Twist1 function in endocardial cushion cell proliferation, migration, and differentiation during heart valve development

Abstract

Twist1 is a bHLH transcription factor that regulates cell proliferation, migration, and differentiation in embryonic progenitor cell populations and transformed tumor cells. While much is known about Twist1's function in a variety of mesenchymal cell types, the role of Twist1 in endocardial cushion cells is unknown. Twist1 gain and loss of function experiments were performed in primary chicken endocardial cushion cells in order to elucidate its role in endocardial cushion development. These studies indicate that Twist1 can induce endocardial cushion cell proliferation as well as promote endocardial cushion cell migration. Furthermore, Twist1 is subject to BMP regulation and can induce expression of cell migration marker genes including Periostin, Cadherin 11, and Mmp2 while repressing markers of valve cell differentiation including Aggrecan. Previously, Tbx20 has been implicated in endocardial cushion cell proliferation and differentiation, and in the current study, Tbx20 also promotes cushion cell migration. Twist1 can induce Tbx20 expression, while Tbx20 does not affect Twist1 expression. Taken together, these data indicate a role for Twist1 upstream of Tbx20 in promoting cell proliferation and migration and repressing differentiation in endocardial cushion cells during embryonic development.

Figure 1. Twist1 and Tbx20 are coordinately expressed with cell migration marker genes in endocardial cushions and remodeling valves

Expression of Twist1, Tbx20, Cad11, Postn, and Mmp2 was examined in sectioned HH stage 25 and HH stage 36 chicken hearts. In situ hybridizations show Twist1 (A), Tbx20 (C), Cad11 (E), and Mmp2 (I) are expressed throughout the entire endocardial cushion. In addition, Twist1, Tbx20, and Cad11 expression was also detected in early epicardially derived cells (arrowheads in panel A, C, and E). In contrast, Postn was more strongly expressed in the ventricular aspect of the cushion near the intreventricular septum (arrow in panel G) and is absent from the subatrial region of the cushion (star in panel G). In remodeling mitral valves, Twist1 (B), Tbx20 (D), and Mmp2 (J) are expressed weakly throughout the entire valve leaflet and in epicardially derived cells (asterisks in panels B, D, and J). In contrast, Cad11 expression is weak and restricted to the distal tips of the valve (arrow in panel F) but is also expressed in the epicardially derived cells (asterisk in panel F). Postn is expressed at the intersection of the valve leaflet and the myocardium (arrow in panel H) with increased expression in the chordae tendineae and the papillary muscle points of insertion (arrowhead in panel H). In contrast, Postn expression is absent in epicardially derived cells (asterisk in panel H). EC, endocardial cushion; MV, mitral valve; IVS, interventricular septum; AS, atrial septum.

Figure 2. Differential Expression of Twist1, Tbx20, and cell migration marker genes during endocardial cushion and remodeling valve stages

Expression of Twist1, Tbx20, Cad11, Postn, and Mmp2 was examined in isolated AV canals from HH stage 25 or HH stage 36 chicken embryos using real time RT-PCR. The expression of Twist1, Tbx20, Cad11, and Mmp2 was decreased at HH stage 36 compared to the expression at HH stage 25. In contrast, the expression of Postn was increased at HH stage 36 compared to the expression at HH stage 25 consistent with increased expression in valve supporting structures. These data are representative of 4 independent real time RT-PCR experiments performed in duplicate (n=4). Statistical significance of observed differences between gene expression levels at HH stage 25 and HH stage 36 is indicated by an asterisk (P<0.05) and error bars represent standard error of the mean.

Figure 3. Twist1-specific siRNA transfected into primary chicken endocardial cushion cells results in decreased Twist1 protein and mRNA expression

Twist1 protein expression was examined by immunohistochemistry with an antibody specific for Twist1 in parallel cultures of siRNA-transfected cells (A–C). Cells transfected with Twist1-specific siRNA (C) had significantly reduced nuclear staining (arrowheads) compared to controls (A, B, arrows indicate Twist1 immunopositive cells). In Twist1-specific siRNA transfected cultures, a small subset of cells remained Twist1 positive (arrows in panel C). Twist1 mRNA expression was quantified using real time RT-PCR (D). Cells transfected with Twist1-specific siRNA had an 80% reduction in Twist1 mRNA relative to untransfected and scrambled siRNA controls. The asterisk indicates a statistically significant difference from the control value and error bars represent standard error of the mean (n=3, P<0.01).

