FoxG1 is an evolutionarily conserved, winged-helix transcriptional repressor that maintains progenitor cells in the vertebrate forebrain. How the activity of FoxG1 is regulated is not known. Here, we report that in the developing Xenopus and mouse forebrain, FoxG1 is nuclear in progenitor cells but cytoplasmic in differentiating cells. The subcellular localisation of FoxG1 is regulated at the post-translational level by casein kinase I (CKI) and fibroblast growth factor (FGF) signalling. CKI phosphorylation of Ser 19 of FoxG1 promotes nuclear import, whereas FGF-induced phosphorylation of Thr 226 promotes nuclear export. Interestingly, FGF-induced phosphorylation of FoxG1 is mediated Akt kinase (also known as protein B kinase, PKB) kinase, rather than the MAPK pathway. Phosphorylation of endogenous FoxG1 is blocked by CKI and Akt inhibitors. In the mouse olfactory placode cell line OP27, and in cortical progenitors, increased FGF signalling causes FoxG1 to exit the nucleus and promotes neuronal differentiation, whereas FGF and Akt inhibitors block this effect. Thus, CKI and FGF signalling converge on an antagonistic regulation of FoxG1, which in turn controls neurogenesis in the forebrain.

Transforming growth factor-b (TGF-b) signals through membrane-bound serine/threonine kinase receptors, which upon stimulation phosphorylate Smad proteins and thereby trigger their nuclear translocation and transcriptional activity. Although the three mammalian isoforms of TGF-b are highly homologous at the level of sequence, analysis of their in vivo function by gene knockouts revealed striking differences, suggesting no signi®cant functional redundancy between TGF-b1, -2 and -3. While signal transduction by TGF-b1 has been well characterized, receptor binding and activation by the TGF-b2 isoform is less well understood. Here, we show that TbRII-B, an alternatively spliced variant of the TGF-b type II receptor, is a TGF-b2 binding receptor, which mediates signalling via the Smad pathway in the absence of any TGF-b type III receptor (TbRIII). L6 cells lacking endogenous TbRIII as well as TbRII-B do not respond to TGF-b2. Transfection of these cells with TbRII-B restores TGF-b2 sensitivity. The expression of TbRII-B is restricted to cells originating from tissues such as bone where the isoform TGF-b2 has a predominant role. This re¯ects the importance of this receptor in TGF-b isoform-speci®c signalling.

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SUMMARYFoxG1 is a conserved transcriptional repressor that plays a key role in the specification, proliferation and differentiation of the telencephalon, and is expressed from the earliest stages of telencephalic development through to the adult. How the interaction with co-factors might influence the multiplicity and diversity of FoxG1 function is not known. Here, we show that interaction of FoxG1 with TLE2, a Xenopus tropicalis co-repressor of the Groucho/TLE family, is crucial for regulating the early activity of FoxG1. We show that TLE2 is co-expressed with FoxG1 in the ventral telencephalon from the early neural plate stage and functionally cooperates with FoxG1 in an ectopic neurogenesis assay. FoxG1 has two potential TLE binding sites: an N-terminal eh1 motif and a C-terminal YWPMSPF motif. Although direct binding seems to be mediated by the N-terminal motif, both motifs appear important for functional synergism. In the neurogenesis assay, mutation of either motif abolishes functional cooperation of TLE2 with FoxG1, whereas in the forebrain deletion of both motifs renders FoxG1 unable to induce the ventral telencephalic marker Nkx2.1. Knocking down either FoxG1 or TLE2 disrupts the development of the ventral telencephalon, supporting the idea that endogenous TLE2 and FoxG1 work together to specify the ventral telencephalon.

