Research ArticleVASCULAR BIOLOGY

Inactivating mutations in Drosha mediate vascular abnormalities similar to hereditary hemorrhagic telangiectasia

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Science Signaling  16 Jan 2018:
Vol. 11, Issue 513, eaan6831
DOI: 10.1126/scisignal.aan6831
  • Fig. 1 Drosha morphants exhibit vascular abnormalities with increased vascular permeability.

    (A) Two different Drosha morpholino oligonucleotides (MO) (MO1 or MO2) or control MO (Ctrl) were injected into Tg(flk1:egfp)s843; Tg(gata1:dsRed)sd2 zebrafish at the one- to two-cell stage. Representative images of intersegmental vessel (ISV) are shown. n = 33 zebrafish for Ctrl MO, 34 zebrafish for MO1, and 35 zebrafish MO2. The fraction (%) of fish with defective ISVs and vascular density are shown as means ± SEM. White arrow, defective ISVs. **P < 0.01, ***P < 0.001, ****P < 0.0001, significant by one-way analysis of variance (ANOVA) with post hoc Tukey’s test. (B and C) Ctrl MO (2 ng) or Drosha MO (1 ng of MO1 + 1 ng of MO2) was injected into Tg(fli1:nEGFP)y7 (B) or Tg(flk1:egfp)s843 (C) zebrafish at the one- to two-cell stage. (B) Representative images of the anterior (A), middle (M), or posterior (P) regions of the ISVs. The number of endothelial cells (ECs) per ISV was shown as means ± SEM. n = 33 zebrafish for Ctrl MO and 28 zebrafish for Drosha MO. (C) Representative angiography images of ISVs and the caudal plexus are shown. Mean fluorescence density of the caudal plexus is presented as means ± SEM. n = 18 zebrafish for Ctrl MO and 20 for Drosha MO. a.u., arbitrary units. *P < 0.05, **P < 0.01, ***P < 0.001, significant by two-tailed unpaired Student’s t test. Scale bars (A to C), 200 μm.

  • Fig. 2 Depletion of Drosha in the endothelium in mice results in embryonic developmental vascular abnormalities similar to HHT.

    (A) Representative images of a whole-mount liver from control (Ctrl) or Drosha cKOEC embryos at embryonic day (E) 12.5 stained with CD31 are shown. Quantitative analysis of the diameter of fetal liver capillaries is shown as means ± SEM. Scale bar, 100 μm. n = 4 Ctrl embryos and 3 cKOEC embryos. (B) β-Galactosidase staining was performed to visualize the dorsal aorta (DA) isolated from Ctrl or cKOEC embryo at E12.5. Quantitative analysis of the DA diameter is shown as means ± SEM. ***P < 0.001, significant by two-tailed unpaired Student’s t test. Scale bar, 50 μm. n = 4 Ctrl embryos and 4 cKOEC embryos. (C) Representative hematoxylin and eosin (H&E) stain images of a 7-μm transverse thoracic section of Ctrl or cKOEC embryos at E14.5. Scale bar, 100 μm. The diameter of DA was quantified by ImageJ and presented as means ± SEM. n = 3 Ctrl embryos and 3 cKOEC embryos. *P < 0.05, significant by two-tailed unpaired Student’s t test. Hemorrhagic telangiectasia, HHT.

  • Fig. 3 Postnatal depletion of Drosha in the endothelium in mice results in HHT-like vascular abnormalities.

    (A) Representative images of lungs from 10-week-old Drosha iKOEC or Ctrl mice. White arrow, dilated capillary. n = 4 mice for each genotype. Scale bar, 1 mm. (B) Representative images of livers from 8-month-old Drosha iKOEC or Ctrl mice. White arrow, dilated capillary. Scale bar, 1 mm. n = 3 mice for each genotype. (C) The diameter of lung and liver capillaries was quantified by ImageJ and presented as means ± SEM. *P < 0.05, significant by two-tailed unpaired Student’s t test. (D) Representative images of intestines from 8-month-old Drosha iKOEC or Ctrl mice and feces from 13-month-old Drosha iKOEC or Ctrl mice. A, artery; V, vein. n = 4 Ctrl and 3 iKOEC mice. Scale bar, 1 mm. (E) Representative images of the subcutaneous vasculature in the back of 8-month-old Drosha iKOEC and Ctrl mouse n = 4 mice for each genotype. Scale bar, 500 μm.

  • Fig. 4 HHT pedigrees with DROSHA nonsynonymous substitution alleles and a map of DROSHA variants.

