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. 2008 Nov 21;103(11):1241-8.
doi: 10.1161/CIRCRESAHA.108.178749. Epub 2008 Oct 16.

Tropomodulin1 is required in the heart but not the yolk sac for mouse embryonic development

Caroline R McKeown  1 , Roberta B Nowak, Jeannette Moyer, Mark A Sussman, Velia M Fowler
Affiliations

Tropomodulin1 is required in the heart but not the yolk sac for mouse embryonic development

Caroline R McKeown et al. Circ Res. .

Abstract

Tropomodulin (Tmod)1 caps the pointed ends of actin filaments in sarcomeres of striated muscle myofibrils and in the erythrocyte membrane skeleton. Targeted deletion of mouse Tmod1 leads to defects in cardiac development, fragility of primitive erythroid cells, and an absence of yolk sac vasculogenesis, followed by embryonic lethality at embryonic day 9.5. The Tmod1-null embryonic hearts do not undergo looping morphogenesis and the cardiomyocytes fail to assemble striated myofibrils with regulated F-actin lengths. To test whether embryonic lethality of Tmod1 nulls results from defects in cardiac myofibrillogenesis and development or from erythroid cell fragility and subsequent defects in yolk sac vasculogenesis, we expressed Tmod1 specifically in the myocardium of the Tmod1-null mice under the control of the alpha-myosin heavy chain promoter Tg(alphaMHC-Tmod1). In contrast to Tmod1-null embryos, which fail to undergo cardiac looping and have defective yolk sac vasculogenesis, both cardiac and yolk sac morphology of Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos are normal at embryonic day 9.5. Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos develop into viable and fertile mice, indicating that expression of Tmod1 in the heart is sufficient to rescue the Tmod1-null embryonic defects. Thus, although loss of Tmod1 results in myriad defects and embryonic lethality, the Tmod1(-/-) primary defect is in the myocardium. Moreover, Tmod1 is not required in erythrocytes for viability, nor do the Tmod1(-/-) fragile primitive erythroid cells affect cardiac development, yolk sac vasculogenesis, or viability in the mouse.

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Conflict of interest statement

Disclosures: None.

