ES cells represent a valuable model for investigating early embryo development and hold promise for future regenerative medicine strategies. The self-renewal of pluripotent mouse ES cells has been shown to require extrinsic stimulation by the bone morphogenetic protein (BMP) and leukemia inhibitory factor signaling pathways and the expression of the transcription factors Oct4 and Nanog. However, the network of interactions among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Here, we show that Nanog expression is up-regulated in mouse ES cells by the binding of T (Brachyury) and STAT3 to an enhancer element in the mouse Nanog gene. We further show that Nanog blocks BMP-induced mesoderm differentiation of ES cells by physically interacting with Smad1 and interfering with the recruitment of coactivators to the active Smad transcriptional complexes. Taken together, our findings illustrate the existence of ES cellspecific regulatory networks that underlie the maintenance of ES cell pluripotency and provide mechanistic insights into the role of Nanog in this process.pluripotency ͉ T (Brachyury) ͉ self-renewal ͉ mesoderm differentiation ͉ leukemia inhibitory factor M ouse ES cells are self-renewing, pluripotent cell lines derived from preimplantation embryos (1, 2). Strict culture conditions must be followed to maintain the self-renewal of pluripotent mouse ES cells. Two extrinsic culture requirements, a feeder layer of fibroblasts and the addition of FBS, have been identified as necessary to sustain proliferation of undifferentiated mouse ES cells and their activities pinpointed to specific molecules (reviewed in ref. 3). Thus, self-renewal of mouse ES cells can be sustained in feeder-free conditions by supplementing the culture media with the cytokine leukemia inhibitory factor (LIF) (4, 5). In the absence of LIF, ES cell colonies flatten and form epithelium-like sheets (4, 5). More recently, the self-renewal promoting activity of animal serum has been identified as being mediated by ligands of specific families of the TGF-␤ superfamily, including the bone morphogenetic protein (BMP) family members BMP2 and BMP4 and the growth and differentiation factor (GDF) family member GDF6 (6). In the absence of BMP͞GDF signals, LIF is not sufficient to prevent the neural differentiation of ES cells, whereas the absence of both BMP͞GDF and LIF stimulation results in a flattened cell phenotype similar to that observed during LIF withdrawal (6).The intracellular signaling cascades initiated by both LIF and BMP͞GDF that sustain self-renewal of mouse ES cells have been worked out to a significant degree of detail (reviewed in ref.3). In summary, binding of LIF to its cognate LIF receptor results in the recruitment of gp130 and the formation of a ternary complex that catalyzes the tyrosine phosphorylation, dimerization, and nuclear translocation of the downstream signal transducer STAT3. BMP͞ GDF, in turn, promotes ES cell self-renewal by inducing the expression of members of the inhibit...

We describe a new methodology for rapid 2D and 3D computer analysis and visualisation of gene expression and gene product pattern in the context of anatomy and tissue architecture. It is based on episcopic imaging of embryos and tissue samples, as they are physically sectioned, thereby producing inherently aligned digital image series and volume data sets, which immediately permit the generation of 3D computer representations. The technique uses resin as embedding medium, eosin for unspecific tissue staining, and colour reactions (beta-galactosidase/Xgal or BCIP/NBT) for specific labelling of gene activity and mRNA pattern. We tested the potential of the method for producing high-resolution volume data sets of adult human and porcine tissue samples and of specifically and unspecifically stained mouse, chick, quail, frog, and zebrafish embryos. The quality of the episcopic images resembles the quality of digital images of true histological sections with respect to resolution and contrast. Specifically labelled structures can be extracted using simple thresholding algorithms. Thus, the method is capable of quickly and precisely detecting molecular signals simultaneously with anatomical details and tissue architecture. It has no tissue restrictions and can be applied for analysis of human tissue samples as well as for analysis of all developmental stages of embryos of a wide variety of biomedically relevant species.

Several components of noncanonical Wnt signaling pathways are involved in the control of convergence and extension (CE) movements during zebrafish and Xenopus gastrulation. However, the complexity of these pathways and the wide patterns of expression and activity displayed by some of their components immediately suggest additional morphogenetic roles beyond the control of CE. Here we show that the key modular intracellular mediator Dishevelled, through a specific activation of RhoA GTPase, controls the process of convergence of endoderm and organ precursors toward the embryonic midline in the zebrafish embryo. We also show that three Wnt noncanonical ligands wnt4a, silberblick/wnt11, and wnt11-related regulate this process by acting in a largely redundant way. The same ligands are also required, nonredundantly, to control specific aspects of CE that involve interaction of Dishevelled with mediators different from that of RhoA GTPase. Overall, our results uncover a late, previously unexpected role of noncanonical Wnt signaling in the control of midline assembly of organ precursors during vertebrate embryo development.[Keywords: Heart; endoderm; noncanonical Wnt signaling; Dishevelled; RhoA] Supplemental material is available at http://genesdev.org.

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Vertebrate segments called somites are generated by periodic segmentation of the anterior extremity of the presomitic mesoderm (PSM). During somite segmentation in zebrafish, mesp-b determines a future somite boundary at position B-2 within the PSM. Heat-shock experiments, however, suggest that an earlier future somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Here, we characterized fibroblast growth factor (FGF) signal activity within the PSM, and demonstrated that an anterior limit of downstream Erk activity corresponds to the future B-5 somite boundary. Moreover, the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. Our results provide the first molecular evidence of the future somite boundary at B-5, and we propose that clock-dependent cyclic inhibition of the FGF/Erk signal is a key mechanism in the generation of perfect repetitive structures in zebrafish development. KEY WORDS: FGF signalling, Clock, Segmentation, Somitogenesis INTRODUCTIONProper formation of a well-proportioned body in multicellular organisms requires spatiotemporal control of multiple biological processes during development. For instance, the regularity of repetitive structures in vertebrate bodies is derived from the vertebrate segments called somites, which are generated by periodic segmentation of the uniform presomitic mesoderm (PSM) (Pourquié, 2001). The periodicity of segmentation is regulated by oscillation of the segmentation clock genes, and the position of segmentation is determined by a gradient of fibroblast growth factor (FGF) (Dubrulle et al., 2001;Dubrulle and Pourquié, 2004;Holley, 2007;Pourquié, 2001;Sawada et al., 2001).The first detectable sign of a future segment boundary, as visualized by the expression of mesoderm posterior homolog-b (mesp-b; mespba -Zebrafish Information Network), appears at a distance of two somite lengths posterior to B0 (B-2, between S-I and S-II; see Fig. 1D for nomenclature) in the anterior PSM in zebrafish (Sawada et al., 2000), although this may not be the earliest event in boundary determination. A brief exposure of zebrafish embryos to heat shock disrupts somite segmentation after four cycles of normal segmentation (Roy et al., 1999), suggesting that the boundaries of at least five somites (from B-1 to B-5, between S-IV and S-V) are predetermined somewhere in the uniform PSM. However, no RESEARCH ARTICLEGene Regulation Research, Nara Institute Science and Technology, 8916-5 Takayama, Nara 630-0101, Japan. RESULTS The fgf8a gradient moves continuously towards the posterior during somitogenesisIn chick, an Fgf8 gradient, generated by restricted transcription and mRNA decay of Fgf8, is essential for setting up the position of the segmentation (Dubrulle and Pourquié, 2004). In zebrafish, segmentation defects can be seen in ace/ace embryos (fgf8a mutants) (supplementary material Fig. S1) (see also Reifers et al., 1998), suggesting that zebrafish fgf8a has a similar role a...