    (A) The pedigree of family 1 carrying the DROSHA P100L variant. Circles and squares indicate females and males, respectively. Black symbols indicate individuals affected with HHT. The + sign indicates carriers of the rare DROSHA variant. (B) The pedigree of family 2 who carry the DROSHA R279L variant. Circles and squares indicate females and males, respectively. The + sign indicates carrier of the DROSHA variant and an ENG c.1311+1G>A mutation. The +’ sign indicates carrier of the DROSHA variant and mosaic carrier of an ENG c.1311+1G>A mutation. (C) Schematic representation of DROSHA protein and variants associated with HHT. Red circles indicate the position of variants and the number of HHT patients. dsRBD, double-stranded RNA–binding domain. Red ovals indicate the number of HHT patients carrying the respective variant. RNase, ribonuclease.

  • Fig. 5 The N-terminal missense mutants of DROSHA are partially inactive.

    (A) Drosha protein abundance in the nuclear extracts of mouse embryonic fibroblasts (MEFs) expressing DROSHA WT, P100L, R279L, or mock was analyzed by immunoblotting. Lamin A/C was used as a loading control. n = 3 independent experiments. (B) The abundance of different microRNAs (miRNAs) (miR-10a, miR-34a, miR-21, miR-103, let-7a, and miR-126) relative to U6 small nuclear RNA was quantitated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in MEFs expressing DROSHA WT, P100L, R279L, or mock (empty vector) and shown as means ± SEM. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, significant by one-way ANOVA with post hoc Tukey’s test. (C) In vivo miRNA processing assay. The nuclear extracts from MEFs expressing DROSHA WT, P100L, R279L, or mock were treated with or without BMP4 for 2 hours. Total RNA was extracted and subjected to qRT-PCR analysis of primary transcripts of miRNAs (pri-miRNA) and precursor miRNAs (pre-miRNA) of miR-21 and miR-100, and DROSHA mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase. RNA abundance relative to nontreated WT MEFs was plotted as means ± SD. n = 3 independent experiments. ***P < 0.001, significant by two-tailed unpaired Student’s t test.

  • Fig. 6 The N-terminal missense mutants of DROSHA show reduced binding activity with cofactors.

    The amount of Drosha cofactors (p68 and Smad1/5/8 associated with Drosha) was examined using nuclear extracts from MEFs stably expressing Flag-tagged WT, P100L, or R279L. Exogenously expressed DROSHA was immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted for p68, p-Smad1/5/8, or Drosha. Representative images are shown. n = 3 independent experiments.

  • Fig. 7 Exogenous expression of DROSHA missense mutants in zebrafish exhibits vascular phenotypes similar to Drosha morphants.

    (A) Control (T7) RNA and DROSHA WT, P100L, or R279L mRNA were injected into Tg(flk1:egfp)s843; Tg(gata1:dsRed)sd2 transgenic zebrafish at the one- to two-cell stage. Representative images of the whole zebrafish (top) and ISVs (bottom) at 54 hours postfertilization (hpf) are shown. n = 36 zebrafish for Ctrl, 30 zebrafish for WT, 28 zebrafish for P100L, and 33 zebrafish for R279L. Scale bar, 50 μm. (B) The fraction (%) of fish with ISV defects (top) and vascular density (bottom) are shown as means ± SEM. *P < 0.05, **P < 0.01, significant by one-way ANOVA with post hoc Tukey’s test. (C) Control (T7) RNA and DROSHA WT, P100L, or R279L mRNA were injected into Tg(flk:mcherry) zebrafish. At 54 hpf, angiography was performed, and representative images are shown (left). Mean fluorescence density was quantified by ImageJ and presented as means ± SEM (right). n = 20 zebrafish for Ctrl, 17 zebrafish for WT, 20 zebrafish for P100L, and 22 zebrafish for R279L. Scale bar, 200 μm. NS, not significant. *P < 0.05, **P < 0.01, significant by one-way ANOVA with post hoc Tukey’s test.

  • Table 1 Rare DROSHA variants identified in HHT patients.

    Sequencing was performed by Sanger sequencing. All nucleotide substitutions are heterozygous. F, family; P, proband; E, epistaxis; T, telangiectasia; GI-T, gastrointestinal telangiectasia; C, cerebral arteriovenous malformation (AVM); H, hepatic AVM; and P, pulmonary AVM. The presence of P32L (rs202053700; frequency, 0.0001), P100L (rs199846087; frequency, 0.0002), and K226E (rs762758438; frequency, 0.00009) was significantly lower in the Exome Aggregation Consortium (ExAC) database (which spans 60,706 unrelated individuals) compared to HHT patients using Fisher’s exact test at P < 0.01. NA, not available.

