Tohru Ishitani

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

The cytokines IL‐1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF‐κB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL‐1‐ and TNF‐induced activation of the NF‐κB pathway. TAB2, a TAK1‐binding protein, is involved in IL‐1‐induced NF‐κB activation by physically linking TAK1 to TRAF6. However, IL‐1‐induced activation of NF‐κB is not impaired in TAB2‐deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2‐like molecule that associates with TAK1 and can activate NF‐κB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL‐1‐ and a TNF‐dependent manner, respectively. Further more, IL‐1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL‐1‐ and TNF‐induced activation of TAK1 and NF‐κB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL‐1 and TNF signal transduction.

Nrarp functions to modulate neural-crest-cell differentiation by regulating LEF1 protein stability

Nrarp (Notch-regulated ankyrin repeat protein) is a small protein that has two ankyrin repeats. Although Nrarp is known to be an inhibitory component of the Notch signalling pathway that operates in different developmental processes, the in vivo roles of Nrarp have not been fully characterized. Here, we show that Nrarp is a positive regulator in the Wnt signalling pathway. In zebrafish, knockdown of Nrarp-a expression by an antisense morpholino oligonucleotide (MO) results in altered Wnt-signalling-dependent neural-crest-cell development. Nrarp stabilizes LEF1 protein, a pivotal transcription factor in the Wnt signalling cascade, by blocking LEF1 ubiquitination. In accordance with this, the knockdown phenotype of lef1 is similar to that of nrarp-a, at least in part, in its effect on the development of multiple tissues in zebrafish. Furthermore, activation of LEF1 does not affect Notch activity or vice versa. These findings reveal that Nrarp independently regulates canonical Wnt and Notch signalling by modulating LEF1 and Notch protein turnover, respectively.

NLK positively regulates Wnt/β-catenin signalling by phosphorylating LEF1 in neural progenitor cells

Nemo‐like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/β‐catenin signalling. However, the roles of NLK in Wnt/β‐catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor‐1 (Lef1)‐mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC‐like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/β‐catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/β‐catenin signalling pathway.

Nemo-like kinase is involved in NGF-induced neurite outgrowth via phosphorylating MAP1B and paxillin

Nerve growth factor (NGF) promotes neurite outgrowth through regulating cytoskeletal organization and cell adhesion. These activities are modulated by protein phosphorylation. Nemo‐like kinase (NLK) is an evolutionarily conserved MAP kinase‐like kinase that phosphorylates several transcription factors. Although NLK is known to be expressed at relatively high levels in the nervous system, its function is not well understood. We found that NGF promotes the translocation of NLK to PC12 cells’ leading edges, and triggers NLK kinase activity in them. Activated NLK directly phosphorylates microtubule‐associated protein‐1B (MAP1B) and the focal adhesion adaptor protein, paxillin. Knockdown of NLK attenuates the phosphorylation of both paxillin and MAP1B and inhibits both the NGF‐induced re‐distribution of F‐actin and neurite outgrowth. We also discovered that NLK is a LiCl‐sensitive kinase. LiCl is known to block NGF‐induced neurite outgrowth and the phosphorylation of MAP1B and paxillin in PC12 cells. Therefore, the effects of LiCl are mediated in part by blocking NLK activity. These results suggest that NLK controls the dynamics of the cytoskeleton downstream of NGF signaling.

Zebrafish Dmrta2 regulates neurogenesis in the telencephalon

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab‐3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB‐3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior–dorsal telencephalon) is markedly reduced in this mutant at the 14‐somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes‐related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over‐expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior–dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior–dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior–dorsal telencephalic cell fate during somitogenesis.

Homodimerization of Nemo-like kinase is essential for activation and nuclear localization

NLK is an evolutionarily conserved protein kinase that phosphorylates several transcription factors. However, the molecular mechanisms that regulate NLK activity have been poorly understood. This study shows that homodimerization of NLK is required for its activation and nuclear localization.

DEAD-box protein Ddx46 is required for the development of the digestive organs and brain in zebrafish

Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing.

Delta1 family members are involved in filopodial actin formation and neuronal cell migration independent of Notch signaling

Delta family proteins are transmembrane molecules that bind Notch receptors and activate downstream signaling events in neighboring cells. In addition to serving as Notch ligands, Notch-independent roles for Delta have been suggested but are not fully understood. Here, we demonstrate a previously unrecognized role for Delta in filopodial actin formation. Delta1 and Delta4, but not Delta3, exhibit filopodial protrusive activity, and this activity is independent of Notch signaling. The filopodial activity of Delta1 does not depend on the PDZ-binding domain at the C-terminus; however, the intracellular membrane-proximal region that is anchored to the plasma membrane plays an important role in filopodial activity. We further identified a Notch-independent role of DeltaD in neuronal cell migration in zebrafish. These findings suggest a possible functional link between Notch-independent filopodial activity of Delta and the control of cell motility.