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

How Wnt signaling builds the brain : bridging development and disease

Wnt/beta-catenin signaling plays a crucial role throughout all stages of brain development and remains important in the adult brain. Accordingly, many neurological disorders have been linked to Wnt signaling. Defects in Wnt signaling during neural development can give rise to birth defects or lead to neurological dysfunction later in life. Developmental signaling events can also be hijacked in the adult and result in disease. Moreover, knowledge about the physiological role of Wnt signaling in the brain might lead to new therapeutic strategies for neurological diseases. Especially, the important role for Wnt signaling in neural differentiation of pluripotent stem cells has received much attention as this might provide a cure for neurodegenerative disorders. In this review, we summarize the versatile role of Wnt/beta-catenin signaling during neural development and discuss some recent studies linking Wnt signaling to neurological disorders

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

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

At E9.5 Actb^−/− embryos show defects in neural crest ontogeny.

<p>(A) Crabp1 whole mount and section ISH (at the indicated planes: a, b, c and d) on Actb^+/+ and Actb^−/− embryos. For the latter all sections show absence of neural crest cells in certain regions of the sections. Actb^−/− neural crest cells are not able to reach the brachial arches (arrows in section d). (B) Sox10 whole mount embryo ISH marks the migrating neural crest. Sox10 ISH shows comparable results as the Crabp1 probe. (C) Pax3 marks the dermomyotome and the cranial neural crest. Compared to Actb^+/+ embryos, the Actb^−/− embryos show a similar expression pattern of pax3 at the level of the dermomyotomes, but a reduction in the cranial region. Panels depicting Actb^+/+ and Actb^−/− embryos are of the same magnification.</p

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

ROCK inhibition rescues impaired migration capacity of Actb^−/− neural crest cells ex vivo.

<p>(A) Neural tube explant assays reveal a migration defect of Actb^−/− neural crest cells ex vivo. After 24 hours the Actb^−/− explant (red line) shows a smaller outgrowth area (blue line) than the Actb^+/+ control neural tube (left panel). After 48 hours, differences between Actb^+/+ and Actb^−/− neural crest cells become more pronounced (right panel). (B) After 24 hours of ROCK inhibition the Actb^−/− explant shows a remarkable difference in migration capacity (left panel, compare with 5A left panel). The regained migration capacity of Actb^−/− neural crest cells is even more obvious after 48 hours (right panel). (C) Quantification of the neural tube outgrowths. A significant reduction of outgrowth area was found for Actb^−/− neural crest cells compared to Actb^+/+ neural crest cells. When treated with ROCK inhibitor Actb^−/− neural crest cells showed increased migration to the level of Actb^+/+ neural crest cells. Bars represent mean ± SEM; **P<0.01, ***P<0.001.</p

At E9.5 Actb^−/− neural crest cells show a significant decrease of cadherin-11 expression.

<p>(A) Quantification of N-cadherin intensities in Actb^+/+ and Actb^−/− migratory cells adjacent to the neural tube at E8.5 and E9.5 (left graph, Y-axis: relative intensities) and colocalization of N-cadherin with p75 in migratory cells at E8.5 and E9.5 (right graph, Y-axis: colocalization index on a scale from 0 to 1). Quantification was done on images such as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085608#pone-0085608-g006" target="_blank">figure 6</a>. No significant differences were observed between Actb^+/+ and Actb^−/− embryos in this region for both stages. (B) Quantification of cadherin-11 intensities in Actb^+/+ and Actb^−/− migratory cells next to the neural tube at E8.5 and E9.5 (left graph, Y-axis: relative intensities) and colocalization of cadherin-11 with p75 in migratory cells at E8.5 and E9.5 (right graph, Y-axis: colocalization index on a scale from 0 to 1). We only observed a significant reduction of cadherin-11 intensities in Actb^−/− neural crest cells at E9.5. At the same stage, a significant decrease of cadherin-11 and p75 colocalization was seen as well in Actb^−/− neural crest cells. (C) Quantification of cadherin-11 intensities specifically in the dorsal neural tube of Actb^+/+ and Actb^−/− embryos at E8.5 and E9.5. This region also showed a significant reduction of cadherin-11 intensity for Actb^−/− embryos. Bars represent mean ± SEM; *P<0.05, ***P<0.001.</p