Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Establishment of the body plan in vertebrates depends on the temporally coordinated patterning of tissues along the body axes. We have previously shown that dorsoventral (DV) tissues are temporally patterned progressively from anterior to posterior by a BMP signaling pathway. Here we report that DV patterning along the zebrafish anteroposterior (AP) axis is temporally coordinated with AP patterning by an identical patterning clock. We altered AP patterning by inhibiting or activating FGF, Wnt or retinoic acid signaling combined with inhibition of BMP signaling at a series of developmental time points, which revealed that the temporal progression of DV patterning is directly coordinated with AP patterning. We investigated how these signaling pathways are integrated and suggest a model for how DV and AP patterning are temporally coordinated. It has been shown that in Xenopus dorsal tissues FGF and Wnt signaling quell BMP signaling by degrading phosphorylated (P) Smad1/5, the BMP pathway signal transducer, via phosphorylation of the Smad1/5 linker region. We show that in zebrafish FGF/MAPK, but not Wnt/GSK3, phosphorylation of the Smad1/5 linker region localizes to a ventral vegetal gastrula region that could coordinate DV patterning with AP patterning ventrally without degrading P-Smad1/5. Furthermore, we demonstrate that alteration of the MAPK phosphorylation sites in the Smad5 linker causes precocious patterning of DV tissues along the AP axis during gastrulation. Thus, DV and AP patterning are intimately coordinated to allow cells to acquire both positional and temporal information simultaneously.

Nodal/Bozozok-independent induction of the dorsal organizer by zebrafish cell lines.

Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades--Bozozok/Dharma (Boz) and Nodal signaling--act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cells themselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers in cells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activated following implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.

Variant BMP receptor mutations causing fibrodysplasia ossificans progressiva (FOP) in humans show BMP ligand-independent receptor activation in zebrafish.

The large majority of cases of the autosomal dominant human disease fibrodysplasia ossificans progressiva (FOP) are caused by gain-of-function Arg206His mutations in the BMP type I receptor ACVR1 (ALK2). The Arg206His mutation is located in the GS domain of the type I receptor. This region is normally phosphorylated by the BMP type II receptor, which activates the type I receptor to phosphorylate its substrate, the signal transducer Smad1/5/8. A small subset of patients with FOP carry variant mutations in ACVR1 altering Gly328 to Trp, Glu or Arg. Since these mutations lie outside the GS domain, the mechanism through which ACVR1 Gly328 mutations cause disease remains unclear. We used a zebrafish embryonic development assay to test the signaling of human ACVR1 Gly328 mutant receptors comparing them to the Arg206His mutant. In this assay increased or decreased BMP pathway activation alters dorsal-ventral axial patterning, providing a sensitive assay for altered BMP signaling levels. We expressed the human ACVR1 Gly328 mutant receptors in zebrafish embryos to investigate their signaling activities. We found that all ACVR1 Gly328 human mutations ventralized wild-type embryos and could partially rescue Bmp7-deficient embryos, indicating that these mutant receptors can activate BMP signaling in a BMP ligand-independent manner. The degree of ventralization or rescue was similar among all three Gly328 mutants. Smad1/5 phosphorylation, a readout of BMP receptor signaling, was mildly increased by ACVR1 Gly328 mutations. Gene expression analyses demonstrate expanded ventral and reciprocal loss of dorsal cell fate markers. This study demonstrates that Gly328 mutants increase receptor activation and BMP ligand-independent signaling through Smad phosphorylation.

Lagrangian chaos and particle diffusion in electroconvection of planar nematic liquid crystals.

Two types of spatiotemporal chaos in the electroconvection of nematic liquid crystals, such as defect turbulence and spatiotemporal intermittency, have been statistically investigated according to the Lagrangian picture. Here fluctuations are traced using the motion of a single particle driven by chaotic convection. In the defect turbulence (fluctuating normal rolls), a particle is mainly trapped in a roll but sometimes jumps to a neighboring roll. Its activation energy is then obtained from the jumping (hopping) rate. This research clarifies that diffusion in the defect turbulence regime in electroconvection can be regarded as a kind of hopping process. The spatiotemporal intermittency appears as a coexistent state of ordered grid domains and turbulent domains. The motion of a single particle shows weak and strong diffusion, respectively, in the ordered and turbulent domains. The diffusion characteristics intermittently change from one to another with certain durations as the domains change. This research has found that the distribution function of the duration that a particle remains in an ordered area has a power-law decay for which the index is different from that obtained by the Eulerian measurement.

An intermediate level of BMP signaling directly specifies cranial neural crest progenitor cells in zebrafish.

The specification of the neural crest progenitor cell (NCPC) population in the early vertebrate embryo requires an elaborate network of signaling pathways, one of which is the Bone Morphogenetic Protein (BMP) pathway. Based on alterations in neural crest gene expression in zebrafish BMP pathway component mutants, we previously proposed a model in which the gastrula BMP morphogen gradient establishes an intermediate level of BMP activity establishing the future NCPC domain. Here, we tested this model and show that an intermediate level of BMP signaling acts directly to specify the NCPC. We quantified the effects of reducing BMP signaling on the number of neural crest cells and show that neural crest cells are significantly increased when BMP signaling is reduced and that this increase is not due to an increase in cell proliferation. In contrast, when BMP signaling is eliminated, NCPC fail to be specified. We modulated BMP signaling levels in BMP pathway mutants with expanded or no NCPCs to demonstrate that an intermediate level of BMP signaling specifies the NCPC. We further investigated the ability of Smad5 to act in a graded fashion by injecting smad5 antisense morpholinos and show that increasing doses first expand the NCPCs and then cause a loss of NCPCs, consistent with Smad5 acting directly in neural crest progenitor specification. Using Western blot analysis, we show that P-Smad5 levels are dose-dependently reduced in smad5 morphants, consistent with an intermediate level of BMP signaling acting through Smad5 to specify the neural crest progenitors. Finally, we performed chimeric analysis to demonstrate for the first time that BMP signal reception is required directly by NCPCs for their specification. Together these results add substantial evidence to a model in which graded BMP signaling acts as a morphogen to pattern the ectoderm, with an intermediate level acting in neural crest specification.