Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis.

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain

Genotypes of offspring from mating of β-MHC-Fhod3-IA × B6.

<p>Genotypes of offspring from mating of β-MHC-Fhod3-IA × B6.</p

Schematic representation of the sarcomeric structure at embryonic stages.

<p>Relative localization of Fhod3 to α-actinin and F-actin shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148472#pone.0148472.g003" target="_blank">3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148472#pone.0148472.g005" target="_blank">5</a> is schematically represented. F-actin content is represented in gray tones. Arrowheads indicate the center of the sarcomere.</p

F-actin organization and α-actinin localization in the assembling myofibrils in the embryonic heart.

<p>Confocal fluorescence micrographs of cardiac myofibrils of <i>Fhod3</i>^{Tg(β-MHC-Fhod3WT)} embryos at E9.5 (A), E10.5 (B), or E13.5 (C), and those of wild-type (<i>Fhod3</i>^<b>+/+</b>) embryos at E9.5 (D, F), or E13.5 (F). Sections of embryonic hearts were subjected to immunofluorescent staining for α-actinin (green) and phalloidin staining for F-actin (magenta). Arrowheads indicate the center of the sarcomere. Scale bars, 5 μm.</p

Rescue effect of transgenic expression of Fhod3 in the embryonic heart of <i>Fhod3</i>^−/− mice.

<p>(A) Whole mount analysis of wild-type (<i>Fhod3</i>^<b>+/+</b>) and <i>Fhod3</i>^{−/−Tg(β-MHC-Fhod3WT)} (<i>Fhod3</i>^−/−;<i>Tg(WT)</i>^<b>+</b>) embryos at E18.5. Scale bars, 1 cm. (B) Histological analysis of wild-type (<i>Fhod3</i>^<b>+/+</b>) and <i>Fhod3</i>^{−/−Tg(β-MHC-Fhod3WT)} (<i>Fhod3</i>^−/−;<i>Tg(WT)</i>^<b>+</b>) embryos at E18.5. LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bars: (left) 300 μm; (right) 10 μm. (C) Confocal fluorescence micrographs of cardiac myofibrils of wild-type (<i>Fhod3</i>^<b>+/+</b>) and <i>Fhod3</i>^{−/−Tg(β-MHC-Fhod3WT)} (<i>Fhod3</i>^−/−;<i>Tg(WT)</i>^<b>+</b>) embryos at E18.5. Sections of embryonic hearts were subjected to immunofluorescent staining for α-actinin (green) and phalloidin staining for F-actin (magenta). Scale bars, 5 μm.</p

Genotypes of embryos from mating of β-MHC-Fhod3-IA#4 × B6.

<p>Genotypes of embryos from mating of β-MHC-Fhod3-IA#4 × B6.</p

Localization of Fhod3 and other sarcomeric components in the assembling myofibrils in the embryonic heart.

<p>Confocal fluorescence micrographs of cardiac myofibrils of <i>Fhod3</i>^{<b>Tg(β-MHC-Fhod3WT)</b>} embryos at E9.5. Sections of embryonic hearts were subjected to immunofluorescent staining for Fhod3 (magenta) and Tmod (green) (A), β-MHC (green) (B), or myomesin (green) (C). Scale bars, 5 μm.</p

Genotypes of embryos from mating of <i>Fhod3</i>^+/–and <i>Fhod3</i>^{+/–Tg(β-MHC-Fhod3WT)}.

<p>Genotypes of embryos from mating of <i>Fhod3</i>^+/–and <i>Fhod3</i>^{+/–Tg(β-MHC-Fhod3WT)}.</p

Sarcomeric localization of wild-type Fhod3 in the assembling myofibrils in the embryonic heart.

<p>Confocal fluorescence micrographs of cardiac myofibrils of <i>Fhod3</i>^{Tg(β-MHC-Fhod3WT)} embryos at E9.5 (A, B), E10.5 (C), or E13.5 (D). Sections of embryonic hearts were subjected to immunofluorescent staining for α-actinin (green) and Fhod3 (magenta) followed by phalloidin staining (not shown in merge). Scale bars, 5 μm.</p

Fhod3 localization and F-actin organization in the assembling myofibrils in the embryonic heart.

<p>Confocal fluorescence micrographs of cardiac myofibrils of <i>Fhod3</i>^{Tg(β-MHC-Fhod3WT)} embryos at E9.5 (A), E10.5 (B), or E13.5 (C). Sections of embryonic hearts were subjected to immunofluorescent staining for Fhod3 (magenta) and phalloidin staining for F-actin (green). Arrowheads indicate the center of the sarcomere. Scale bars, 5 μm.</p