Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear ß-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.

PIAS-like protein Zimp7 is required for the restriction of the zebrafish organizer and mesoderm development.

The Zmiz2 (Zimp7) protein and its homolog Zmiz1 (Zimp10) were initially identified in humans as androgen receptor co-activators. Sequence analysis revealed the presence of an SP-RING/Miz domain, which is highly conserved in members of the PIAS family and confers SUMO-conjugating activity. Zimp7 has been shown to interact with components of the Wnt/ß-Catenin signaling pathway and with Brg1 and BAF57, components of the ATP-dependent mammalian SWI/SNF-like BAF chromatin-remodeling complexes. In this work, we analyze the role of zygotic Zimp7 in zebrafish development. We describe evidence indicating that Zimp7 is required for mesoderm development and dorsoventral patterning. Morpholino-mediated reduction of zygotic Zimp7 produced axial mesodermal defects that were preceded by up-regulation of organizer genes such as bozozok, goosecoid and floating head at the onset of gastrulation and by down-regulation of the ventral markers vox, vent and eve1 indicating loss of the ventrolateral mesoderm. Consistently, embryos overexpressing zimp7 RNA exhibited midline defects such as loss of forebrain and cyclopia accompanied by transcriptional changes directly opposite of those found in the morphants. In addition, the patterning of ventralized embryos produced by the overexpression of vox and vent was restored by a reduction of Zimp7 activity. Altogether, our findings indicate that Zimp7 is involved in transcriptional regulation of factors that are essential for patterning in the dorsoventral axis.

Expression and evolution of the Tiki1 and Tiki2 genes in vertebrates.

Tiki1 is a Wnt protease and antagonist specifically expressed in the Spemann-Mangold Organizer and is required for head formation in Xenopus embryos. Here we report neighbor-joining phylogenetic analysis of vertebrate Tiki genes and their mRNA expression patterns in chick, mouse, and rabbit embryos. Tiki1 and Tiki2 orthologues are highly conserved, and exhibit similar but also different developmental expression patterns among the vertebrate/mammalian species analyzed. The Tiki1 gene is noticeably absent in the rodent lineage, but is present in lagomorphs and all other vertebrate/mammalian species examined. Expression in Hensen's node, the equivalent of the Xenopus Organizer, was observed for Chick Tiki2 and Rabbit Tiki1 and Tiki2. Mouse Tiki2 was detected at low levels at gastrulation and head fold stages, but not in the node. Mouse Tiki2 and chick Tiki1 display similar expression in the dorsal spinal cord. Chick Tiki1 expression was also detected in the surface ectoderm and maxillary bud, while chick Tiki2 was found in the anterior intestinal portal, head mesenchyme and primitive atrium. Our expression analyses provide evidence that Tiki1 and Tiki2 are evolutionarily conserved among vertebrate species and their expression in the Organizer and other regions suggests contributions of these Wnt inhibitors to embryonic patterning, as well as organogenesis. Our analyses further reveal mis-regulation of TIKI1 and TIKI2 in human cancer and diseases.

The homeoprotein Xiro1 is required for midbrain-hindbrain boundary formation.

The isthmic organizer, which patterns the anterior hindbrain and midbrain, is one of the most studied secondary organizers. In recent years, new insights have been reported on the molecular nature of its morphogenetic activity. Studies in chick, mouse and zebrafish have converged to show that mutually repressive interactions between the homeoproteins encoded by Otx and Gbx genes position this organizer in the neural primordia. We present evidence that equivalent, in addition to novel, interactions between these and other genes operate in Xenopus embryos to position the isthmic organizer. We made use of fusion proteins in which we combined Otx2 or Gbx2 homeodomains with the E1A activation domain or the EnR repressor element which were then injected into embryos. Our results show that Otx2 and Gbx2 are likely to be transcriptional repressors, and that these two proteins repress each other transcription. Our experiments show that the interaction between these two proteins is required for the positioning of the isthmic organizer genes Fgf8, Pax2 and En2. In this study we also developed a novel in vitro assay for the study of the formation of this organizer. We show that conjugating animal caps previously injected with Otx2 and Gbx2 mRNAs recreate the interactions required for the induction of the isthmic organizer. We have used this assay to determine which cells produce and which cells receive the Fgf signal. Finally, we have added a novel genetic element to this process, Xiro1, which encode another homeoprotein. We show that the Xiro1 expression domain overlaps with territories expressing Otx2, Gbx2 and Fgf8. By expressing wild-type or dominant negative forms of Xiro1, we show that this gene activates the expression of Gbx2 in the hindbrain. In addition, Xiro1 is required in the Otx2 territory to allow cells within this region to respond to the signals produced by adjacent Gbx2 cells. Moreover, Xiro1 is absolutely required for Fgf8 expression at the isthmic organizer. We discuss a model where Xiro1 plays different roles in regulating the genetic cascade of interactions between Otx2 and Gbx2 that are necessary for the specification of the isthmic organizer.