Harumasa Okamoto

Regulation of Slug transcription in embryonic ectoderm by β‐catenin‐Lef/Tcf and BMP‐Smad signaling

Neural crest is formed at the boundary of epidermal and neural ectoderm. To understand the molecular mechanism of neural crest formation, we focused on the transcriptional regulation of the Slug gene. In the upstream sequence of the chicken Slug gene, we have identified potential binding sites for transcription factors, such as Lef/Tcf and Smad1. Transgenic mouse embryos carrying the chicken Slug promoter‐reporter gene showed a crest‐specific activation of the reporter, suggesting the isolated sequence included the cis‐regulatory elements to receive Slug‐inducing signals in the mouse neural crest. While these potential cis‐regulatory elements could be recognized and activated by corresponding transcription factors, such as Lef1 and Smad1, Wnt‐Lef‐β‐catenin signal failed to induce endogenous Slug expression in quail neural plate tissue prepared from forebrain and midbrain levels. In contrast, Slug expression and subsequent epithelial‐mesenchymal transition were effectively induced by BMP4. Consistently, while we could detect phosphorylation of Smad1 in the ectoderm including the neural plate and the neural fold region, the activation of a reporter gene for a detection of canonical Wnt signal activation was below the level of detection at the forebrain and midbrain levels. These observations indicated that in the anterior ectoderm BMP signal has a predominant role for Slug expression.

Integration of multiple signal transducing pathways on Fgf response elements of the Xenopus caudal homologue Xcad3.

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. We investigate the nature of Fgf response element (FRE) in a posterior neural gene, Xcad3 (Xenopus caudal homologue) that plays a crucial role of posterior neural development. We provide evidence that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s).

A Xenopus homologue of the human β-amyloid precursor protein: Development regulation of its gene expression

Abstract Complimentary DNA clones have been isolated from Xenopus larva to delineate a protein highly homologous to the human β-amyloid precursor protein (APP). Developmental change of Xenopus APP gene expression has been analyzed with molecular probes. From early oogenesis, there is a high accumulation of maternal APP. After fertilization, the mRNA is degraded, reaching a minimum level around the gastrula stage. Then zygotic transcription appears to be initiated, and this continues during the subsequent embryonic and larval stages. Splicing patterns differ between the maternal and zygotic transcripts. The ratio of mRNA including the protease inhibitor domain (PID) sequence is extremely low for the transcript of maternal origin as compared to that for the transcript of zygotic origin. These results suggest some roles for the APP molecule in Xenopus early development.

Melatonin signaling regulates locomotion behavior and homeostatic states through distinct receptor pathways in Caenorhabditis elegans

Melatonin is a hormone that controls circadian rhythms and seasonal behavioral changes in vertebrates. Recent studies indicate that melatonin participates in diverse physiological functions including the modulation of neural activities. Melatonin is also detected in many other organisms that do not exhibit obvious circadian rhythms, but their precise functions are not known. To understand the role of melatonin and its genetic pathway in vivo, we examined the effects of melatonin and its receptor antagonists on various behaviors in Caenorhabditis elegans. Exogenously applied melatonin specifically decreased locomotion rates in 15-min treatments, suggesting that melatonin directly regulates neural activities for locomotion. This melatonin signaling functions through MT1-like melatonin receptors, because the MT1/2 receptor antagonist luzindole effectively blocked the effect of melatonin on locomotion, while MT2-specific antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT) and MT3-selective antagonist prazosin had no effect. Alternatively, long-term treatment with prazosin specifically altered homeostatic states of the worm, suggesting another melatonin-signaling pathway through MT3-like receptors. We also found that two G-protein alpha subunit mutants and newly isolated five mutants exhibited defects in response to melatonin. Our findings imply that melatonin acts as a neuromodulator by regulating locomotion behavior and as a ligand for homeostatic control through distinct receptor pathways in C. elegans.

Affixin activates Rac1 via βPIX in C2C12 myoblast

— MINT‐6179203, MINT‐6179212, MINT‐6178859, MINT‐6178812, MINT‐6178832, MINT‐6178843: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by coimmunoprecipitation (MI:0019) — MINT‐6179221: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with αpix (uniprotkb:Q8K4I3) by coimmunoprecipitation (MI:0019) — MINT‐6178962, MINT‐6178983: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) — MINT‐6179002, MINT‐6179021: Affixin (uniprotkb:Q9HBI1) binds (MI:0407) βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) — MINT‐6179039: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Rac1 (uniprotkb:P63001) by pull-down (MI:0096) — MINT‐6179054: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Cdc42 (uniprotkb:P70766) by pull-down (MI:0096) — MINT‐6178790: Affixin (uniprotkb:Q9HBI1) and αpix (uniprotkb:Q8K4I3) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178760: Affixin (uniprotkb:Q9HBI1) and βpix (uniprotkb:Q9ES28) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178801: Affixin (uniprotkb:Q9HBI1) and dysferlin (uniprotkb:Q9ESD7) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178779: Affixin (uniprotkb:Q9HBI1) and ILK (uniprotkb:O55222) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Binding of scorpion toxin to sodium channels in vitro and its modification by β‐bungarotoxin

1. Binding of a purified scorpion toxin to membrane fragments isolated from electroplaque of an electric eel Electrophorus electricus was studied using a radio‐iodinated toxin.

