Coordination of Cell Proliferation and Cell Fate Determination by CES-1 Snail

<div><p>The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. We present evidence that the Snail-related transcription factor CES-1 of <i>Caenorhabditis elegans</i> coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene <i>egl-1</i>, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. We now demonstrate that CES-1 also affects cell cycle progression in this lineage. Specifically, we found that CES-1 can repress the transcription of the <i>cdc-25.2</i> gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of <i>cya-1</i>, which encodes Cyclin A. Our results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the <i>ces-1</i> gene leads to incorrect regulation of this functional ‘module’. Finally, we provide evidence that <i>dnj-11</i> MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. Our findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination.</p></div

<i>ces-1(n703</i>gf<i>)</i>; <i>cya-1(bc416)</i> causes temperature-sensitive embryonic lethality.

<p>(A) The percentages of embryonic lethality at 15°C and 25°C. The numbers above the bars represent the percentage of embryonic lethality. For each genotype, around 1000 embryos were scored. DIC images of embryos arrested during the elongation stage of embryogenesis (B, D, E) or during the first larval stage (L1) (C) when grown at 25°C are shown. White arrows point to abnormalities in the hypodermis. All strains analyzed were homozygous for <i>bcIs66</i>. RNAi was performed by injection.</p

<i>cdc-25.2</i> expression is down-regulated by <i>ces-1</i> over-expression.

<p>Transgenic animals carrying an extra-chromosomal array of <i>ces-1</i> heat-shock plasmids and coinjection marker were used as the sample group (P_HS<i>ces-1</i>), while transgenic animals carrying an extra-chromosomal array of only coinjection marker were used as control (<i>+/+</i>). Relative expression levels of <i>cdc-25</i> genes and <i>cya-1</i> gene in control animals (<i>+/+</i>) and animals over-expressing <i>ces-1</i> (P_HS<i>ces-1</i>) were determined by real-time PCR (qPCR). Data are represented as fold change relative to control. Data shown are the means ± SEM from four independent repeats. Paired t-test was used to determine significance. The level of <i>cdc-25.2</i> in P_HS<i>ces-1</i> is significantly lower than in control. The level of <i>cdc-25.3</i> in P_HS<i>ces-1</i> is significantly higher than in control. The levels of <i>cdc-25.1</i>, <i>cdc-25.4</i>, <i>cya-1</i> are not significantly changed in response to <i>ces-1</i> over-expression. *p<0.05, **p<0.01 significantly different from the control.</p

<i>ces-1(n703</i>gf<i>); cya-1(bc416)</i> affects the number of ‘NSM-like’ cells.

<p><i>ces-1(n703</i>gf<i>); cya-1(bc416)</i> affects the number of ‘NSM-like’ cells.</p

<i>ces-1</i> Snail represents a functional link between cell cycle progression, cell polarity and apoptosis in the NSM lineage.

<p>Genetic model of <i>ces-1</i> Snail functions in the NSM neuroblast (top), the NSM and the NSM sister cell (bottom). In the NSM neuroblast, <i>ces-1</i> function is negatively regulated by the genes <i>dnj-11</i> MIDA1 and <i>ces-2</i> bZIP. <i>ces-1</i> affects cell cycle progression in the NSM neuroblast by negatively regulating <i>cdc-25.2</i> Cdc25. <i>ces-1</i> also affects the polarity of the NSM neuroblast. However, to date, it is unclear through what mechanism. After the asymmetric division of the NSM neuroblast, the level of <i>ces-1</i> activity is high in the larger NSM (left) and low in the smaller NSM sister cell (right). The activity of <i>ces-1</i> in the NSM is sufficient to block the function of <i>hlh-2/3</i> bHLH, thereby resulting in a level of <i>egl-1</i> BH3-only activity that is too low to induce apoptosis. Conversely, in the NSM sister cell, the activity of <i>ces-1</i> is not sufficient to block the function of <i>hlh-2/3</i>, thereby resulting in a level of <i>egl-1</i> activity that is high enough to induce apoptosis. See text for details and molecular interpretations.</p

<i>ces-1(n703</i>gf<i>)</i>; <i>cya-1(bc416)</i> blocks cell divisions in the ABarp, C and E lineages.

<p>All strains analyzed were homozygous for <i>bcIs66</i>. Lineage analyses were performed for two (wild-type, <i>+/+</i>), three (<i>ces-1(n703</i>gf<i>); cya-1(bc416)</i>) and three (<i>cdc-25.2(RNAi)</i>) embryos raised at 25°C. The ABarp, C and E lineages are shown. Vertical axis indicates approximate time in min after the 1^st round of embryonic division, in which P0 divides into AB and P1. In the case of <i>ces-1(n703</i>gf<i>); cya-1(bc416)</i>, cell division defects observed in three out of three embryos are depicted in red, defects found in two out of three embryos are depicted in blue, and defects found in one out of three embryos are depicted in orange. In the case of <i>cdc-25.2(RNAi)</i>, RNAi was carried out by injection. Since there is some variability of the RNAi effect, the lineage shown here was derived from the embryo with the strongest phenotype (cell division defects observed in this embryo are depicted in green), and the lineages from the other two <i>cdc-25.2(RNAi)</i> embryos are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003884#pgen.1003884.s006" target="_blank">Figure S6</a>. The severe cell division defects in the ABarp, C and E lineages were seen in all three <i>cdc-25.2(RNAi)</i> embryos. The cell death in the ABarp lineage is labeled with the cross. The defects in the C lineage and ABarp lineage result in a defect in the formation of the hypodermis (the mitoses that generate hyp7, hyp5, hyp11, H0, H1, H2, V1, V2, V4, and V6 fail to occur).</p

CES-1 binds to an upstream region of the <i>cdc-25.2</i> locus.

<p>The genome-wide binding sites of the CES-1 protein were identified using ChIP-seq. Shown are the distributions of CES-1-bound regions around the genomic loci of the four <i>cdc-25</i> genes, whose transcription units are indicated by blue arrows. The black boxes correspond to the gene exons. The red arrow points to the CES-1-bound region upstream of <i>cdc-25.2</i>. Data was visualized using Integrated Genome Browser based on genome WS190 of <i>C. elegans</i> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003884#pgen.1003884-Nicol1" target="_blank">[39]</a>.</p

Additional file 2: Figure S1. of Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165

Examples of hematoxylin-eosin stained sections of mADSC CS attached to mouse muscle. Low magnification images show presence of CS on the femoral quadriceps muscle at day 14 (black arrows). (TIFF 3564 kb

Additional file 1: Video 1. of Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165

Detachment and manipulation of cell sheets from mADSC. Brief introduction to two critical points in ADSC CS preparation – detachment using a pipette tip and manipulation in PBS using a cell-strainer mesh glued to a steel spatula. (MP4 19296 kb