Rana AA et al. (2011), Loss of Xenopus tropicalis EMSY causes impairme...

XB-ART-42194

N Biotechnol 2011 Jul 01;284:334-41. doi: 10.1016/j.nbt.2010.10.010.

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Loss of Xenopus tropicalis EMSY causes impairment of gastrulation and upregulation of p53.

Rana AA , Roper SJ , Palmer EA , Smith JC .

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EMSY interacts directly with BRCA2 and links the BRCA2 pathway to sporadic breast and ovarian cancer. It also interacts with BS69 and HP1b, both of which are involved in chromatin remodelling, and with NIF-1 and DBC-1 in the regulation of nuclear receptor-mediated transcription. Here we investigate the function of EMSY during amphibian development, and in doing so provide the first loss-of-function analysis of this protein. Injection of Xenopus tropicalis embryos with antisense morpholino oligonucleotides targeting XtEMSY impairs gastrulation movements, disrupts dorsal structures, and kills embryos by tailbud stages. Consistent with these observations, regional markers such as Xbra, Chd, Gsc, Shh, Sox3 and Sox17 are downregulated. In contrast to these regional markers, expression of p53 is upregulated in such embryos, and at later stages Bax expression is elevated and apoptotic cells can be detected. Our results demonstrate that EMSY has an essential role in development and they provide an in vivo loss-of-function model that might be used to explore the biochemical functions of this protein in more detail.

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Species referenced: Xenopus tropicalis
Genes referenced: bax brca2 chrd emsy ets2 gsc neff1 odc1 shh sox17a sox17b.2 sox3 tbxt tp53
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	Figure 1. Genomic structure of the XtEMSY locus and of XtEMSY isoforms. The XtEMSY locus comprises 22 exons, of which the first 21 encode a protein of 1291 amino acids. A short transcript comprising exons 1–7 and exon 22, encoding a protein of 269 amino acids, was detected in early embryos of Xenopus tropicalis. ESTs representing this transcript were found in egg, gastrula and tadpole cDNA libraries: AL870737.2 (egg); AL871852.2 (egg); AL652745.2 (gastrula); and CX371164.1 (tadpole). The short and the long forms of XtEMSY both contain the ENT domain and putative HP1β and BS69 binding sites.
	Figure 2. Expression of XtEMSY during development. RT-PCR confirms that the long form of XtEMSY is expressed in adult brain and testis (a), and that the short form is expressed at the 2-cell stage and at embryonic stages 8.5, 10.5, 13, 18, 24 and 40 (b). Primers specific for EF1α were used as a control.
	Figure 3. Analysis of the expression of XtEMSY and of some of its putative interactors. Gastrula stages are shown in panels (a, e, i, m, q, u), neurula stages in panels (b, f, j, n, r, v), tailbud stages in panels (c, g, k, o, s, w) and tadpole stages in panels (d, h, l, p, t, x). (a–d) Use of an XtEMSY sense probe shows no staining. (e–h) XtEMSY is expressed throughout the ectoderm at the gastrula stage (e) and is then expressed most strongly in the neural plate and dorsal regions at the neurula stage (f). By tailbud stages XtEMSY is expressed most strongly in the head and along the dorsal axis (g). At tadpole stage XtEMSY staining is observed in the entire head region, including the brain, eye, otic vesicle and branchial arches and along the dorsal axis in the neural tube and somites (h). The expression patterns of BRCA2 (i–l), BS69 (m–p), Ets-2 (q–t) and HP1β (u–x) resemble those of XtEMSY.
	Figure 4. Loss of XtEMSY function causes defects in gastrulation and neurulation. Embryos injected with GeneTools control MO develop normally (a and b), whereas those injected with XtEMSY MO1 (c and d) or XtEMSY MO2 (g and f) fail to gastrulate normally and form a truncated axis. (g–o) Embryos injected with antisense morpholino oligonucleotides carrying five bases mismatches do not perturb development. (g–i) Uninjected embryos at gastrula, neurula and tailbud stages. (j–l) Embryos at the same stages injected with XtEMSY mMO1, and (m–o) show embryos injected with XtEMSY mMO2. (p–y) A time course experiment shows that in comparison with controls (p–t) embryos injected with XtEMSY MO1 (u–y) first show abnormalities at the early gastrula stage (compare q and v).
	Figure 5. Expression of various regional markers is down regulated in embryos injected with XtEMSY MO1. (a–x) The expression patterns of the indicated genes at gastrula and tailbud stages in control embryos and embryos injected with XtEMSY MO1. Note the impairment of gastrulation and the downregulation of, particularly, Shh (m–p). The spatial expression patterns of the analysed genes are only slightly affected. (y) Real-time RT-PCR shows that expression of Xbra and Chd is slightly decreased by loss of XtEMSY function, while expression of Gsc and Shh is down regulated more strongly.
	Figure 6. Loss of XtEMSY function causes upregulation of p53. (a and b)In situ hybridisation analysis at the late neurula stage reveals that p53 is upregulated in embryos injected with XtEMSY MO1 (b) compared with controls. (c) Temporal RT-PCR analysis of p53 and Bax expression in embryos injected with XtEMSY MO1. Expression levels are normalised to those of ornithine decarboxylase and levels in control MO injected embryos are defined as 100%. Note that expression of p53 is elevated between stages 18 and 24 (dark blue), while Bax is upregulated slightly later than this.
	Figure 7. Apoptosis in embryos injected with XtEMSY MO1. Control embryos (a and b) or embryos injected with XtEMSY MO1 (c and d) were subjected to TUNEL staining. No apoptosis was detected at stage 11 (a and b), but apoptotic cells were present by tailbud stages in dorsal regions (c and d).

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