Takaaki Matsui

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin‐6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45+ hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.

Sp8 and Sp9, two closely related buttonhead-like transcription factors, regulate Fgf8 expression and limb outgrowth in vertebrate embryos.

Initiation and maintenance of signaling centers is a key issue during embryonic development. The apical ectodermal ridge, a specialized epithelial structure and source of Fgf8, is a pivotal signaling center for limb outgrowth. We show that two closely related buttonhead-like zinc-finger transcription factors, Sp8 and Sp9, are expressed in the AER, and regulate Fgf8 expression and limb outgrowth. Embryological and genetic analyses have revealed that Sp8 and Sp9 are ectodermal targets of Fgf10 signaling from the mesenchyme. We also found that Wnt/β-catenin signaling positively regulates Sp8, but not Sp9. Overexpression functional analyses in chick unveiled their role as positive regulators of Fgf8 expression. Moreover, a dominant-negative approach in chick and knockdown analysis with morpholinos in zebrafish revealed their requirement for Fgf8 expression and limb outgrowth, and further indicate that they have a coordinated action on Fgf8 expression. Our study demonstrates that Sp8 and Sp9, via Fgf8, are involved in mediating the actions of Fgf10 and Wnt/β-catenin signaling during vertebrate limb outgrowth.

K-Ras mediates cytokine-induced formation of E-cadherin-based adherens junctions during liver development.

The E‐cadherin‐based adherens junction (AJ) is essential for organogenesis of epithelial tissues including the liver, although the regulatory mechanism of AJ formation during development remains unknown. Using a primary culture system of fetal hepatocytes in which oncostatin M (OSM) induces differentiation, we show here that OSM induces AJ formation by altering the subcellular localization of AJ components including E‐cadherin and catenins. By retroviral expression of dominant‐negative forms of signaling molecules, Ras was shown to be required for the OSM‐induced AJ formation. Fetal hepatocytes derived from K‐Ras knockout (K‐Ras−/−) mice failed to form AJs in response to OSM, whereas AJ formation was induced normally by OSM in mutant hepatocytes lacking both H‐Ras and N‐Ras. Moreover, the defective phenotype of K‐Ras−/− hepatocytes was restored by expression of K‐Ras, but not by H‐Ras and N‐Ras. Finally, pull‐down assays using the Ras‐binding domain of Raf1 demonstrated that OSM directly activates K‐Ras in fetal hepatocytes. These results indicate that K‐Ras specifically mediates cytokine signaling for formation of AJs during liver development.

Maintenance of embryonic stem cell pluripotency by Nanog-mediated reversal of mesoderm specification

Embryonic stem cells (ESCs) can be propagated indefinitely in culture, while retaining the ability to differentiate into any cell type in the organism. The molecular and cellular mechanisms underlying ESC pluripotency are, however, poorly understood. We characterize a population of early mesoderm-specified (EM) progenitors that is generated from mouse ESCs by bone morphogenetic protein stimulation. We further show that pluripotent ESCs are actively regenerated from EM progenitors by the action of the divergent homeodomain-containing protein Nanog, which, in turn, is upregulated in EM progenitors by the combined action of leukemia inhibitory factor and the early mesoderm transcription factor T/Brachyury. These findings uncover specific roles of leukemia inhibitory factor, Nanog, and bone morphogenetic protein in the self-renewal of ESCs and provide novel insights into the cellular bases of ESC pluripotency.

Zebrafish IκB Kinase 1 Negatively Regulates NF-κB Activity

The IkappaB kinase (IKK) activity is critical for processing IkappaB inhibitory proteins and activating the NF-kappaB signaling, which is involved in a series of physiological and developmental steps in vertebrates. The IKK activity resides in two catalytic subunits, IKK1 and IKK2, and two regulatory subunits, NEMO and ELKS. IKK2 is the major cytokine-responsive IkappaB kinase because depletion of IKK1 does not interfere with the IKK activity. In fact, IKK1-/- mice display morphological abnormalities that are independent of its kinase activity and NF-kappaB activation. Hence, using zebrafish (Danio rerio) as a model, we examined the evolutionary role of IKK1 in modulating NF-kappaB. Ikk1-/- zebrafish embryos present head and tail malformations and, surprisingly, show upregulation of NF-kappaB-responsive genes and increased NF-kappaB-dependent apoptosis. Overexpression of ikk1 leads to midline structure defects that resemble NF-kappaB blockage in vivo. Zebrafish Ikk1 forms complexes with NEMO that represses NF-kappaB in vertebrate cells. Indeed, truncation of its NEMO binding domain (NBD) restores NF-kappaB-dependent transcriptional activity and, consequently, the ikk1-overexpressing phenotype. Here, we report that Ikk1 negatively regulates NF-kappaB by sequestering NEMO from active IKK complexes, indicating that IKK1 can function as a repressor of NF-kappaB.

Maintenance of Embryonic Stem Cell Pluripotency by Nanog-Mediated Dedifferentiation of Committed Mesoderm Progenitors

Embryonic stem (ES) cells can be propagated indefinitely in culture while retaining the ability to differentiate into any cell type in the organism. The molecular and cellular mechanisms underlying ES cell pluripotency are, however, poorly understood. Here, we characterize a population of early mesoderm-committed (EM) progenitors that is generated from mouse ES cells by bone morphogenetic protein (BMP) stimulation. We further show that EM progenitors are actively dedifferentiated to ES cells by the action of Nanog, which, in turn, is directly up-regulated in EM progenitors by the combined action of leukemia inhibitory factor (LIF) and the early mesoderm transcription factor T/Brachyury. Finally, we demonstrate that this negative feedback mechanism contributes to the maintenance of ES cell pluripotency. These findings uncover specific roles of LIF, Nanog, and BMP in the self-renewal of ES cells and provide novel insights into the cellular bases of ES cell pluripotency.