. Two-sided Wilcoxon rank sum test, All genes with coverage in RRBS (n=12620 genes) vs genes associated with hypo-DMRs (n=169) or hyper-DMRs (n=161). h-i, Neurogenic gene transcription bias in Dnmt3a W326R/W326R NPCs. ( h ) log2 CPM ratios of genes for Dnmt3a W326R/W326R versus wild-type 9 day-differentiated NPCs. All, all genes n=13,022; and gene sets, upregulated (n=3,864 genes), unchanged (n=3,516) and downregulated genes (n=3,281) during differentiation from mESCs to neurons. Box, 25 th -75 th percentile; whiskers, 1.5x interquartile range from box; centre line, median. Two-sided Wilcoxon rank sum test, log2 W326R/WT for All vs up or downregulated gene sets. ( i ) Schematic: Gene sets defined on basis of published dataset of mESC differentiation to terminally differentiated neurons . Downregulated and upregulated gene sets defined as those genes with reduced and increased transcripts respectively in neurons relative to ES cells. The downregulated set therefore contains pluripotency-related genes (light blue) and the upregulated set, neuronal differentiation genes (light red).

Article Snippet: Immunoblotting was performed using antibodies to Dnmt3a (Novus Biologicals NB120–13888; 1:500), EZH2 (Cell Signaling #5246S; 1:1000) and Actin (Sigma A2066; 1:5000).

Techniques: DNA Methylation Assay, Cell Differentiation, CRISPR, Methylation Sequencing, Methylation, ChIP-sequencing, Expressing, RNA Sequencing, Clone Assay

Journal: Nature genetics

Article Title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions

doi: 10.1038/s41588-018-0274-x

Figure Lengend Snippet: a, 10-week old Dnmt3a W326R/+ mouse next to wild-type littermate. ( b ) Body weight for 6 week-old Dnmt3a W326R/+ mice compared to wild type littermates. Males, n=14 wildtype and n=18 Dnmt3a W326R/+ animals. Females, n= 16 wildtype and n=23 Dnmt3a W326R/+ animals. ( c ) Brain weight of female Dnmt3a W326R/+ mice compared to wild type litter mates at 5 months of age. n=7 wildtype and 9 Dnmt3a W326R/+ animals. h,i P -values, two-tailed t-test. Horizontal bar, mean weight per group. ( d ) Locus-specific ( Hoxc13 ) bisulfite sequencing for cortex and liver samples from Dnmt3a W326R/+ and wild-type littermates (n=3/group; female, age 8 weeks). e , Proposed model linking disruption of the H3K36me2/3<--> PWWP interaction with DMV DNA methylation. WT-DNMT3A is normally targeted to H3K36me2 and H3K36me3, marks present widely in the genome , , but rarely coexist with H3K27me3 , . This limits availability of free-DNMT3A to bind at other locations. When the PWWP-H3K36me2/3 interaction is disrupted, sufficient free DNMT3A is available to methylate genomic DNA at DMVs. Enzymatic activity of DNMT3A and DNA methylation impair PRC2 chromatin binding , , leading to secondary loss of H3K27me3. Notably, the long isoform of DNMT3A (DNMT3A1) localises to the edge of Polycomb domains , . When mutated it is therefore well placed to methylate these regions. DNMT3A1 is also the major isoform expressed after ESC differentiation , potentially explaining timing of hypermethylation. Filled circles methylated CpG, open circles unmethylated CpG. Diamonds, H3K36me2/3 modified histones.

Article Snippet: Immunoblotting was performed using antibodies to Dnmt3a (Novus Biologicals NB120–13888; 1:500), EZH2 (Cell Signaling #5246S; 1:1000) and Actin (Sigma A2066; 1:5000).

Techniques: Two Tailed Test, Methylation Sequencing, Disruption, DNA Methylation Assay, Activity Assay, Binding Assay, Methylation, Modification