In mammals, organ-extrinsic influences such as nutritional status, circulating growth factors, and hormones have a large impact on organ size control (1
). Several lines of evidence indicate that there are also organ-intrinsic mechanisms to modulate organ size (1
). Insight into genetic pathways regulating organ size has come from Drosophila
, where two main organ-size control pathways are bone morphogenetic protein (Bmp) and Hippo signaling pathways (3
). Whether these growth control mechanisms are broadly conserved in mammalian organs remains unclear.
Organ size is important in cardiac development, because the heart must be large enough to generate a physiological cardiac output but not so large as to block cardiac outflow, as in obstructive cardiomyopathies. The mammalian core Hippo signaling components include Ste20 family kinases Mst1 and Mst2, which are homologous to Drosophila Hippo. Mst kinases form an active complex with WW repeat scaffolding protein Salvador (Salv), also called WW45, that phosphorylates large tumor suppressor homolog (Lats) kinase. Mammals have two Lats genes, Lats1 and Lats2, which are homologous to Drosophila Warts. Lats kinases complex with Mob to phosphorylate Yap and Taz, two related transcriptional coactivators. Upon phosphorylation, Yap and Taz, the most downstream Hippo signaling components, are excluded from the nucleus and are transcriptionally inactive.
To determine whether the Hippo signaling pathway functions in the determination of mammalian heart size, we inactivated the single mammalian Salv
ortholog in the mouse heart by using a Salv
conditional null allele and the Nkx2.5 cre
allele that directs cardiac cre activity (5
). Yap phosphorylation at Ser127
(pYAP) was examined in Nkx2.5cre:Salvf/f
) mutants via Western blot and immunofluorescence (IF) (fig. S1
). IF of embryonic day 9.5 (E9.5) sagittal sections revealed pYap signal in both second heart field (SHF) progenitors and outflow tract and ventricular cardiomyocytes (fig. S1, A to D
). Salv CKO
hearts had reduced pYAP but no change in total Yap, revealing that Hippo signaling is active in the developing heart and that Salv
deletion reduces cardiac Hippo activity (fig. S1, E and F
mutants survived development, but most mutants expired postnatally with obvious heart enlargement or cardiomegaly (, and table S1
). Histological examination of Salv CKO
mutant hearts revealed that, although organ size is affected, arterioventricular connections and arrangement of chambers and valves were unaffected (), consistent with preserved patterning observed in Hippo mutant imaginal discs (4
). Hearts of some Salv CKO
mutant animals had a ventricular septal defect (VSD), indicating that Hippo signaling regulates ventricular septation (). Because VSD can cause heart failure, we confined further analysis of Salv CKO
mutants to stages before ventricular septation is completed, E14.5 and earlier stages.
Fig. 1 Salvador mutant cardiomegaly. (A to D) Control [(A) and (C)] and Salv CKO [(B) and (D)] P2 neonate hearts. ra indicates right atrium; la, left atrium; rv, right ventricle; lv, left ventricle. Hearts in (A) and (B) were sectioned and stained with hematoxylin (more ...) Salv CKO
mutant hearts had expansion of trabecular and subcompact ventricular myocardial layers, thickened ventricular walls, and enlarged ventricular chambers without a change in myocardial cell size (, and fig. S2
and Mst1/2 CKO
E11.5 mutant hearts had similar myocardial expansion phenotypes (fig. S3
We investigated cardiomyocyte proliferation by double immunostaining with phosphorylated Ser10
histone H3 (pHH3) antibodies to detect mitotic cells and sarcomeric myosin (α-MF20) (, top and middle). Less than 1% of control ventricular cardiomyocytes were pHH3-positive, whereas about 4.5% of Salv CKO
mutant cardiomyocytes were pHH3-positive, indicating excessive cardiac proliferation in cardiomyocytes (, bottom). Cardiomyocyte proliferation was elevated in both left and right Salv CKO
ventricles. Cell counting indicated elevated ventricular cardiomyocte number in Salv CKO
mutants but no changes in ventricular smooth muscle (α-SMA) or cardiac fibroblasts (α-DDR2) (fig. S4
Fig. 2 Cardiomyocyte proliferation in Salvador mutant ventricles. E12.5 control (top) and Salv CKO (middle) coronal sections of left ventricles: stain with TO-PRO-3, blue; α-pHH3, red; and α-MF20, green. Arrows, pHH3/MF20-positive cells. (Bottom (more ...)
