Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.

Nodal, a member of the transforming growth factor ␤ (TGF-␤) superfamily, is implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Here we define the intracellular signaling pathway induced by recombinant nodal protein treatment of P19 embryonal carcinoma cells. Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-␤. However, nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. We also demonstrate that nodal induces p(CAGA) 12 , a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduces the level of luciferase reporter activity induced by nodal treatment. Finally, we show that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide the first direct biochemical evidence that nodal signaling is mediated by both activin-TGF-␤ pathway Smads, Smad2 and Smad3. We also show here that the extracellular cripto protein is required for nodal signaling, making it distinct from activin or TGF-␤ signaling. Members of the transforming growth factor ␤ (TGF-␤)1 superfamily of intercellular signaling factors regulate cell fate and behavior during development and in the adult (1). The three major subgroups based on sequence similarity are the TGF-␤s, activins and inhibins, and bone morphogenetic proteins (BMPs; Ref. 1). Nodal and related factors form a separate subgroup and are implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification (2).Signaling by TGF-␤ and related ligands uses two types of receptors, type I and type II transmembrane serine-threonine kinases. Ligand binding results in the formation of heteromeric receptor complexes, in which type II receptors phosphorylate type I receptors (1, 3). Downstream signal transduction events are mediated by the intracellular Smad proteins. One class, the receptor-regulated Smads (R-Smads), are directly phosphorylated by activated type I receptors on a C-terminal SSXS motif. Upon phosphorylation, R-Smads form complexes with the coSmad, Smad4 and then translocate to the nucleus and regulate transcription of target genes. Biochemical and biological studies have established that the R-Smads used by TGF-␤ and activin signaling, Smad2 and Smad3, are distinct from those used by BMP signaling, Smad1, Smad5, and Smad8 (1, 3, 4).The nodal signaling pathway awaits characterization at the biochemical level. However, mutational studies in the mouse and zebrafish and ectopic expression studies in Xenopus and zebrafish suggest that the nodal and activin signaling pathways may share receptors and Smads. Targeted mutations in the mouse Smad2 gene (5-8) and the activin type IB receptor gene (9) and combined mutations of the activin type IIA and IIB receptor genes (10) show gastrulation phenotypes resem...

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Ras-like small GTPases are involved in the regulation of many processes essential for the specification of the vertebrate body plan. Recently, we identified the gene of novel small GTPase Ras-dva, which is specifically expressed at the anterior margin of the neural plate of the Xenopus laevis embryo. Now, we demonstrate that Ras-dva and its homologs in other species constitute a novel protein family, distinct from the previously known families of small GTPases. We show that the expression of Ras-dvabegins during gastrulation throughout the anterior ectoderm and is activated by the homeodomain transcription factor Otx2; however, later on, Ras-dva expression is inhibited in the anterior neural plate by another homeodomain factor Xanf1. Downregulation of Ras-dva functioning by the dominant-negative mutant or by the antisense morpholino oligonucleotides results in severe malformations of the forebrain and derivatives of the cranial placodes. Importantly, although the observed abnormalities can be rescued by co-injection of the Ras-dva mRNA, they cannot be rescued by the mRNA of the closest Ras-dva homolog from another family of small GTPases, Ras. This fact indicates functional specificity of the Ras-dva signaling pathway. At the molecular level, downregulation of Ras-dva inhibits the expression of several regulators of the anterior neural plate and folds patterning, such as Otx2, BF-1 (also known as Foxg1), Xag2, Pax6, Slug and Sox9, and interferes with FGF8 signaling within the anterior ectoderm. By contrast, expression of the epidermal regulator BMP4 and its target genes, Vent1, Vent2band Msx1, is upregulated. Together, the data obtained indicate that Ras-dva is an essential component of the signaling network that patterns the early anterior neural plate and the adjacent ectoderm in the Xenopus laevis embryos.