    IndividualClinical descriptionNucleotide
    change
    Protein changePresence in
    population*
    Family
    segregation
    Predicted effectOther mutation
    P1P, mother with H, sister and
    grandmother have E
    c.95C>Tp.P32L0.0001NADamagingNone
    F1-I-1Severe E, (cauterized age 10)c.299C>Tp.P100L0.0002YesDamagingNone
    F1-II-4Severe E, Tc.299C>Tp.P100L0.0002YesDamagingNone
    F1-III-1E, Tc.299C>Tp.P100L0.0002YesDamagingNone
    F1-III-2E, C (ruptured)c.299C>Tp.P100L0.0002YesDamagingNone
    P4E, Tc.299C>Tp.P100L0.0002NADamagingNone
    P5E, T, P,c.676A>Gp.K226E0.00009NADamaging§None
    F2-I-2E, T, GI-T, P, H, liver shuntsc.836G>Tp.R279LAbsentYesDamagingENG
    F2-II-1E, T, GI-T, multiple Pc.836G>Tp.R279LAbsentYesDamagingENG

    *Presence in population refers to the presence of the variant in the ExAC database.

    †Mutation is predicted to be damaging by SIFT.

    ‡Mutation is predicted to be damaging by SIFT, PolyPhen-2, and Mutation Taster.

    §Mutation is predicted to be damaging by Mutation Taster.

    ¶Mutation is predicted to be damaging by PolyPhen-2 and Mutation Taster.

    ║Affected individuals in family 2 that carries a mosaic ENG c.1311+1G>A splice site mutation.

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/11/513/eaan6831/DC1

      Fig. S1. Drosha morphants develop angiogenesis defects.

      Fig. S2. Generating Drosha mutant fish by CRISPR/Cas9.

      Fig. S3. Drosha morphants showed vascular leakiness.

      Fig. S4. Drosha cKOEC embryos showed mild angiogenesis defects and a sign of hemorrhage.

      Fig. S5. Drosha cKOEC embryos showed disorganized and dilated vasculature.

      Fig. S6. Drosha iKOEC mice show vascular leakage.

      Fig. S7. The distribution of the tight junction protein ZO-1 is disrupted in the endothelial cells of Drosha iKOEC mice.

      Fig. S8. In vitro processing assay of pri-miR-21 and the BMP-Smad signaling pathway in cells expressing DROSHA mutants or depleted of Drosha.

      Fig. S9. ISV defects mediated by the knockdown of Drosha by morpholino oligos are rescued by coinjection of wild-type (WT) Drosha mRNA, but not by mutant mRNA.

    • Supplementary Materials for:

      Inactivating mutations in Drosha mediate vascular abnormalities similar to hereditary hemorrhagic telangiectasia

      Xuan Jiang, Whitney L. Wooderchak-Donahue, Jamie McDonald, Prajakta Ghatpande, Mai Baalbaki, Melissa Sandoval, Daniel Hart, Hilary Clay, Shaun Coughlin, Giorgio Lagna, Pinar Bayrak-Toydemir,* Akiko Hata*

      *Corresponding author. Email: pinar.bayrak-toydemir{at}aruplab.com (P.B.-T.); akiko.hata{at}ucsf.edu (A.H.)

      This PDF file includes:

      • Fig. S1. Drosha morphants develop angiogenesis defects.
      • Fig. S2. Generating Drosha mutant fish by CRISPR/Cas9.
      • Fig. S3. Drosha morphants showed vascular leakiness.
      • Fig. S4. Drosha cKOEC embryos showed mild angiogenesis defects and a sign of hemorrhage.
      • Fig. S5. Drosha cKOEC embryos showed disorganized and dilated vasculature.
      • Fig. S6. Drosha iKOEC mice show vascular leakage.
      • Fig. S7. The distribution of the tight junction protein ZO-1 is disrupted in the endothelial cells of Drosha iKOEC mice.
      • Fig. S8. In vitro processing assay of pri-miR-21 and the BMP-Smad signaling pathway in cells expressing DROSHA mutants or depleted of Drosha.
      • Fig. S9. ISV defects mediated by the knockdown of Drosha by morpholino oligos are rescued by coinjection of wild-type (WT) Drosha mRNA, but not by mutant mRNA.

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      Citation: X. Jiang, W. L. Wooderchak-Donahue, J. McDonald, P. Ghatpande, M. Baalbaki, M. Sandoval, D. Hart, H. Clay, S. Coughlin, G. Lagna, P. Bayrak-Toydemir, A. Hata, Inactivating mutations in Drosha mediate vascular abnormalities similar to hereditary hemorrhagic telangiectasia. Sci. Signal. 11, eaan6831 (2018).

      © 2018 American Association for the Advancement of Science

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