Figures

Figure 1
Figure 1. Tmod1 is present in Tmod1-/-Tg(αMHC-Tmod1), but not Tmod1-/- embryonic hearts
(A) Western blot of E8.5 mouse embryos generated from a Tmod1+/- × Tmod1+/-Tg(αMHC-Tmod1) mating. Each lane contains a single embryo. Blots were probed for Tmod1 (top) and Actin as a loading control (bottom, note actin is partially proteolyzed resulting in a doublet). (B) Whole-mount immunofluorescent confocal stacks of E8.5 wild-type (13 somite pairs; i-iii), Tmod1-/- (11 somite pairs; iv-vi), andTmod1-/-Tg(αMHC-Tmod1) (13 somite pairs; vii-ix) embryos stained for Tmod1 (i, iv, vii), sarcomeric α-actinin (ii, v, viii), and actin (iii, vi, ix). The Tmod1 transgene staining is restricted to the heart (vii). Scale bar is 100 μm.
Figure 2
Figure 2. Tg(αMHC-Tmod1) rescues the cardiac looping defect of the Tmod1 nulls
(A, B) Gross morphology of E7.5 mouse embryos, ventral view depicting first right inward looping of the embryonic heart tube at 7 somite pairs in Tmod1+/+ (A), but not in Tmod1-/- (B). (C-F) Gross morphology of E9.5 Tmod1+/+ (C), Tmod1+/+Tg(αMHC-Tmod1) (D), Tmod1-/- (E), and Tmod1-/-Tg(αMHC-Tmod1) (F) mouse embryos, side view. The cardiac morphology of the Tmod1-/-Tg(αMHC-Tmod1) (F) is similar to the Tmod1+/+ (C). Note that overexpression of Tmod1 does not affect cardiac looping in the wild-type embryo (D). Scale bar is 200μm in A,B and 500 μm in C,D,E,F.
Figure 3
Figure 3. Tg(αMHC-Tmod1) rescues the cardiac myofibril assembly defect of the Tmod1 nulls
Immunofluorescence of the cardiomyocytes in the outer wall of the heart from Tmod1+/+ (A-C), Tmod1-/-(D-F), and Tmod1-/-Tg(αMHC-Tmod1) (G-I) E8.5 mouse embryos. Staining for sarcomeric α-actinin (A,D,G, green in merge) and actin (B,E,H, red in merge) show typical striated myofibrils in wild-type embryos (A-C). Tmod1-/- embryos lack any discernable myofibrils and instead display aberrant aggregates of sarcomeric α-actinin and F-actin (D-F, arrows). The Tg(αMHC-Tmod1) restores myofibrillogenesis in the Tmod1-/- embryonic hearts (G-I). Nuclei are stained with Hoechst dye and appear blue in merged images (C,F,I). Note that the cardiomyocytes in the developing heart are a heterogeneous population, images presented are from comparable regions. Images are digital projections of confocal stacks. Scale bar is 20 μm.
Figure 4
Figure 4. Tmod1 protein expressed from the Tg(αMHC-Tmod1) transgene localizes to pointed ends of thin filaments in cardiac myofibrils
Immunofluorescence of single cardiac myofibrils from Tmod1+/+ (A-D) and Tmod1-/-Tg(αMHC-Tmod1) (E-H) E9.5 mouse embryonic hearts. Embryos are staged similarly at 12-13 somite pairs. Staining for F-actin (B,F, red in merge) and sarcomeric α-actinin (C,G, green in merge) show typical striated myofibrils in both Tmod1+/+ (A-D) and Tmod1-/-Tg(αMHC-Tmod1) (E-H) embryos. Arrowheads indicate the H-zone where actin pointed ends (B,F) are capped by Tmod1 (A,E, blue in merge). Images are single optical sections (0.6 μm thick). Scale bar is 4 μm.
Figure 5
Figure 5. Tg(αMHC-Tmod1) rescues the yolk sac vasculogenesis defect of Tmod1 nulls but is not expressed in the yolk sac or in the circulating erythrocytes
(A-C) Gross morphology of E8.5 embryonic yolk sacs from Tmod1+/+ (A,A'), Tmod1-/- (B,B'), and Tmod1-/-Tg(αMHC-Tmod1) (C,C'). Wild-type embryos have a highly vascularized yolk sac (A'), while Tmod1-/- embryonic yolk sacs do not undergo vascular remodeling (B'). Vascularization of the Tmod1-/-Tg(αMHC-Tmod1) embryonic yolk sac (C') is similar to Tmod1+/+ (A'). (D) Western blot of embryonic yolk sacs; each lane contains a single E8.5 yolk sac. Blots were probed for Tmod1 (top) and Actin as a loading control (bottom). The Tg(αMHC-Tmod1) does not express Tmod1 in the yolk sac (last 3 lanes). Note that the faint band in last lane has altered mobility relative to Tmod1 and is likely a cross-reacting band. Also note that actin shadow is due to a high signal for actin and shifting of blot during exposure. (E) Western blot of isolated erythrocytes from E15.5 embryos. Blots were probed for Tmod1 (top) and α-adducin (bottom) as a loading control. The Tg(αMHC-Tmod1) does not express Tmod1 in the definitive erythrocytes (last lane).
Figure 6
Figure 6. Tg(αMHC-Tmod1) rescues the embryonic lethality of the Tmod1 nulls
(A, B) Gross morphology of E15.5 littermates demonstrates that Tmod1-/-Tg(αMHC-Tmod1) embryos (B) are indistinguishable from their wild-type counterparts (A). (C) PCR genotyping of a typical Tmod1+/- x Tmod1+/-Tg(αMHC-Tmod1) litter. Tg(αMHC-Tmod1) band (top) indicates that the transgene is present, Tmod1 band (middle) indicates that the wild-type endogenous Tmod1 genomic locus is intact, and LacZ band (bottom) is a marker for the Tmod1 targeting event. The 9th lane shows that the Tmod1-/-Tg(αMHC-Tmod1) embryos are positive for the transgene and positive for the LacZ targeting event, but null for the Tmod1 genomic locus.
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