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. 2011 May 13;286(19):17193-204.
doi: 10.1074/jbc.M110.205146. Epub 2011 Mar 14.

Endothelial Jarid2/Jumonji is required for normal cardiac development and proper Notch1 expression

Matthew R Mysliwiec  1 , Emery H Bresnick, Youngsook Lee
Affiliations

Endothelial Jarid2/Jumonji is required for normal cardiac development and proper Notch1 expression

Matthew R Mysliwiec et al. J Biol Chem. .

Abstract

Jarid2/Jumonji critically regulates developmental processes including cardiovascular development. Jarid2 knock-out mice exhibit cardiac defects including hypertrabeculation with noncompaction of the ventricular wall. However, molecular mechanisms underlying Jarid2-mediated cardiac development remain unknown. To determine the cardiac lineage-specific roles of Jarid2, we generated myocardial, epicardial, cardiac neural crest, or endothelial conditional Jarid2 knock-out mice using Cre-loxP technology. Only mice with an endothelial deletion of Jarid2 recapitulate phenotypic defects observed in whole body mutants including hypertrabeculation and noncompaction of the ventricle. To identify potential targets of Jarid2, combinatorial approaches using microarray and candidate gene analyses were employed on Jarid2 knock-out embryonic hearts. Whole body or endothelial deletion of Jarid2 leads to increased endocardial Notch1 expression in the developing ventricle, resulting in increased Notch1-dependent signaling to the adjacent myocardium. Using quantitative chromatin immunoprecipitation analysis, Jarid2 was found to occupy a specific region on the endogenous Notch1 locus. We propose that failure to properly regulate Notch signaling in Jarid2 mutants likely leads to the defects in the developing ventricular chamber. The identification of Jarid2 as a potential regulator of Notch1 signaling has broad implications for many cellular processes including development, stem cell maintenance, and tumor formation.

