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. 2015 Sep 1;142(17):2962-71.
doi: 10.1242/dev.125807. Epub 2015 Aug 7.

Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest

Lauren J Manderfield  1 , Haig Aghajanian  1 , Kurt A Engleka  1 , Lillian Y Lim  1 , Feiyan Liu  1 , Rajan Jain  1 , Li Li  1 , Eric N Olson  2 , Jonathan A Epstein  3
Affiliations

Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest

Lauren J Manderfield et al. Development. .

Abstract

Notch signaling has well-defined roles in the assembly of arterial walls and in the development of the endothelium and smooth muscle of the vasculature. Hippo signaling regulates cellular growth in many tissues, and contributes to regulation of organ size, in addition to other functions. Here, we show that the Notch and Hippo pathways converge to regulate smooth muscle differentiation of the neural crest, which is crucial for normal development of the aortic arch arteries and cranial vasculature during embryonic development. Neural crest-specific deletion of the Hippo effectors Yap and Taz produces neural crest precursors that migrate normally, but fail to produce vascular smooth muscle, and Notch target genes such as Jagged1 fail to activate normally. We show that Yap is normally recruited to a tissue-specific Jagged1 enhancer by directly interacting with the Notch intracellular domain (NICD). The Yap-NICD complex is recruited to chromatin by the DNA-binding protein Rbp-J in a Tead-independent fashion. Thus, Hippo signaling can modulate Notch signaling outputs, and components of the Hippo and Notch pathways physically interact. Convergence of Hippo and Notch pathways by the mechanisms described here might be relevant for the function of these signaling cascades in many tissues and in diseases such as cancer.

Keywords: Hippo signaling; Jagged1; Mouse; Neural crest; Notch signaling; Taz; Vascular development; Yap.

