<p><b>A</b>. A timeline of experiments of AG1478 treatment. Embryos were soaked... more <p><b>A</b>. A timeline of experiments of AG1478 treatment. Embryos were soaked into 30 μM AG1478 solution or the control 2% DMSO from 26 to 45 hpf. Then, they were washed and grown in a fresh medium from 45 hpf. Embryos at 44, 48 and 54 hpf were collected for analyses. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s011" target="_blank">S3 Movie</a> on the recovery of neurogenesis after removal of AG1478. <b>B</b>. pH3-positive mitotic cells in the OT. Removal of AG1478 at 45 hpf enhanced mitoses in the SVZ at 48 hpf prior to recovery of GFP-expressing neurons in the OT of Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>) at 54 hpf. Yellow arrows, pH3-positive cells in the SVZ. Scale bar, 10 μm. <b>C</b>. Quantification of the number of pH3-positive cells in the VZ (blue) and the SVZ (red) of AG1478-treated embryos and the control (white) embryos (mean ± s.e.m.; *<i>P</i> < 0.05, ***<i>P</i> < 0.001; n = 3, 5, 5 for AG1478, n = 6, 4, 6 for DMSO at 44, 48, 54 hpf, respectively). <b>D</b>. Quantification of GFP intensity in the OT (mean ± s.e.m.; *<i>P</i> < 0.05, ****<i>P</i> < 0.0001; n = 6, 4, 5 for AG1478, n = 6, 4, 6 for DMSO at 44, 48, 54 hpf, respectively). <b>E</b>. Time-lapse imaging of NPCs stochastically labeled by co-injection of <i>UAS</i>:<i>mCherry</i>/<i>UAS</i>:<i>memb</i>:<i>GFP</i> plasmids into Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>;<i>SAGFF(LF)81C</i>) embryos. After the removal of AG1478, two NPCs (1, 2; arrowheads) divided once in the SVZ (asterisk) to generate two daughter cells (a, b), and then, these cells were incorporated into the presumptive neuronal layer (NL, green bar, above the thin dotted line). Note that mitoses of these NPCs were not preceded by mitoses in the apical VZ and those NPCs began to express cytoplasmic GFP (right). An apical mitosis of another NPC or RGC labeled with membGFP was also observed in this imaging (arrow). Scale bar, 10 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s012" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s013" target="_blank">S5</a> Movies.</p
<p><b>A</b>. Representative Tg(<i>pou4f1-hsp70l</i>:<i>GFP;-8... more <p><b>A</b>. Representative Tg(<i>pou4f1-hsp70l</i>:<i>GFP;-8</i>.<i>4neurog1</i>:<i>nRFP</i>) embryos in a dorsal view. GFP-positive neurons gradually expand from the outside (basal) to the inside (apical) region. The <i>-8</i>.<i>4neurog1</i>:nRFP is weakly expressed in a subset of NPCs, and highly expressed in pigment cells on the surface of the brain. Dotted circle, optic tectum (OT); v, ventricle. Scale bars, 50 μm. <b>B</b>. Quantification of <i>pou4f1-hsp70l</i>:GFP intensity in the OT (mean ± s.e.m.; n = 3, 5, 5 for 36, 42, 48 hpf, respectively). A.U., arbitrary units. Representative Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>) embryos at 48 hpf in a lateral view (right). Dotted circle, OT; e, eye. Scale bar, 100 μm. <b>C</b>. Schematics of confocal images from lateral (left) and dorsal (middle) views of the OT, and a image at a higher magnification from a dorsal view (right). Dotted circle, OT; fb, forebrain; hb, hindbrain; A, apical; B, basal; NL, neuronal layer; SVZ, sub-ventricular zone; VZ, ventricular zone. <b>D</b>. The OT of Tg(BAC(<i>neurod</i>:<i>GFP</i>);<i>-8</i>.<i>4neurog1</i>:<i>nRFP</i>) embryos in dorsal views. Dotted line, apical surface of the VZ; asterisk, pigment cell in the skin. Scale bar, 20 μm. <b>E</b>. (top) A timeline of experiments for UV irradiation. Embryos were subjected to UV irradiation at 36, 42 and 48 hpf. Neurons expressing Kaede were fluorescently labeled in red by the irradiation and neurons generated after the irradiation were labeled in green. (bottom) A lateral view of Tg(<i>elavl3</i>:<i>Kaede</i>) embryos irradiated with UV light at 36 hpf. Dotted circle, OT. Scale bar, 50 μm. <b>F</b>. Accumulation of older post-mitotic neurons (Kaede<sup>R</sup>) from the basal to apical regions of the OT in Tg(<i>elavl3</i>:<i>Kaede</i>). Dotted line, apical surface of the VZ. Scale bar, 10 μm. <b>G</b>. pH3-positive mitotic cells in the OT of Tg(BAC(<i>neurod</i>:<i>GFP</i>);<i>-8</i>.