SHF cardiac progenitors that contribute to right ventricle had normal cellular proliferation levels in Salv CKO, Lats2 CKO
, and Mst1/2 CKO
mutants, indicating that Salv CKO
mutant cardiomegaly was unlikely to be due to elevated cardiac progenitor number (fig. S5
Microarray revealed that known canonical Wnt target genes were up-regulated in Salv CKO
embryos (). This Wnt-Hippo signature included Sox2
, which has been implicated in cardiac repair and cell reprogramming (8
, a tumorigenesis factor having roles in epithelial-mesenchymal transition and cell growth (10
); and the anti-apoptosis gene Birc5/Survivin
Fig. 3 Salvador deletion potentiates canonical Wnt signaling. Heat map (A) and qRT-PCR validation (B) showing relative transcript levels of Wnt/β-catenin target genes. Values were determined as a mean of three samples ± SD with glyceraldehyde-3-phosphate (more ...)
Reverse transcription quantitative polymerase chain reaction (qRT-PCR) validated up-regulated expression of Sox2
, as well as cdc20, l-Myc, Birc2
, and Birc5
, in Salv CKO
embryonic hearts (). With the exception of l-Myc
, reduced expression was observed in Mef2ccre
mutants (). Other β-catenin–regulated genes, such as Fgf10
, were unchanged in Salv CKO
hearts, indicating incomplete overlap between Yap targets and β-catenin targets (fig. S6
). In situ hybridization revealed Sox2
up-regulation in Salv CKO
E12.5 hearts (fig. S6
E12.5 hearts were immunostained with β-catenin–specific antibodies to assay the nuclear β-catenin index, a readout for cells receiving a Wnt signal. Whereas β-catenin localized to plasma membrane junctions and cytosol of control myocardium, Salv CKO mutants had a fourfold increase in nuclear β-catenin staining (). These findings indicate that canonical Wnt signaling in cardiomyocytes is derepressed upon Salv deletion and support the notion that Hippo signaling inhibits Wnt/β-catenin to regulate heart size.
To obtain genetic evidence that Hippo signaling negatively regulates Wnt/ β-catenin–dependent cardiac growth, we crossed Salv CKO mice to a β-catenin conditional null allele to generate Nkx2.5cre; Salvf/f;βcatf/+ (Salv/βcatf/+ CKO) embryos that are Hippo-deficient with reduced β-catenin dosage.
Myocardial thickness of Salv
hearts was significantly reduced by comparison to Salv CKO
(figs. S7, B and C
, and S2
). Ventricular proliferation rates, as well as subcompact and trabecular myocardial thickness, in Salv
; mutants resemble those of control ( and figs. S2
and S7, A and C
). qRT-PCR indicated that Sox2, Snai2, Birc2
, and Cdc20
expression levels reverted to control or were lower in hearts with reduced β-catenin dosage (). Suppression of the Hippo myocardial overgrowth phenotype by reduced β-catenin
indicates that Wnt signaling is required for up-regulated cardiomyocyte proliferation and cardiomegaly in Hippo mutants.
Fig. 4 Yap interacts with β-catenin. (A) Quantification of pHH3 indices in control, salv CKO, and Nkx2.5cre; Salvf/f; β-catenin F/+ E12.5 hearts. (B) qRT-PCR with indicated genes. (C) Immunoprecipitation/Western with indicated antibodies. IB, (more ...)