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Figures

Fig. 1.
Fig. 1.
Deletion of Yap and Taz in neural crest results in impaired smooth muscle differentiation. (A) Wnt1-Cre; Taz flox/+;Yap flox/+; R26Tom/+ E10.5 embryo imaged in bright-field and fluorescence. (B-H) Transverse sections of E10.5 Wnt1-Cre; Tazflox/+;Yapflox/+; R26Tom/+ embryos stained for tdTomato (RFP) and SM22α. (I) Wnt1-Cre; Tazflox/flox;Yapflox/flox;R26Tom/+ embryo imaged in bright-field and fluorescence. (J-P) Transverse sections of E10.5 Wnt1-Cre; Tazflox/flox;Yapflox/flox;R26Tom/+ embryos stained for tdTomato (RFP) and SM22α. Arrowheads denote sites of decreased smooth muscle differentiation. Branchial arches (ba) are invested with red-fluorescing Wnt1-derived neural crest (A,I). iii, third aortic arch artery; Ao, aortic sac. Images in B,E,H,J,M,P were merged by combining respective red and green channels using Photoshop software (Adobe). Scale bars: 100 μm.
Fig. 2.
Fig. 2.
Deletion of Yap and Taz in neural crest results in impaired smooth muscle expression. (A-I) Transverse sections of E10.5 Wnt1-Cre; Tazflox/+;Yapflox/+; R26Tom/+ embryos stained for tdTomato (RFP, A), SMA (B), merged RFP/SMA (C), tdTomato (RFP, D), desmin (E) or merged RFP/desmin (F). Yellow arrows denote RFP/desmin double-positive cells. Serial sections stained for tdTomato and Hoechst (RFP, G), smMyosin (H) or smMyosin and Hoechst (I). White arrows denote presumptive RFP/smMyosin double-positive cells. (J-R) Transverse sections of E10.5 Wnt1-Cre; Tazflox/flox;Yapflox/flox;R26Tom/+ embryos stained for tdTomato (RFP, J), SMA (K), merged RFP/SMA (L), tdTomato (RFP, M), desmin (N) or merged RFP/desmin (O). Serial sections stained for tdTomato and Hoechst (RFP, P), smMyosin (Q) or smMyosin and Hoechst (R). White arrows highlight RFP–, smMyosin+ cells. Merged images were generated by combining respective red and green channels using Photoshop (C,F,L,O). iii, third aortic arch artery. Scale bars: 100 μm.
Fig. 3.
Fig. 3.
Mesenchyme adjacent the third aortic arch artery expresses Yap. (A-E) Transverse sections of wild-type E10.5 embryos stained for Yap (A), Yap, eNOS and Hoechst (B), phospho-Yap (pYap, C), pYap, eNOS and Hoechst (D), pYap and Hoechst (E) or eNOS and Hoechst (F). White arrows denote pYap/eNOS double-positive cells. The boxed area in D is shown at higher magnification with single stains in E,F. iii, third aortic arch artery. Scale bars: 100 μm in A-D; 20 μm in E,F.
Fig. 4.
Fig. 4.
Decreased Notch activity following Yap/Taz deletion. (A-D) Transverse sections of E10.5 Tazflox/flox;Yapflox/flox embryos immunostained for Jagged1 (A), Jagged1 with eNOS (B), NICD (C) or NICD with eNOS (D). (E-H) Transverse sections of E10.5 Wnt1-Cre; Tazflox/flox;Yapflox/flox embryos immunostained for Jagged1 (E), Jagged1 with eNOS (F), NICD (G) or NICD with eNOS (H). (I) Anti-Yap and anti-Taz immunoblots from Tazflox/flox;Yapflox/flox MEFs untreated (0) or treated with increasing AAV-CMV-Cre doses [500 genome copies per cell (GC), 1000 GC or 2000 GC]. In parallel, immunoblots of the same protein lysates were probed with anti-actin to demonstrate equivalent protein loading. (J) qRT-PCR of untreated or AAV-CMV-Cre virus-treated (1000 GC) Tazflox/flox;Yapflox/flox MEFs for c-myc, Hrt1, Hrt2, Hrt3 and Jagged1. Data depicted in J are mean+s.e.m. Statistics were completed using Student's t-test. **P<0.01, ***P<0.001. iii, third aortic arch artery. Scale bars: 100 μm.
Fig. 5.
Fig. 5.
NICD transcriptional activity is increased in the presence of Yap and the activation is Tead1 independent. Results of dual luciferase reporter assays in HEK293T cells are shown. (A) Hes1 reporter assay in the presence (+) or absence (−) of NICD, Yap or Dntead1, n=3. Complete ANOVA results are included in supplementary material Table S1. (B) 8× GTIIC-Tead-reporter luciferase assay in the presence (+) or absence (−) of Yap or Dntead1, n=3. (C) Jagged1 enhancer element (ECR6)-luciferase reporter assay in the presence (+) or absence (−) of NICD, Yap or Dntead1, n=3. Complete ANOVA results are included in supplementary material Table S2. (D) ECR6-luciferase reporter assay in the presence (+) or absence (−) of NICD, Yap, Mst1 or a kinase-inactive form of Mst1, Mst1-KI, n=4. Complete ANOVA results are included in supplementary material Table S3. All experiments were performed in duplicate for a minimum of three individual occasions, with specific replicate numbers shown in each legend. Data depicted are mean+s.e.m. Statistics were completed using ANOVA with a Tukey–Kramer post-hoc comparison test. ***P<0.001, **P<0.01, *P<0.05.