<i>4neurog1</i>:<i>nRFP</i>). Yellow arrows, pH3-positive cells in the sub-basal zone/SVZ. Thick dotted line, apical surface of the VZ; thin dotted line, approximate boundary between the VZ and the SVZ. <b>H</b>. Quantification of the number of pH3-positive cells in the VZ (blue) and the SVZ (red) of the developing OT (mean ± s.e.m.; *<i>P</i> < 0.05, ***<i>P</i> < 0.001; n = 3 per group). <b>I</b>. Time-lapse imaging of NPCs stochastically labeled by co-injection of <i>-8</i>.<i>4neurog1</i>:<i>Gal4VP16</i>/<i>UAS</i>:<i>memb</i>:<i>GFP</i>/<i>UAS</i>:<i>H2ARFP</i> plasmids into TgBAC(<i>neurod</i>:<i>EGFP</i>) embryos. Two NPCs (1, 2) underwent mitoses in the SVZ (asterisk) to produce two daughter cells (a, b) that ultimately differentiated into <i>neurod</i>:GFP-expressing post-mitotic neurons in the neuronal layer (NL; 1a, 1b, 2a). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s009" target="_blank">S1 Movie</a>.</p
Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living ... more Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living organisms and is influenced primarily by several factors: motion artifacts, optical properties and spatial resolution. Conventional imag
Integrins, transmembrane molecules that facilitate cell-to-cell and cell-toextracellular matrix i... more Integrins, transmembrane molecules that facilitate cell-to-cell and cell-toextracellular matrix interactions, are heterodimers that consist of an α-and β-subunit. The integrin α4 gene (itgα 4) is expressed in various type of cells and tissues. Its biochemical functions and physiological roles have been revealed using cultured cell assays. In contrast, the primary effect caused by itgα 4 deletion on vertebrate development is poorly understood, because knockout mice exhibit multiple defects that can lead to embryonic lethality in the uterus. Zebrafish are a convenient vertebrate model to investigate morphogenesis during embryogenesis, because of their external fertilization and subsequent development outside the female's body. Here, we generated a zebrafish mutant line named itgα 4 ko108 using the CRISPR/Cas9 genome editing system; the mutant genome harbored an approximately 2.0-kb deletion in the itgα 4 locus. A truncated transcript was detected in itgα 4 (+ / −) or (− / −) fish but not in (+ / +) fish. The mutant transcript was hypothesized to encode a truncated Itgα4 protein due to a premature stop codon. itgα 4 (− / −) embryos obtained from the mating of heterozygous parents exhibited no apparent phenotype during development at 24 hours post-fertilization (hpf). However, approximately half of them exhibited cephalic hemorrhage at 48 hpf. The incidence ratio was significantly higher than that in (+ / +) or (+ / −) embryos. Embryonic hemorrhage has also been reported previously in Itgα4 knockout mice. In contrast, embryonic lethality with the other defects reported in the knockout mice was not observed in our zebrafish model. Therefore, the mutant line itgα 4 ko108 should be a useful model to investigate a physiological function for Itgα 4 in the blood circulation system.
Integrins are transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matr... more Integrins are transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matrix (ECM) interactions. Integrin molecules are heterodimers that consist of α-and β-subunits. The integrin β1 gene is widely expressed in vivo and is the major β molecule in many tissues; however, tissue-specific roles of integrin β1 are still elusive. In this study, we investigated integrin β1 function in endothelial cells of zebrafish. An integrin β1b mutant zebrafish exhibited morphological abnormalities in blood vessel formation, cephalic hemorrhage and a decreased responsiveness to tactile stimulation during development. To determine the role of integrin β1b in vascular formation, we developed a Gal4/UAS-mediated conditional inactivation of integrin β1 by expressing the cytoplasmic region of integrin β1 that acts as a dominant-negative (DN) isoform. Expression of integrin β1 DN in endothelial cells induced blood vessel abnormalities as in integrin β1b mutants. These results show that endothelial cells require integrin activity for the formation and/or maintenance of blood vessels in zebrafish. Furthermore, our time-lapse recording visualized the breakpoint of cephalic vessels and the hemorrhage onset. Taken together, our tissue-specific inactivation of integrin β1 in zebrafish is powerful tools for functional analysis of integrin β1 in developing tissues.
Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, proce... more Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broadspectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2017
During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this pro... more During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this process remain elusive. Here, we addressed this issue by functionally analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. Loss-of-function studies of SALL4 showed that this transcription factor is required for the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also up-regulated integrin gene expression. The impaired cell migration observed in SALL4 knockdown cells was restored by overexpression of integrin α6 and β1. In addition, we clarified that integrin α6 and β1 formed a heterodimer. At the molecular level, loss of the SALL4-integrin α6β1 network lost focal adhesion dynamics, which impairs cell migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho activation was suppressed by integrin α6β1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4-integrin α6β1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated Highlights SALL4 up-regulates integrin α6β1 expression at the transcription level. The SALL4-integrin α6β1 network is required for a spindle-shaped morphology. The SALL4-integrin α6β1 network activates focal adhesion dynamics. The SALL4-integrin α6β1 network modulates Rho activation for cell migration.
CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic respo... more CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.
... Isaac, A., Rodriguez-Esteban, C., Ryan, A., Altabef, M., Tsukui, T., Patel, K., Tickle, C. an... more ... Isaac, A., Rodriguez-Esteban, C., Ryan, A., Altabef, M., Tsukui, T., Patel, K., Tickle, C. and Izpisua-Belmonte, JC, 1998. Tbx genes and limb identity in chick embryo development. Development 125, pp. 867875. Papaioannou, VE and Silver, LM, 1998. The T-box gene family. ...
Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise... more Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise to the membranous septa and valves. Here we show that Meltrin h/ADAM19, a novel metalloprotease-disintegrin, participates in the development of the endocardial cushion. Mice lacking Meltrin h exhibit ventricular septal defect (VSD) and immature valves, and most of the animals die soon after birth. During development of the endocardial cushion, epithelial-mesenchymal transformation (EMT) of endocardial epithelial cells generates most of the cushion mesenchymes that constitute the main components of the septa and valves. Meltrin h is expressed in both the epithelia and the mesenchymes of the endocardial cushion. In the absence of Meltrin h, the cushion is small or thin in the septum-forming region and show poor remodeling of cardiac jelly components; both of these characteristics suggest impaired growth and differentiation of the endocardial cushion. When embryonic fibroblasts are cultured sparsely, Meltrin h-lacking cells exhibit aberrant ectodomain shedding of type I Neuregulin, one of the ErbB ligands expressed in endocardial cells. These results suggest the necessity of proteolytic regulation of ErbB ligands by Meltrin h for proper heart development.
Meltrin /ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of... more Meltrin /ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern of meltrin  during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin  participates in the proteolytic processing of membrane-anchored NRGs. When NRG-1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin , which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-1. Furthermore, expression of protease-deficient mutants of meltrin  exerted dominant negative effects on the basal processing of NRG-1. These results indicate that meltrin  participates in the processing of NRG-1. Since meltrin  affected the processing of NRG-4 but not that of NRG-␣2, meltrin  was considered to have a preference for -type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin  participates in the intracellular processing of NRGs rather than the cleavage on the cell surface. Various intracellular signaling and adhesion molecules govern the cell-cell interactions during the development of multicellular organisms. The actions of these molecules are regulated not only by transcriptional and translational controls but also by post-translational modifications such as phosphorylations and proteolytic processings. Numerous membrane-anchored signaling molecules are subjected to proteolytic processing to release their extracellular domains. Such modifications may cause qualitative and irreversible changes in the functions of these molecules.
Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of post... more Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously. Principal Findings: We report here that Meltrin b, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor a mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin b–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin b–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 k...
Biochemical and biophysical research communications, 2005
Maturation of the neuromuscular junction is accompanied by molecular switching of acetylcholine r... more Maturation of the neuromuscular junction is accompanied by molecular switching of acetylcholine receptor (AChR) channels from embryonic types with gamma-subunits to adult ones with epsilon-subunits after birth. As a step toward understanding the molecular mechanisms of the gamma-to-epsilon switch, we addressed the question of whether embryonic- and adult-type AChRs constitute different endplates during the transitional period. From analyses with double- or triple-staining with anti-gamma- and/or anti-epsilon-antibodies together with alpha-bungarotoxin, which binds to alpha-subunits, we demonstrated that during neonatal stages in mice, adult-type AChRs are incorporated into individual endplates expressing embryonic-AChRs and replace these embryonic-AChRs gradually. The main period of AChR transition in the mouse diaphragm was between postnatal days 5 (P5) and P7, similar to the period described previously in which endplates shift from multi-axon to single-axon innervation. This findi...
Meltrin beta (ADAM19) is a metalloprotease-disintegrin expressed in the peripheral nervous system... more Meltrin beta (ADAM19) is a metalloprotease-disintegrin expressed in the peripheral nervous system and other organs during embryogenesis. We report here an alternatively spliced isoform, meltrin beta mini, that lacks the prodomain, metalloprotease and disintegrin domains. A comparison of the cDNA and genomic sequences suggested the existence of a new exon. This isoform was detected in murine dorsal root ganglion and neuronal cell lines by RT-PCR. Overexpression of meltrin beta mini but not meltrin beta induced neurite outgrowth in neuronal cells. These studies suggest that the novel meltrin beta isoform has a distinct function related to neurogenesis.
During muscle regeneration, extracellular signal-regulated kinase (ERK) promotes both proliferati... more During muscle regeneration, extracellular signal-regulated kinase (ERK) promotes both proliferation and migration. However, the relationship between proliferation and migration is poorly understood in this context. To elucidate this complex relationship on a physiological level, we established an intravital imaging system for measuring ERK activity, migration speed, and cell-cycle phases in mouse muscle satellite cells. We found that in vivo, ERK was maximally activated in satellite cells two days after injury, and this is then followed by increases in cell number and motility. With limited effects of immediate ERK activity on migration, we hypothesized that ERK increases migration speed in the later phase by promoting cell-cycle progression. Our cell-cycle analysis further revealed that in satellite cells, ERK activity is critical for the G1/S transition, and cells migrate more rapidly in the S/G2 phase three days after injury. Finally, migration speed of satellite cells was suppre...
Biochemical and Biophysical Research Communications
The Ras homologous (Rho) proteins are a family of small GTPases, which regulate the cytoskeleton ... more The Ras homologous (Rho) proteins are a family of small GTPases, which regulate the cytoskeleton and are related to stress fibers and focal adhesion. The Rho-associated protein kinases (ROCK) constitute part of the Rho effectors that regulate cell shape and movement via phosphorylation of the myosin light chain and actin depolymerizing factor/cofilin. ROCK members are widely expressed and play roles in various cell types during vertebrate development and morphogenesis; therefore, ROCK-knockout animals exhibit multiple defects mostly initiated at the embryonic stage. Analyzing the distinct roles of ROCK in cell shape and movement during the embryonic stages using live mammalian models is difficult. Here, we inhibited the Rho/ROCK pathway in zebrafish, which is a small fish that can be conveniently used as a developmental animal model in place of mammals. To inhibit the Rho/ROCK pathway, we designed a dominant-negative ROCK-2 (dnROCK-2) that lacked the kinase domain and was under the control of an upstream activation sequence (UAS). To evaluate the effects of expression of dnROCK-2, transgenic zebrafish lines were generated by mating strains expressing the construct with counterpart strains expressing the Gal4 activator in target tissues. In this study, we crossed the dnROCK-2-expressing line with two such Gal4-expressing lines; (1) SAGFF(LF)73A for expression in the whole body, and (2) Tg(fli1a: Gal4FF)ubs4 for endothelial cell-specific expression. The phenotypes of the fish obtained were observed by fluorescent stereomicroscopy or confocal microscopy. Overexpression of dnROCK-2 in the whole body resulted in an inhibition of development, notably in cephalic formation, at 1-day post-fertilization (dpf). Confocal microscopy revealed that Hensen's zone became unclear in the trunk muscle fibers expressing dnROCK-2. Endothelial cell-specific expression of dnROCK-2 caused abnormalities in cardiovascular formation at 2-dpf. These results suggest that dnROCK-2 can act as a dominant negative construct of the Rho/ROCK pathway to affect regulation of the cytoskeleton. This construct could be a convenient tool to investigate the function of ROCK members in other vertebrate cell types.
American Journal of Physiology-Heart and Circulatory Physiology
A disintegrin and metalloproteinase (ADAM)12 is considered to promote cardiac dysfunction based o... more A disintegrin and metalloproteinase (ADAM)12 is considered to promote cardiac dysfunction based on the finding that a small-molecule ADAM12 inhibitor, KB-R7785, ameliorated cardiac function in a transverse aortic constriction (TAC) model by inhibiting the proteolytic activation of heparin-binding-EGF signaling. However, this compound has poor selectivity for ADAM12, and the role of ADAM12 in cardiac dysfunction has not yet been investigated using genetic loss-of-function mice. We revealed that ADAM12 knockout mice showed significantly more advanced cardiac hypertrophy and higher mortality rates than wild-type mice 4 wk after TAC surgery. An ADAM12 deficiency resulted in significantly more expanded cardiac fibrosis accompanied by increased collagen-related gene expression in failing hearts. The results of a genome-wide transcriptional analysis suggested a strongly enhanced focal adhesion- and fibrosis-related signaling pathway in ADAM12 knockout hearts. The loss of ADAM12 increased t...
Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intraepithelial lymph... more Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intraepithelial lymphocytes (IELs), especially CD8αα+ IELs (CD8αα IELs). In the intestine, IL-15 is produced by intestinal epithelial cells (IECs), blood vascular endothelial cells (BECs) and hematopoietic cells. However, the precise role of intestinal IL-15 on IELs is still unknown. To address the question, we generated two kinds of IL-15 conditional knockout (IL-15cKO) mice: villin-Cre (Vil-Cre) and Tie2-Cre IL-15cKO mice. IEC-derived IL-15 was specifically deleted in Vil-Cre IL-15cKO mice, whereas IL-15 produced by BECs and hematopoietic cells is deleted in Tie2-Cre IL-15cKO mice. The cell number and frequency of CD8αα IELs and NK IELs were significantly reduced in Vil-Cre IL-15cKO mice. By contrast, CD8αα IELs were unchanged in Tie2-Cre IL-15cKO mice, indicating that IL-15 produced by BECs and hematopoietic cells is dispensable for CD8αα IELs. Expression of an anti-apoptotic factor, Bcl-2, was decreased,...
<p><b>A</b>. A timeline of experiments of AG1478 treatment. Embryos were soaked... more <p><b>A</b>. A timeline of experiments of AG1478 treatment. Embryos were soaked into 30 μM AG1478 solution or the control 2% DMSO from 26 to 45 hpf. Then, they were washed and grown in a fresh medium from 45 hpf. Embryos at 44, 48 and 54 hpf were collected for analyses. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s011" target="_blank">S3 Movie</a> on the recovery of neurogenesis after removal of AG1478. <b>B</b>. pH3-positive mitotic cells in the OT. Removal of AG1478 at 45 hpf enhanced mitoses in the SVZ at 48 hpf prior to recovery of GFP-expressing neurons in the OT of Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>) at 54 hpf. Yellow arrows, pH3-positive cells in the SVZ. Scale bar, 10 μm. <b>C</b>. Quantification of the number of pH3-positive cells in the VZ (blue) and the SVZ (red) of AG1478-treated embryos and the control (white) embryos (mean ± s.e.m.; *<i>P</i> < 0.05, ***<i>P</i> < 0.001; n = 3, 5, 5 for AG1478, n = 6, 4, 6 for DMSO at 44, 48, 54 hpf, respectively). <b>D</b>. Quantification of GFP intensity in the OT (mean ± s.e.m.; *<i>P</i> < 0.05, ****<i>P</i> < 0.0001; n = 6, 4, 5 for AG1478, n = 6, 4, 6 for DMSO at 44, 48, 54 hpf, respectively). <b>E</b>. Time-lapse imaging of NPCs stochastically labeled by co-injection of <i>UAS</i>:<i>mCherry</i>/<i>UAS</i>:<i>memb</i>:<i>GFP</i> plasmids into Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>;<i>SAGFF(LF)81C</i>) embryos. After the removal of AG1478, two NPCs (1, 2; arrowheads) divided once in the SVZ (asterisk) to generate two daughter cells (a, b), and then, these cells were incorporated into the presumptive neuronal layer (NL, green bar, above the thin dotted line). Note that mitoses of these NPCs were not preceded by mitoses in the apical VZ and those NPCs began to express cytoplasmic GFP (right). An apical mitosis of another NPC or RGC labeled with membGFP was also observed in this imaging (arrow). Scale bar, 10 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s012" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s013" target="_blank">S5</a> Movies.</p