The molecular mechanisms that underlie the development of primitive myeloid cells in vertebrate embryos are not well understood. Here we characterize the role of cebpa during primitive myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Lossand gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally, we use live imaging to show that cebpa-expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus, C/EPB␣ is the first known single factor capable of initiating an entire myelopoiesis pathway in pluripotent cells in the embryo. (Blood. 2009;114:40-48) IntroductionHematopoiesis occurs in 2 distinct phases during development. [1][2][3][4] The first wave, also known as primitive hematopoiesis, often occurs in extraembryonic hemogenic sites and provides the embryos with a transient population of blood cells. The second wave gives rise to blood progenitors, which persist into late development and adulthood in successive hemogenic sites, and is therefore called definitive hematopoiesis. Whereas much is known about the molecular and cellular pathways responsible for definitive hematopoiesis, relatively little is known about the pathways responsible for the specification of the primitive blood lineages.In the past decade, aquatic vertebrate species have emerged as powerful model organisms for the investigation of primitive and definitive hematopoiesis. [5][6][7][8] Favored aquatic model organisms for hematopoiesis include the teleost fish, zebrafish, and the amphibian, Xenopus laevis, and its diploid relative, X tropicalis. 9,10 Fish and frog embryos can be produced in large numbers, and they develop externally, allowing the visualization of the development and behavior of blood lineages in vivo. Because the embryos are large, embryologic manipulations, such as tissue transplantations, can be performed with relative ease. In addition, the molecular and genetic basis of hematopoiesis in these organisms is largely conserved with those of mammals. [5][6][7][8] Studies in zebrafish have shown that primitive hematopoiesis consists of 2 separate events: primitive myelopoiesis and primitive erythropoiesis, which take place in 2 distinct hematopoietic compartments. 11,12 Similarly in Xenopus, the primitive myeloidforming compartment is located in the anterior ventral blood islands (aVBIs), which are derived from dorsal/anterior gastrula mesoderm, while primitive erythropoiesis occurs primarily in the posterior ventral blood islands, which are derived from ventral/ posterior ga...

Phosphorylation of Smads is a crucial regulatory step in the signal transduction pathway initiated by bone morphogenetic proteins (BMPs). Although the dephosphorylation events terminating the pathway in the nucleus have been characterized, little is known about the dephosphorylation of Smads in the cytoplasm. In a proteomic screen for proteins interacting with the BMP type-II receptor, we found the regulatory Bβ subunit of PP2A. PP2A is one of the major serine/threonine phosphatases involved in cell-cycle regulation and signal transduction. Here, we present data showing that the Bβ subunit of PP2A interacts with both BMP type-I and type-II receptors. Furthermore, we demonstrate that several B subunits can associate with the BMP type-II receptor, independently of the kinase activity of the receptor and the catalytic subunit of PP2A. By contrast, the PP2A catalytic subunit is required for PP2A function at the receptor complex. This function of PP2A is the dephosphorylation of Smad1, mainly in the linker region. PP2A-mediated dephosphorylation of the BMP-Smad linker region leads to increased nuclear translocation of Smads and overall amplification of the BMP signal. Although other phosphatases identified within the BMP pathway are all shown to inhibit signalling, PP2A is the first example for a signalling stimulatory phosphatase within this pathway.

optomotor-blind (omb) and optomotor-blind related-1 (org-1) encode T-domain DNA binding proteins in Drosophila. Members of this family of transcription factors play widely varying roles during early development and organogenesis in both vertebrates and invertebrates. Functional specificity differs in spite of similar DNA binding preferences of all family members. Using a series of domain swap chimeras, in which different parts of OMB and ORG-1 were mutually exchanged, we investigated the relevance of individual domains in vitro and in vivo. In cell culture transfection assays, ORG-1 was a strong transcriptional activator, whereas OMB appeared neutral. The main transcriptional activation function was identified in the C-terminal part of ORG-1. Also in vivo, OMB and ORG-1 showed qualitative differences when the proteins were ectopically expressed during development. Gain-of-function expression of OMB is known to counteract eye formation and resulted in the loss of the arista, whereas ORG-1 had little effect on eye development but caused antenna-to-leg transformations and shortened legs in the corresponding gain-of-function situations. The functional properties of OMB/ORG-1 chimeras in several developmental contexts was dominated by the origin of the C-terminal region, suggesting that the transcriptional activation potential can be one major determinant of developmental specificity. In late eye development, we observed, however, a strong influence of the T-domain on ommatidial differentiation. The specificity of chimeric omb/org-1transgenes, thus, depended on the cellular context in which they were expressed. This suggests that both transcriptional activation/repression properties as well as intrinsic DNA binding specificity can contribute to the functional characteristics of T-domain factors.

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