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Figures

FIGURE 1.
FIGURE 1.
Jarid2 is deleted in the endocardium of Jarid2en mice. A and B, E12.5 Jarid2F/F mouse heart displays Jarid2 expression in the myocardium and endocardium (arrows) as indicated by red fluorescent staining. C, Hoechst staining to indicate nuclei. B and C, the arrows indicate the nuclei of endocardial cells positive for Jarid2 staining. D and E, Jarid2en mice display a lack of red fluorescent staining in the endocardium, indicating a lack of Jarid2 expression. F, Hoechst staining to indicate nuclei. E and F, the arrows indicate the nuclei of endocardial cells negative for Jarid2 staining. The scale bar in A represents 100 μm.
FIGURE 2.
FIGURE 2.
Morphological analysis of Jarid2 mutants. Cardiac transverse sections were hematoxylin and eosin stained at various stages of development. A–F, E10.5 wild type (A and B), Jarid2 KO (C and D), and Jarid2en (E and F) sections. B, D, and F are higher magnifications of A, C, and E, respectively. G–L, E12.5 wild type (G and H), Jarid2 KO (I and J), and Jarid2en (K and L) sections. H, J, and L are higher magnifications of G, I, and K, respectively. M–U, E14 wild type (M–O), Jarid2 KO (P–R), and Jarid2en (S–U) sections. N, Q, and T are higher magnifications of M, P, and S, respectively. O, R, and U show the boxed regions in M, P, and S at a higher magnification. # sign indicates fused septum (M) or VSD (P and S). The spaces between dotted lines indicate compact layers (O, R, and U). The arrowheads indicate endocardium. In C, TM, trabecular myocardium; E, endocardium; CJ, cardiac jelly; CL, compact layer. The scale bars in A, G, and M represent 100, 200, and 300 μm, respectively.
FIGURE 3.
FIGURE 3.
Dysregulated pathways in Jarid2 mutant hearts. A–D, in situ hybridization was performed using DIG-labeled αMHC (A and B) and ANF (C and D) riboprobes on E17.5 control (A and C) and mutant Jarid2en (B and D) mice. The scale bar in A represents 500 μm. E, analyses of all up-regulated genes by DAVID indicate that the Notch signaling pathway is the most significantly up-regulated pathway in Jarid2 KO versus wild type hearts at E17. The numbers in parentheses represent the number of up-regulated genes in the pathway, and # indicates pathways where Notch1 is up-regulated. The p values from DAVID are listed next to the bars. The x axis is relative significance based on inverse p values (1/p).
FIGURE 4.
FIGURE 4.
Identification of genes dysregulated in Jarid2 mutant mice. A–C, qRT-PCR was performed on wild type and Jarid2 KO hearts at E10.5 (A), E12.5 (B), and E15.5 (C) for Notch1, Nrg1, Nrg4, Jagged1, ErbB2, ErbB4, Notch4, Hey1, Hey2, and Delta4. For all qRT-PCR, at least three hearts for each stage were used, and qRT-PCR was performed in duplicate. An asterisk indicates a significant difference when p values are less than 0.05 from a Student's paired t test, and error bars represent the standard error of the mean. D–K, section in situ hybridization was performed using DIG-labeled Notch1 riboprobes on wild type and Jarid2 KO mice at E10.5 (D–G) and E12.5 (H–K). Jarid2 KO mice exhibit higher levels of Notch1, and arrows indicate deeper invagination of endocardium into compact layer in Jarid2 KO heart (K) as compared with wild type (J). rv, right ventricle; lv, left ventricle. L–O, section in situ hybridization was performed using DIG-labeled Nrg1 riboprobes on wild type (L and N) and Jarid2 KO (M and O) at E13.5. Jarid2 KO mice exhibit higher levels of Nrg1. rv, right ventricle; lv, left ventricle. P–S, section in situ hybridization was performed on E13 control (P and R) and Jarid2en (Q and S) mice using a DIG-labeled Notch1 riboprobe. Jarid2en mice exhibit higher levels of Notch1 than a littermate control. The scale bars in D, H, L, and P represent 200 μm.
FIGURE 5.
FIGURE 5.
Notch1 and ErbB2/4 are elevated in Jarid2 KO hearts. A–H, immunostaining for Notch1 was performed on wild type and Jarid2 KO mice at both E10.5 (C and D) and E12.5 (G and H). The arrows indicate higher levels of Notch1 in the endocardium of Jarid2 KO mice (D and H) as compared with wild type mice (C and G). Hoechst staining was done to stain for nuclei (A, B, E, and F). I–L, immunostaining for ErbB2/4 was performed on Jarid2 KO (L) and wild type mice (K) at E15.5. Jarid2 KO mice display higher levels of ErbB2/4 in the myocardium versus wild type. Hoechst staining was performed to stain nuclei (I and J). M–P, Western blotting was performed on E14.5 wild type or Jarid2 KO hearts for N1ICD (M and N; n = 3) or ErbB4 (O and P; n = 5). Protein levels were standardized to β-actin. An asterisk indicates a significant difference when p values are less than 0.05 from a Student's paired t test, and error bars represent the standard error of the mean.
FIGURE 6.
FIGURE 6.
Jarid2 occupies a specific site at the Notch1 locus. A, a VISTA alignment was performed on the Notch1 locus for mouse, monkey, human, and rat to determine conserved regions. In the bottom panel, gray bars indicate regions with greater than 75% sequence conservation, whereas black bars indicate highly conserved exon regions. Arrowheads indicate primer sites in conserved regions. B, Jarid2 occupies a specific region at +1150 bp of the Notch1 locus. qChIP was performed four times on 10 pooled E17 wild type mouse hearts using either preimmune (Pre-IM, gray bar) or a Jarid2 specific antibody (Jarid2 antibody, black bar). IP, immunoprecipitation. C, Jarid2 does not accumulate at the +1150-bp region of the Notch1 locus when Jarid2 is deleted in the endocardium. qChIP was performed three times on three pooled E17 control (gray bar) or Jarid2en (black bar) mouse hearts using a Jarid2 antibody. D, luciferase reporter gene assay. pGL3, pGL3-Notch1 −500 to +350 (N+350), and pGL3-Notch1 −500 to +1750 (N+1750) were transfected into HEK293 cells without or with two doses of Jarid2. The asterisk indicates a significant difference when p values are less than 0.01 from a Student's paired t test, and the error bars represent the standard error of the mean.
FIGURE 7.
FIGURE 7.
Proposed model of how endocardial Jarid2 expression regulates myocardial development. A, in the wild type mouse at later stages of development, Jarid2 represses Notch1 by binding to the Notch1 +1150-bp locus. Notch1 signaling to the myocardium ceases, and there is a decrease in trabecular proliferation and normal terminal differentiation. B, in the Jarid2 KO mouse at later stages of development, Jarid2 does not bind to the +1150-bp Notch1 locus, and Notch1 remains active. The prolonged activation of the Notch1 pathway results in increased trabecular proliferation and defective terminal differentiation.
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