To investigate whether Yap and β-catenin are in the same molecular complex, we immunoprecipitated protein extracts from E14.5 hearts with Yap and pYAP-specific antibodies (). Western blotting with antibodies against β-catenin revealed that β-catenin forms a complex only with nuclear, nonphosphorylated Yap, indicating that the Yap/β-catenin molecular interaction is nuclear ().
Because Yap associates with DNA-binding Tead/Tef transcription factors whereas β-catenin binds to Lef/Tcf factors (12
), we examined surrounding genomic sequence of genes within the Wnt-Hippo expression signature for Tead/Tef and Lef/Tcf binding sites. For Sox2
, candidate Yap/Tead binding sites were identified both upstream and downstream of open reading frames (fig. S7D
). Conserved Tcf/Lef binding elements (CTTTG) were in close proximity to Sox2
downstream candidate Yap/Tead sites (fig. S7D
Chromatin immunoprecipitation (ChIP) revealed that Yap and β-catenin were recruited to Sox2 and Snai2 downstream regions but not upstream sites (). Sequential ChIP revealed that Yap and β-catenin concurrently occupied Sox2 and Snai2, suggesting that Yap and β-catenin are contained within a common regulatory complex on Sox2 and Snai2 ().
Transfection experiments indicated that Yap and β-catenin transfection along with their DNA binding co-factors induced luciferase expression from both Sox2 and Snai2 reporter plasmids. Luciferase reporter activity was significantly reduced by preventing Yap or β-catenin recruitment to the reporter either individually or concurrently (). These findings support the model that Yap and β-catenin recruitment to Sox2 and Snai2 chromatin through their respective DNA binding partners potentiates transcriptional activity.
The Yap and β-catenin interaction on genes such as Snai2 and Sox2 uncovers a nuclear mechanism for antagonistic control of cardiomyocyte growth by Hippo and canonical Wnt signaling. Our model is supported by genetic suppression of Hippo-enlarged hearts by reduced β-catenin. Hippo signaling inhibits a pro-growth Wnt/β-catenin-Yap interaction in differentiating cardiomyocytes as the heart transitions from rapid progenitor cell growth to more measured growth in the maturing heart.
Although our pYap data indicate that there is some Hippo activity in E9.5 SHF cardiac progenitors, cell proliferation in Hippo mutant SHF was unchanged from control. This may reflect Salv
-independent Hippo activity and perhaps overlapping functions with other Hippo pathway kinases (i.e., Lats1
). Our data support the previous observation that Hippo signaling was low in E10.5 myocardium (14
). Although Salv CKO
cardiomyocyte cell size was unchanged, this question requires further study under stressed conditions (15
Hippo regulates growth and progenitor genes like Sox2, Snai2, Ccdn1, Cdc20
, and l-Myc
in cardiomyocytes. In livers overexpressing Yap and Hippo
loss-of-function mutants, expression of c-Myc
is up-regulated, suggesting shared mechanisms between liver and heart (4
). Although apoptosis inhibitors Birc2
were up-regulated in Salv CKO
mutant hearts, apoptosis was unchanged.
Important recent work uncovered a repressive cytoplasmic interaction between pTaz and Dvl in kidney (16
). Our findings, uncovering a nuclear interaction between Wnt and Hippo, suggest a two-tiered mechanism by which Hippo negatively modulates Wnt signaling in multiple contexts (fig. S8
Wnt signaling has distinct functions in the two cardiac progenitor fields (13
). Nonetheless, we find no proliferation difference between first heart field–derived left ventricle and SHF-derived right ventricle in Salv CKO
hearts, indicating shared organ size-control mechanisms for the two myocardial lineages. Also Wnt signaling is low in myocardium even though Wnt ligands are expressed (17
). Our findings suggest that, in Hippo-low cardiac progenitors, progrowth genes regulated by Hippo and Wnt are actively transcribed. In cardiomyocytes, Hippo signaling restricts Yap from the nucleus, resulting in diminution of Hippo-Wnt–regulated genes.