Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 67 (9), 564-72

Delayed Embryonic Development and Impaired Cell Growth and Survival in Actg1 Null Mice

Affiliations

Delayed Embryonic Development and Impaired Cell Growth and Survival in Actg1 Null Mice

Tina M Bunnell et al. Cytoskeleton (Hoboken).

Abstract

Actins are among the most highly expressed proteins in eukaryotes and play a central role in nearly all aspects of cell biology. While the intricate process of development undoubtedly requires a properly regulated actin cytoskeleton, little is known about the contributions of different actin isoforms during embryogenesis. Of the six actin isoforms, only the two cytoplasmic actins, beta(cyto)- and gamma(cyto)-actin, are ubiquitously expressed. We found that gamma(cyto)-actin null (Actg1(-/-)) mice were fully viable during embryonic development, but most died within 48 h of birth due to respiratory failure and cannibalization by the parents. While no morphogenetic defects were identified, Actg1(-/-) mice exhibited stunted growth during embryonic and postnatal development as well as delayed cardiac outflow tract formation that resolved by birth. Using primary mouse embryonic fibroblasts, we confirm that gamma(cyto)-actin is not required for cell migration. The Actg1(-/-) cells, however, exhibited growth impairment and reduced cell viability, defects which perhaps contribute to the stunted growth and developmental delays observed in Actg1(-/-) embryos. Since the total amount of actin protein was maintained in Actg1(-/-) cells, our data suggests a distinct requirement for gamma(cyto)-actin in cell growth and survival.

Figures

Figure 1
Figure 1. Stunted growth in Actg1−/− mice
(A) Shown are representative images of E18.5 Actg1+/− and Actg1−/− embryos. Scale bar: 5mm. (B) Crown-rump length and (C) body mass measurements during embryonic and postnatal development. Actg1−/− mice were significantly (one-way ANOVA, p<0.05) smaller than Actg1+/+ and Actg1+/− controls from E11.5 onward. n ≥ 4 for each genotype/timepoint; error bars represent s.e.m.
Figure 2
Figure 2. Delayed development of the cardiac outflow tract in Actg1−/− embryos
Shown are typical hematoxylin and eosin stained transverse sections through the embryonic heart. (A) At E12.5 the aorta was observed to be in communication with the right ventricle in both Actg1+/− and Actg1−/− embryos. (B) By E13.5 the aorta was observed in exclusive communication with the left ventricle in Actg1+/− embryos but remained in communication with the right ventricle in all six Actg1−/− embryos examined. (C) By E14.5, however, the aorta had been walled into the left ventricle in six out of seven Actg1−/− embryos. (D) At E14.5 in Actg1+/− embryos, but not in the seven Actg1−/− embryos examined (arrow), the ventricular septum was completely formed along the entire anterior-posterior axis. (E) By E18.5 the ventricular septum had fully developed in all Actg1−/− embryos. Scale bars: 500 µm. Ao, aorta; LV, left ventricle; RV, right ventricle; VS, ventricular septum.
Figure 3
Figure 3. Partially penetrant respiratory failure observed in Actg1−/− newborns
Shown are representative transverse sections of the developing lung. (A) Hematoxylin and eosin staining of Actg1−/− lungs at E16.5 (top row) and E18.5 (middle row) revealed comparable morphology to wild-type. Immunohistochemistry using an antibody to surfactant protein B as a marker of lung development showed no differences in expression between Actg1+/− and Actg1−/− lungs at E18.5 (bottom row). (B) In Actg1−/− lungs at P0 the extent of saccular inflation was variable and corresponds with observed variations in ability to breath at the time of birth. Scale bars: 100 µm.
Figure 4
Figure 4. Altered actin isoform expression with no change in levels of total actin in Actg1−/− MEFs
(A) Representative immunoblots of cell lysates from Actg1+/+, Actg1+/− and Actg1−/− MEFs probed with actin isoform specific antibodies. (B) Protein levels were quantified and are expressed relative to wild-type levels (n ≥ 3, mean ± s.e.m.). (C) Quantification of G- to F-actin ratios in Actg1+/+, Actg1+/− and Actg1−/− MEFs (n ≥ 3, mean ± s.e.m.).
Figure 5
Figure 5. Comparable rates of cell migration between wild-type and Actg1−/− MEFs
Still images from time-lapse video microscopy showing the wound edge in yellow at time 0 and 200 minutes from both Actg1+/+ (A, A’) and Actg1−/− (B, B’) MEF cultures. (C) Rates of cell migration were calculated as the change in wound area divided by the change in time (n ≥ 3, mean ± s.e.m.). Scale bar: 100 µm.
Figure 6
Figure 6. Impaired growth due to decreased cell survival in Actg1−/− MEFs
(A) Growth kinetics revealed impaired growth in Actg1−/− MEFs. n = 4 for each genotype; error bars represent s.e.m. Asterisks denote significant differences (two-way ANOVA, p<0.05). (B) Reduced cell viability in Actg1−/− MEFs as determined by an automated trypan blue exclusion assay (n = 13, mean ± s.e.m.). Asterisk denotes significant differences (one-way ANOVA, p<0.05). (C) Mean MEF cell size as determined by the ViCELL Series’ automated contrast imaging and analysis (n = 13, mean ± s.e.m.). (D) Representative cell cycle profiles. (E) Mean percentage of cells in the G1, S, and G2 phases of the cell cycle (n = 3, mean ± s.e.m.). (F) Dot plots from FITC-Annexin V flow cytometric analyses. The lower-right box represents early apoptotic cells (Annexin V-FITC Positive/PI Negative) while the upper right box represents dead cells (Annexin V-FITC Positive/PI Positive). (G) Mean percentage of apoptotic cells, defined as FITC-positive and PI-negative (n ≥ 5, mean ± s.e.m.). CPT, camptothecin.

Similar articles

See all similar articles

Cited by 34 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback