Xnrs and Activin Regulate Distinct Genes during Xenopus Development: Activin Regulates Cell Division Joana M. Ramis, Clara Collart, James C. Smith* Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge, United Kindgom Background. The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type b family such as Vg1, activin B, the nodal-related proteins and derrie ` re. Methodology and Principle Findings. Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17a/b, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited. Conclusions/Significance. These observations reveal distinct functions for these two classes of the TGF-b family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation. Citation: Ramis JM, Collart C, Smith JC (2007) Xnrs and Activin Regulate Distinct Genes during Xenopus Development: Activin Regulates Cell Division. PLoS ONE 2(2): e213. doi:10.1371/journal.pone.0000213 INTRODUCTION The mesoderm of the amphibian embryo arises through an inductive interaction in which cells of the vegetal hemisphere act on overlying equatorial cells [1]. Of the several mesoderm- inducing factors that have been discovered, most are members of the transforming growth factor type b family. These include activin [2–4], Vg1 [5,6], five nodal-related proteins [7–9], and derrie `re [10]. Although these factors have similar abilities to induce gene expression in isolated animal pole regions, they are differently expressed in the embryo (see above references) and under some experimental conditions have different abilities to exert long-range effects [11,12]. In addition, each exerts different effects at different concentrations [7,13]. The challenge now is to elucidate the individual roles of these proteins within the embryo and to ask how their actions are coordinated. Some attempts along these lines have been made, and it proves that although each of the factors is essential for normal development, their roles differ. For example, ablation of the maternal transcripts encoding Vg1 causes a reduction in anterior and dorsal development and the down-regulation of genes such as chordin, cerberus and noggin [6]. Of the zygotically-expressed inducing factors, depletion of activin also causes axial defects [3,14,15], although these are less severe than those caused by loss of Vg1, and inhibition of derrie `re activity causes just posterior defects [10]. Simultaneous inhibition of the activities of all the nodal related proteins, by expression of Cerberus-short, causes loss of mesoderm [16,17] and the down regulation of genes such as Chordin and Pintallavis [18]. The requirements of the individual nodal related proteins have not been studied in detail, although injection of antisense morpholino oligonucleotides directed against Xnr1 causes defects in left-right axis determination [19]. Here we perform microarray analyses of gene expression in embryos in which activin or nodal-related signalling has been inhibited. We find that activin and the nodal-related proteins regulate distinct and almost completely non-overlapping sets of genes, with those regulated by the nodal-related genes tending to be expressed in a more restricted pattern than those regulated by activin. It further proved that the nodal-related proteins often regulate the expression of genes involved in regional specification, while activin particularly regulates genes involved in the control of the cell cycle. Consistent with this observation, we find that inhibition of activin B in the early embryo causes dorsal axial mesodermal cells to fail to exit the cell cycle: the results of others [20–22] suggest that it is the continued proliferation of these cells that underlies the gastrulation defects observed in such embryos. RESULTS Microarray results In an effort to understand the different requirements for activin B and the nodal-related genes during Xenopus development, we have carried out microarray analyses of gene expression in embryos in which signalling by the two classes of factor has been disrupted. Activin signalling was blocked using an antisense morpholino oligonucleotide [3], and nodal-related signalling by Cerberus- short, a truncated form of Cerberus [17]. Our microarray slides comprise 10,898 probes designed to recognise sequences derived from a large scale Xenopus tropicalis EST project [23]. These arrays also recognise X. laevis transcripts [24]. For each series of experiments Xenopus laevis embryos from three different spawnings were injected with RNA encoding Cerberus- short (150 pg into each blastomere at the four-cell stage) or with Academic Editor: Thomas Zwaka, Baylor College of Medicine, United States of America Received January 16, 2007; Accepted January 19, 2007; Published February 14, 2007 Copyright: ß 2007 Ramis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by the Wellcome Trust, an EC Marie Curie Individual Fellowship to JMR, and the EC Network of Excellence ‘Cells into Organs’. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: jim@gurdon.cam.ac.uk PLoS ONE | www.plosone.org 1 February 2007 | Issue 2 | e213