Fig. S1. Cell proliferation and apoptosis in Geminin overexpressing and knockdown embryos. Embryos were injected with mRNA encoding a Geminin variant deleted for the Cdt1-binding central coiled-coil (Gemininδcoil) (Seo et al., 2005) or with a Geminin antisense morpholino oligo (MO) (Seo et al., 2005) at doses used for subsequent experiments. We have previously found that both full-length Geminin and two Cdt1 non-binding variants (the N-terminal half or Gemininδcoil) had similar effects on cell fate: overexpression of full-length Geminin at high levels in embryos was cytotoxic, but injecting lower doses (20-30 pg full-length Geminin RNA) or injecting Cdt1-non-binding Geminin variants expanded the neural plate and suppressed epidermis (Kroll et al., 1998; Seo et al., 2005). Likewise, Geminin MO-mediated knockdown could induce epidermal differentiation at the expense of neural tissue formation (Seo et al., 2005). (A) To assess whether Gemininδcoil or MO affected cell proliferation, we performed immunostaining for phosphorylated Histone H3 (PH3), which marks mitotic cells in mid-gastrula embryos (stage 11-11.5; A). Immunostaining was performed as described previously (Saka and Smith, 2001), using a 1:1000 dilution of the primary antibody (Millipore/06-570). (B) In each embryo, the number of phosphorylated Histone H3-positive cells located in the X-Gal-labeled injected region was compared with an uninjected region of equivalent size (e.g. A, top right-hand panel). Paired t-tests (brackets) showed no significant difference in PH3-positive cell numbers between injected and uninjected tissue. (C) TUNEL assays were performed as described previously (Hensey and Gautier, 1998) under the same conditions to detect apoptotic cells. (D) TUNEL-positive cells in injected embryos were counted. Most embryos had few or no TUNEL-positive cells, as expected (Hensey and Gautier, 1998); to illustrate the assay, an embryo with several TUNEL-positive cells is shown in C, bottom right-hand panel.
Image published in: Lim JW et al. (2011)
Copyright © 2011. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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