Fig. 6.
Fig. 6.
NICD and Yap specifically co-occupy Jagged1 ECR6 and physically interact. (A) Chromatin immunoprecipitation (ChIP) for NICD (FLAG) or Yap from cells transfected with a 3× FLAG-tagged NICD construct, a Yap expression construct and either an ECR1 expression plasmid, an ECR6 expression plasmid or a mutant ECR6 expression plasmid (ECR6*). Data are reported as fold enrichment over an IgG ChIP performed in parallel with the same samples. (B) Results of dual luciferase reporter assays in HEK293T cells with a Jagged1 enhancer element (ECR6), using either a wild-type ECR6-luciferase reporter or a mutant ECR6-luciferase reporter (ECR6* reporter) with a mutated Rbp-J binding site in the presence (+) or absence (−) of NICD or Yap, n=3. (C) Western blots demonstrating co-immunoprecipitation of Yap and NICD. Lysates were immunoprecipitated with either a control IgG or FLAG antibody in the presence (+) or absence (−) of Yap and NICD. The Yap construct contains an N-terminal FLAG epitope tag. The NICD construct contains a C-terminal V5 epitope tag. Input immunoblots confirmed NICD (V5) and Yap (FLAG) expression in specified samples. β-actin immunoblot confirmed protein expression in all samples. (D) ChIP for endogenous Yap from non-transfected MDA-MB-231 cells at Jagged1 genomic loci, ECR1 and ECR6, and the Hes1 promoter. Data are reported as fold enrichment over an IgG ChIP performed in parallel with the same samples. Dual luciferase experiments were performed in duplicate for a minimum of three individual occasions. ChIP experiments were also completed in biological triplicate. Data depicted are mean+s.e.m. Statistics were completed using ANOVA with a Tukey–Kramer post-hoc comparison test. ***P<0.001, **P<0.01, *P<0.05.
Fig. 7.
Fig. 7.
NICD and Yap interact and modulate transcription in smooth muscle cells. (A) RT-PCR from three biological replicates of MOVAS cells. (B) qRT-PCR of vehicle (DMSO)- or DAPT (100 µM)-treated MOVAS cells. Data depicted are mean+s.e.m. and the statistics were completed using Student's t-test; ***P<0.001. (C) Anti-myc (Lats2) and anti-pYap immunoblots from MOVAS cells transfected with increasing amounts of myc-tagged Lats2. In parallel, immunoblots of the same protein lysates were probed with anti-actin to demonstrate equivalent protein loading. (D) qRT-PCR of non-transfected (NT) or Lats2 (2 µg)-transfected MOVAS cells. Data depicted are mean+s.e.m. and the statistics were completed using Student's t-test; *P<0.05. (E) Western blot demonstrating co-immunoprecipitation of Yap and NICD in MOVAS cells. Lysates were immunoprecipitated with either a control IgG or NICD antibody. Input immunoblots confirmed NICD and Yap expression. (F) ChIP for endogenous Yap and NICD in non-transfected MOVAS cells at Jagged1 genomic loci, ECR1 and ECR6, and the Hes1 promoter. Data are reported as fold enrichment over an IgG ChIP performed in parallel with the same samples. Data depicted are mean+s.e.m. The statistics were completed using Student's t-test; **P<0.01, *P<0.05. qRT-PCR and ChIP experiments were completed in biological triplicate.
Fig. 8.
Fig. 8.
NICD-Yap interaction requires the first Yap WW domain. (A) Results of dual luciferase reporter assays in HEK293T cells with an ECR6-luciferase reporter in the presence (+) or absence (−) of NICD, Yap or Yap mutants, Yap-WW1, Yap-WW2 or Yap-WW1WW2, n=4. Complete ANOVA results are included in supplementary material Table S4. (B) Results of dual luciferase reporter assays in HEK293T cells with 8× GTIIC-Tead luciferase reporter in the presence (+) or absence (−) of Yap or Yap mutants, Yap-WW1, Yap-WW2 or Yap-WW1WW2, n=3. (C) Anti-FLAG and anti-Yap immunoblots from HEK293T cells transfected with FLAG-tagged Yap or FLAG-tagged Yap mutants. (D) Examination of NICD-Yap interaction using Duolink reagents. Interaction is documented as red fluorescence. Samples were co-transfected in the presence (+) or absence (−) of the specified plasmids. NICD-V5+Rbp-J served as positive control. The samples in the bottom row were not transfected with NICD-V5, but did include the V5 antibody to serve as negative controls. Scale bars: 100 µm. Dual luciferase experiments were performed in duplicate for a minimum of three individual occasions. Data depicted are mean+s.e.m. Statistics were completed using ANOVA with a Tukey–Kramer post-hoc comparison test. ***P<0.001, **P<0.01.
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