Beta-actin is required for proper mouse neural crest ontogeny

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11

Phenotypes induced by NM causing α-skeletal muscle actin mutants in fibroblasts, Sol 8 myoblasts and myotubes

<p>Abstract</p> <p>Background</p> <p>Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with α-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects.</p> <p>Findings</p> <p>We conducted the same biochemical experiments for twelve new actin mutants associated with nemaline myopathy. We observed folding and polymerization defects. Immunostainings of these and eight other mutants in transfected cells revealed typical cellular defects such as nemaline rods or aggregates, decreased incorporation in F-actin structures, membrane blebbing, the formation of thickened actin fibres and cell membrane blebbing in myotubes.</p> <p>Conclusion</p> <p>Our results confirm that NM associated α-actin mutations induce a range of defects at the biochemical level as well as in cultured fibroblasts and muscle cells.</p

Beta-actin as a regulator of crucial developmental processes : focus on a nuclear role for beta-actin

In mammals the actin family consists of 6 isoforms, and although the roles of actin isforms have been rigorously studied in adult life, the functions of actin family members during development are poorly understood. Especially the emerging role of beta-actin in the nucleus remains enigmatic. To investigate the role of beta-actin in mammalian development, a beta-actin knockout mouse model was used. Homozygous beta-actin knockout mice are lethal at embryonic day (E)10.5. At E10.25 beta-actin knockout embryos are growth retarded and display a pale yolk sac and embryo proper that is suggestive of altered erythropoiesis. Using a combined strategy involving bioinformatics, biochemical, and novel genetic rescue approaches, we report that lack of beta-actin resulted in a block of primitive and definitive hematopoiesis. Reduced levels of the transcription factor Gata2 were associated with this phenotype. Consistent with this, ChIP analysis revealed multiple binding sites for beta-actin in the Gata2 promoter. Gata2 mRNA levels could almost completely be restored by expression of an erythroid lineage restricted ROSA26-promotor based GATA2 transgene. As a result, erythroid differentiation was restored and the knockout embryos showed a significant improvement of their vascularization pattern. We here provide evidence for an association of betaactin with a developmentally important gene, Gata2. We define beta-actin as a novel modulator of Gata2 levels during early mammalian hematopoiesis. We thereby contribute to the unraveling of beta-actin’s specific nuclear functions as well as providing new molecular insights for a novel and essential function of beta-actin in embryonic development

ROCK inhibition affects cadherin expression in Actb^−/− neural crest cells ex vivo.

<p>(A) N-cadherin expression in Actb^+/+ (upper panel) and Actb^−/− (lower panel) neural crest cells after 48 hours of neural tube culture. Actb^+/+ and Actb^−/− neural crest cells showed comparable N-cadherin expression levels in the cytoplasm. Actb^+/+, but not Actb^−/−, neural crest cells display expression of membrane-bound N-cadherin (indicated by white arrows). (B) Cadherin-11 expression in Actb^+/+ (upper panel) and Actb^−/− (lower panel) neural crest cells after 48 hours of neural tube culture. Actb^−/− (lower panel) neural crest cells display no cadherin-11 expression compared to Actb^+/+ (upper panel) neural crest cells. (C) N-cadherin expression in Actb^+/+ (upper panel) and Actb^−/− (lower panel) neural crest cells after 48 hours ROCK treatment of neural tube culture. ROCK inhibition had no effect on N-cadherin expression of Actb^−/− neural crest cells. However, membrane-bound N-cadherin was absent from cell-cell contacts of ROCK inhibitor treated Actb^+/+ neural crest cells. (D) Cadherin-11 expression in Actb^+/+ (upper panel) and Actb^−/− (lower panel) neural crest cells after 48 hours ROCK treatment of neural tube culture. Expression of cadherin-11 in Actb^−/− neural crest cells (lower panel) was increased after ROCK treatment relative to non-treated cells. Note that for both Actb^+/+ and Actb^−/− neural crest cells ROCK inhibition resulted in the expected disruption of stress fibers. Imaging of fluorescence was done at similar laser settings.</p

Actb^−/− embryos display abnormal peripheral nervous system at E10.25.

<p>(A) Neurofilament expression in Actb^+/+ and Actb^−/− embryos. Aberrations of neurofilament expression in Actb^−/− embryos are visible at the level of all nerves. (B) Beta-III tubulin expression in Actb^+/+ and Actb^−/− embryos. Aberrations of beta-III tubulin expression in Actb^−/− embryos are visible at the level of IX and X nerves and the dorsal root ganglia. (C) Western Blot analysis showing presence of GFP expression (driven by the endogenous promotor) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085608#pone.0085608-Shmerling1" target="_blank">[11]</a> and the lack of beta-actin expression in Actb^−/− embryos. V: trigeminal nerve; VII: facial nerve; IX: glossopharyngeal nerve; X: vagus nerve; max.b: maxillary branch; man.b: mandibular branch; op.b: ophthalmic branch; drg: dorsal root ganglia. Panels depicting Actb^+/+ and Actb^−/− embryos are of the same magnification.</p

Actb^−/− neural crest cells display decreased cadherin-11 expression and migrate in clusters.

<p>Schematic view of N-cadherin and cadherin-11 expression in the Actb^+/+ and Actb^−/− embryos at E9.5. Neural crest cells in the dorsal neural tube express both N-cadherin and cadherin-11, migrating neural crest cells express high levels of cadherin-11 while maintaining N-cadherin levels. Actb^−/− neural crest cells in the dorsal neural tube show very low levels of cadherin-11. Migrating Actb^−/− neural crest cells show decreased cadherin-11 expression levels and therefore mainly express N-cadherin. Most migrating Actb^−/− neural crest cells were organized as clusters rather than distinct streams as Actb^+/+ neural crest cells. n: notochord; nt: neural tube; d: dermomyotome; scl: sclerotome.</p

Actb^−/− neural crest cells express the mesenchymal marker vimentin in vivo.

<p>(A) Vimentin immunohistochemistry on Actb^+/+ and Actb^−/− embryo sections at E9.5. p75 and DAPI were used as markers for neural crest cells and nuclei respectively. Both Actb^+/+ and Actb^−/− sections show comparable levels of vimentin expression in migratory neural crest cells of both regions (indicated by white arrows). (B) Quantification of Vimentin intensities in migratory neural crest cells adjacent to the neural tube (region A) and in the dorsal neural tube (region B). There is no significant difference between Actb^+/+ and Actb^−/− embryo sections. Bars represent mean ± SEM.</p

Increased apoptosis in Actb^−/− neural crest cells in vivo.

<p>(A) TUNEL analysis on Actb^+/+ and Actb^−/− embryo sections stained with p75 at E9.5. Actb^−/− sections show increased apoptosis, both in the dorsal neural tube and migratory neural crest cells (indicated by white arrows) compared to Actb^+/+ sections. (B) Phospho-Histone H3 immunohistochemistry on embryo sections stained with p75 at E9.5. Both Actb^+/+ and Actb^−/− sections show similar staining pattern (indicated by white arrows). In (A) and (B) DAPI was used to reveal nuclear staining. (C) Quantification of TUNEL and phospho-Histone H3 intensities in migratory neural crest cells adjacent to the neural tube (region A) and in the dorsal neural tube (region B). Whereas there is no significant difference for phospho-Histone H3, there is a significant increase of TUNEL staining in Actb^−/− sections compared to Actb^+/+ sections. Bars represent mean ± SEM; **P<0.01, ***P<0.001.</p