Morpholinos for splice modificatio

Morpholinos for splice modification

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Fkbp1a controls ventricular myocardium trabeculation and compaction by regulating endocardial Notch1 activity
Hanying Chen, Wenjun Zhang, Xiaoxin Sun, Momoko Yoshimoto, Zhuang Chen, Wuqiang Zhu, Jijia Liu, Yadan Shen, Weidong Yong, Deqiang Li, Jin Zhang, Yang Lin, Baiyan Li, Nathan J. VanDusen, Paige Snider, Robert J. Schwartz, Simon J. Conway, Loren J. Field, Mervin C. Yoder, Anthony B. Firulli, Nadia Carlesso, Jeffrey A. Towbin, Weinian Shou
  1. Fig. 1.

    Genetic ablation of Fkbp1a using Nkx2.5cre. (A) Comparison of gross morphology of Fkbp1aflox/-:Nkx2.5cre and control mouse embryos (top) and hearts (bottom) at E14.5. Fkbp1aflox/-:Nkx2.5cre embryos appear edematous (red arrow) and die in utero. Gray arrow indicates formation of the ventricular groove in the control heart. (B) Histological analysis of Fkbp1aflox/-:Nkx2.5cre and control hearts at E14.5. Arrows indicate the overgrowth of the trabecular myocardium in Fkbp1a mutant hearts. The width of the ventricular compact wall is indicated. The boxed regions are enlarged beneath. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; VSD, ventricular septal defect.

  2. Fig. 2.

    Cardiomyocyte-restricted ablation of Fkbp1a using H1-Ncx-cre transgenic mice. (A) Dual immunofluorescence analyses confirm the genetic ablation of Fkbp1a in developing myocardium at E12.5. Representative confocal images of heart sections co-stained with Alexa Fluor 647-conjugated anti-Fkbp1a antibody (red) and Alexa Fluor 488-conjugated anti-myosin heavy chain antibody (MF-20; green). There is a significant reduction in Fkbp1a in Fkbp1aflox/-:H1-Ncx-cre myocardium. The boxed regions of the merge are enlarged to the right. (B) Western blot confirms the efficient removal of Fkbp1a in Fkbp1aflox/-:H1-Ncx-cre heart. (C) Morphological and histological analysis of Fkbp1aflox/-:H1-Ncx-cre and control hearts at E14.5. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle.

  3. Fig. 3.

    Endothelial-restricted ablation of Fkbp1a using Tie2-cre transgenic mice. (A) Dual immunofluorescence and PCR analyses to confirm the genetic ablation of Fkbp1a in developing endocardial/endothelial cells at E12.5. Representative confocal images of heart sections are co-stained with Alexa Fluor 647-conjugated anti-Fkbp1a antibody (red) and Alexa Fluor 488-conjugated anti-CD31 antibody (green). There is a significant reduction of Fkbp1a in the CD31-positive endothelial cells in Fkbp1aflox/-:Tie2-cre hearts. Beneath is shown a diagnostic genomic PCR analysis of DNA samples isolated from E13.5 heart alongside a schematic of the Fkbp1aflox allele showing the location of diagnostic primers p1, p2 and p3; E, exon. A specific p3-p2 band represents the allele after Cre-loxP-mediated recombination. (B) Morphological and histological analysis of Fkbp1aflox/-:Tie2-cre and control embryos and hearts at E14.5. Fkbp1aflox/-:Tie2-cre embryos are edematous (red arrow). The mutant hearts lack a normal ventricular groove (yellow arrows) and demonstrate hypertrabeculation and noncompaction (black arrows). RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; VSD, ventricular septal defect.

  4. Fig. 4.

    Histological and functional analyses of adult Fkbp1aflox/-:Tie2-cre mice. (A) (Left) Restricted growth of surviving Fkbp1aflox/-:Tie2-cre mice. (Right) Abnormal ventricular wall structure in Fkbp1aflox/-:Tie2-cre hearts, with hypertrabeculation and noncompaction. Arrows indicate abnormal trabecular myocardia and ventricular septum. (B) M-mode echocardiographic analysis of 2-month-old adult males. One-second traces are shown. Both fractional shortening and ejection fraction are severely compromised in Fkbp1aflox/-:Tie2-cre mutants. Values indicate mean ± s.e.m.; n=12; *P<0.05. LVIDd, left ventricular internal diameter diastolic; LVIDs, left ventricular internal diameter systolic; FS%, fractional shortening; LV vol d, left ventricular volume diastolic; LV vol s, left ventricular volume systolic; EF%, ejection fraction; HR, heart rate.

  5. Fig. 5.

    Assessment of Notch1-mediated signaling in the developing endocardium of Fkbp1a-deficient and Fkbp1aflox/-:Tie2-cre mouse hearts. (A) Immunohistological analysis using antibody specific to activated Notch1 (N1ICD). In control heart (WT), nuclear N1ICD is mainly located in endothelial cells at the proximal end of trabeculae (arrows in a′). By contrast, N1ICD was found throughout endothelial cells in both Fkbp1a-deficient and Fkbp1aflox/-:Tie2-cre mutant hearts (arrows in b′ and c′). The boxed regions in a-c are magnified in a′-c′. (B) In situ hybridization of downstream targets of Notch1 in Fkbp1aflox/-:Tie2-cre and control hearts at E12.5. Ephrin B2 (Efnb2), neuregulin 1 (Nrg1) and Hey1 are upregulated in endocardial cells in Fkbp1aflox/-:Tie2-cre hearts. Bmp10 is upregulated in both trabecular and compact myocardium. Interestingly, Hey2 expression is upregulated in trabecular myocardium compared with controls. Arrows indicate positive signals. (C) qRT-PCR confirms the expression levels of Notch1, Efnb2, Nrg1, Hey1, Hey2 and Bmp10 in Fkbp1aflox/-:Tie2-cre hearts at E13.5. Interestingly, despite the altered expression pattern, the overall expression level of Hey2 is not altered. Error bars indicate s.e.m. LV, left ventricle.

  6. Fig. 6.

    Biochemical analyses of altered Notch1 activity in Fkbp1a mutant cells. (A) (Left) Luciferase assay to determine Notch1 activity in Fkbp1a-deficient and control mouse embryonic fibroblasts (MEFs) in which Hes1-luciferase reporter was transfected together with Notch1, Dll4, Fkbp1a and eGFP as indicated. Fkbp1a-deficient cells maintain significantly higher luciferase activity, which is reduced by Fkbp1a re-introduction. (Right) Western blot analysis of endogenous N1ICD levels in Fkbp1a-deficient and wild-type MEFs. (B) (Left) Luciferase assay to determine Notch1 activity in Fkbp1a-deficient and control mouse endothelial cells. (Right) Western blot shows that the N1ICD protein level is significantly higher in Fkbp1a-deficient cells. (C) (Top) Western blot showing that the N1ICD protein level is downregulated in mouse endothelial cells overexpressing human FKBP1A. (Bottom) Luciferase assay to determine Notch1 activity in FKBP1A-overexpressing and control endothelial cells. (D) qRT-PCR analysis shows that Notch1 mRNA levels are not altered in Fkbp1a-deficient or FKBP1A-overexpressing endothelial cells. However, Efnb2, Hey1 and Hey2 expression levels are altered. Error bars indicate s.e.m.

  7. Fig. 7.

    Biochemical analysis of N1ICD stability and degradation. (A) Assessment of the N1ICD degradation rate in HEK293 versus FKBP1A-HEK293 cells and in N1ICD-Fkbp1aflox/flox versus N1ICD-Fkbp1aflox/flox/Cre mouse endothelial cells. Cells were treated with the indicated concentrations of cycloheximide (CHX). Representative western blots show the N1ICD protein level at different time points after CHX treatment in HEK293 cells and endothelial cells (EC) of different genotype (a and c), and representative analyses of N1ICD degradation in FKBP1A-HEK293 cells versus control HEK293 cells (b) and N1ICD-Fkbp1aflox/flox versus N1ICD-Fkbp1aflox/flox/Cre endothelial cells (d). (B) Representative western blot of three independent experiments showing that N1ICD protein synthesis is not affected in FKBP1A-HEK293 cells. (C) Ubiquitylated N1ICD is significantly more abundant in FKBP1A-HEK293 cells. To detect the ubiquitylated N1ICD protein, 6× histidine-tagged ubiquitin (His-ubiquitin) plasmid was co-transfected with N1ICD expression plasmid into HEK293 or FKBP1A-HEK293 cells. Transfected cells were incubated with the proteasome inhibitor MG-132 (10 μmol) for 8 hours before cell lysis and Ni-NTA pulldown assay. Error bars indicate s.e.m.

  8. Fig. 8.

    Rescue of Fkbp1a-deficient mice with the γ-secretase inhibitor DBZ. (A) Representative images of E13.5 hearts isolated from DBZ-treated and control mouse embryos. (B) Quantification of the thickness of trabeculae (a), the thickness of the compact wall (b), the ratio of the thickness of trabeculae and compact wall (c), and the ratio of overall trabecular area versus the length of the compact wall (d). The data demonstrate that the thickness of ventricular trabeculae is reduced in DBZ-treated Fkbp1a-deficient hearts, and the ratio of trabecular thickness versus compact wall thickness is close to normal when compared with controls. Error bars indicate s.e.m. (C) Representative images of three independent sets of Efnb2, Nrg1 and Bmp10 in situ hybridization and Ki67 immunohistological staining of E13.5 hearts from DBZ-treated or vehicle-treated Fkbp1a-deficient and control Fkbp1a heterozygous embryos. LV, left ventricle; RV, right ventricle.