Figure 4. Twist1 promotes endocardial cushion cell proliferation

Primary endocardial cushion cells were infected with adenoviruses expressing β-gal (Adβ-gal) or Twist1 (AdTwist1) for gain of function or transfected with Twist1-specific siRNA for loss of function. Proliferation was measured using immunohistochemistry for BrdU incorporation. Cells infected with AdTwist1 (B) exhibit increased BrdU incorporation compared to control cells infected with Adβ-gal (A) (arrows indicate positively stained cells). Cells transfected with Twist1-specific siRNA (D) have less BrdU reactivity than untransfected control cells (C). The percent of BrdU positive cells relative to total nuclei in each group is quantified (E). The asterisk represents a statistically significant difference compared to the Adβ-gal control group, while the cross represents a statistically significant difference compared to the no siRNA control group (n=3, P<0.01). Error bars represent standard error of the mean.

Figure 5. Tbx20 and Twist1 promote endocardial cushion cell migration

Primary endocardial cushion cells were infected with adenoviruses expressing β-gal (Adβ-gal), Tbx20 (AdTbx20), or Twist1 (AdTwist1) for gain of function or transfected with Tbx20-specific or Twist1-specific siRNA for loss of function. Cells were stained with Giemsa Stain and migration was measured by counting the number of cells that migrated through the pores (visible in panels A–F) of a modified Boyden chamber membrane. The number of cells observed on the underside of the membrane was increased in cultures infected with AdTbx20 (B) or AdTwist1 (C) as compared to control cultures infected with Adβ-gal (A). In addition, the number of cells observed on the underside of the membrane was decreased in cultures transfected with Tbx20-specific siRNA (E) or Twist1-specific siRNA (F) as compared to untransfected control cultures (D). The fold change in the number of cells that migrated through the membrane relative to the number of control cells that migrated is quantified (G). Asterisks represent a statistically significant difference compared to the Adβ-gal control group, while crosses represent a statistically significant difference compared to the no siRNA control group (n=3, P<0.01). Error bars represent standard error of the mean.

Figure 6. Altered Twist1 function affects the expression of cell migration and differentiation marker genes in endocardial cushion cells

Primary endocardial cushion cells were infected with adenoviruses expressing β-gal (Adβ-gal) or Twist1 (AdTwist1) for gain of function or transfected with Twist1-specific siRNA for loss of function. The expression of Postn, Cad11, and Mmp2, genes associated with cell migration, and Agg, a gene associated with cushion cell differentiation, was measured by real time RT-PCR. Cells transfected with Twist1-specific siRNA had decreased Postn (A), Cad11 (B), and Mmp2 (C) expression and increased Agg (D) expression compared to untransfected control cells. In contrast, cells infected with AdTwist1 had increased Postn (A), Cad11 (B), and Mmp2 (C) expression and decreased Agg (D) expression compared to Adβ-gal infected control cells. Asterisks represent a statistically significant difference compared to the no siRNA control group (n=4, P<0.01). Error bars represent standard error of the mean.

Figure 7. BMP2 induces Twist1, Cad11, Postn, and Mmp2 expression in endocardial cushion cells

Endocardial cushion cells were cultured with or without the addition of soluble recombinant BMP2 or Noggin, a BMP inhibitor. Addition of BMP2 resulted in increased Twist1, Cad11, Postn, and Mmp2 expression relative to untreated controls, while addition of Noggin attenuated that response as measured by real time RT-PCR. Asterisks represent a statistically significant difference compared to untreated control groups (n=4, P<0.01). Error bars represent standard error of the mean.

Figure 8. Twist1 promotes Tbx20 expression in endocardial cushion cells

Primary endocardial cushion cells were infected with adenoviruses expressing β-gal (Adβ-gal), Twist1 (AdTwist1), or Tbx20 (AdTbx20) or transfected with a scrambled control siRNA, Twist1-specific siRNA, or Tbx20-specific siRNA. Real time RT-PCR was used to measure the expression of Tbx20 and Twist1 on each experimental group. Cells infected with AdTwist1 had increased Tbx20 expression relative to controls, while cells transfected with Twist1-specific siRNA had decreased Tbx20 expression relative to controls (A). In contrast, cells with altered Tbx20 function showed no significant change in Twist1 expression relative to controls (B). The asterisk represents a statistically significant difference compared to the Adβ-gal control group, while the crosses represents a statistically significant difference compared to the no siRNA control group (n=4, P<0.01). Error bars represent standard error of the mean.

Figure 9. Model for Twist1 function in endocardial cushion cells

BMP2 can induce Twist1 expression, which can promote Tbx20 expression. Twist1 and Tbx20 can then promote endocardial cushion cell proliferation and migration and repress cushion extracellular matrix organization. High levels of Twist1 and Tbx20 in endocardial cushions relative to remodeling valves is consistent with Twist1 and Tbx20 functioning to promote and maintain the proliferative, migratory, and undifferentiated nature of endocardial cushion mesenchyme.