Caenorhabditis elegans Rab escort protein (REP-1) differently regulates each Rab protein function and localization in a tissue-dependent manner.

Rab proteins play a critical role in intracellular vesicle trafficking and require post‐translational modification by adding lipids at the C‐terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue‐specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep‐1 gene and found that a rep‐1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab‐3 or rab‐27 suggested that rep‐1 functions only in the RAB‐27 pathway, and not in the RAB‐3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP‐1 did not cause defecation defects compared to severe defects in either RAB‐27 or RabGGTase disruption, suggesting that REP‐1 is not essential for RAB‐27 signaling in defection. Some Rab proteins did not physically interact with REP‐1, and localization of these Rab proteins was not severely affected by REP‐1 disruption. These findings suggest that REP‐1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP‐1.

Expression cloning of Xenopus zygote arrest 2 (Xzar2) as a novel epidermalization‐promoting factor in early embryos of Xenopus laevis

In vertebrates, BMPs are known to induce epidermal fate at the expense of neural fate. To further explore the molecular mechanisms of epidermal differentiation, we have developed an expression cloning system for isolating cDNAs that encode intrinsic proteins with epidermal‐inducing activity. Under our conditions, 92.5% of the dissociated animal cap cells treated with the conditioned medium from H2O‐injected control oocytes differentiated into neural tissue, which developed neural fibers and expressed a neural marker (NCAM). In contrast, when dissociated animal cap cells were treated with the supernatant collected from the culture of BMP‐4 mRNA‐injected oocytes, the microcultures differentiated into epidermal tissue, which developed cilium. The cells expressed an epidermal marker (keratin), but not NCAM. Using the dissociated animal cap cells in a functional screening system, we cloned a cDNA encoding a novel polypeptide, Xenopus zygote arrest 2 (Xzar2). Over‐expression of Xzar2 caused anterior defects and suppressed expressions of the neural markers. The epidermalization‐promoting activity of Xzar2 was substantially not affected by over‐expression of the BMP signaling antagonists Smad6 and 7, and a dominant negative receptor for BMP (tBR). Our results suggest that Xzar2 is involved in epidermal fate determination mainly through signaling pathways distinct from that of BMP‐Smad during early embryogenesis.

Developmental Regulation of Xenopus β-Amyloid Precursor Protein Gene Expression

We isolated cDNA clones from Xenopus larvae, which encode a protein highly homologous to the mammalian β-amyloid precursor protein (APP), and analyzed developmental change of XenopuIs APP gene expression. The maternal APP mRNA is accumulated from early oogenesis, but after fertilization, this mRNA is degraded reaching a minimum level around the gastrula stage. Zygotic transcription appears to start after the gastrula stage and to continue during subsequent embryonic and larval stages. These results suggest some fundamental roles of the APP molecule in Xenopus early development.

Involvement of a Xenopus nuclear GTP‐binding protein in optic primordia formation

Using a subtracted Xenopus cDNA library based on the differential sensitivity of anterior and posterior genes to retinoic acid, we isolated a novel Xenopus nuclear GTP‐binding protein (XGB). XGB is expressed prominently in the optic primordia at the tailbud stage. The N‐terminal region of XGB contains a set of GTP‐binding protein motifs, and the C‐terminal region contains two putative nuclear localization signals and two coiled regions. A GFP‐XGB fusion protein was expressed in the nucleus of NIH3T3 cells where it bound to subnuclear structures. Truncated C‐terminal constructs of XGB containing both nuclear localization signal(s) and coiled region(s) suppressed eye formation, whereas neither the N‐terminal construct nor constructs with a mutated GTP‐binding protein motif affected eye formation. Expression of Pax6 and Rx1 genes, which are crucial for eye development, was reduced in embryos overexpressing the C‐terminal constructs of XGB. Suppression of Pax6 and Rx1 at earlier developmental stages as well as perturbation of eye formation at later stages was counteracted by co‐expression of wild‐type XGB. We conclude that XGB plays a role in the formation of optic primordia through activation of at least two eye field transcription factors.