<p><b>A</b>. Representative Tg(<i>pou4f1-hsp70l</i>:<i>GFP;-8... more <p><b>A</b>. Representative Tg(<i>pou4f1-hsp70l</i>:<i>GFP;-8</i>.<i>4neurog1</i>:<i>nRFP</i>) embryos in a dorsal view. GFP-positive neurons gradually expand from the outside (basal) to the inside (apical) region. The <i>-8</i>.<i>4neurog1</i>:nRFP is weakly expressed in a subset of NPCs, and highly expressed in pigment cells on the surface of the brain. Dotted circle, optic tectum (OT); v, ventricle. Scale bars, 50 μm. <b>B</b>. Quantification of <i>pou4f1-hsp70l</i>:GFP intensity in the OT (mean ± s.e.m.; n = 3, 5, 5 for 36, 42, 48 hpf, respectively). A.U., arbitrary units. Representative Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>) embryos at 48 hpf in a lateral view (right). Dotted circle, OT; e, eye. Scale bar, 100 μm. <b>C</b>. Schematics of confocal images from lateral (left) and dorsal (middle) views of the OT, and a image at a higher magnification from a dorsal view (right). Dotted circle, OT; fb, forebrain; hb, hindbrain; A, apical; B, basal; NL, neuronal layer; SVZ, sub-ventricular zone; VZ, ventricular zone. <b>D</b>. The OT of Tg(BAC(<i>neurod</i>:<i>GFP</i>);<i>-8</i>.<i>4neurog1</i>:<i>nRFP</i>) embryos in dorsal views. Dotted line, apical surface of the VZ; asterisk, pigment cell in the skin. Scale bar, 20 μm. <b>E</b>. (top) A timeline of experiments for UV irradiation. Embryos were subjected to UV irradiation at 36, 42 and 48 hpf. Neurons expressing Kaede were fluorescently labeled in red by the irradiation and neurons generated after the irradiation were labeled in green. (bottom) A lateral view of Tg(<i>elavl3</i>:<i>Kaede</i>) embryos irradiated with UV light at 36 hpf. Dotted circle, OT. Scale bar, 50 μm. <b>F</b>. Accumulation of older post-mitotic neurons (Kaede<sup>R</sup>) from the basal to apical regions of the OT in Tg(<i>elavl3</i>:<i>Kaede</i>). Dotted line, apical surface of the VZ. Scale bar, 10 μm. <b>G</b>. pH3-positive mitotic cells in the OT of Tg(BAC(<i>neurod</i>:<i>GFP</i>);<i>-8</i>.<i>4neurog1</i>:<i>nRFP</i>). Yellow arrows, pH3-positive cells in the sub-basal zone/SVZ. Thick dotted line, apical surface of the VZ; thin dotted line, approximate boundary between the VZ and the SVZ. <b>H</b>. Quantification of the number of pH3-positive cells in the VZ (blue) and the SVZ (red) of the developing OT (mean ± s.e.m.; *<i>P</i> < 0.05, ***<i>P</i> < 0.001; n = 3 per group). <b>I</b>. Time-lapse imaging of NPCs stochastically labeled by co-injection of <i>-8</i>.<i>4neurog1</i>:<i>Gal4VP16</i>/<i>UAS</i>:<i>memb</i>:<i>GFP</i>/<i>UAS</i>:<i>H2ARFP</i> plasmids into TgBAC(<i>neurod</i>:<i>EGFP</i>) embryos. Two NPCs (1, 2) underwent mitoses in the SVZ (asterisk) to produce two daughter cells (a, b) that ultimately differentiated into <i>neurod</i>:GFP-expressing post-mitotic neurons in the neuronal layer (NL; 1a, 1b, 2a). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127360#pone.0127360.s009" target="_blank">S1 Movie</a>.</p
Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living ... more Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living organisms and is influenced primarily by several factors: motion artifacts, optical properties and spatial resolution. Conventional imag
Integrins, transmembrane molecules that facilitate cell-to-cell and cell-toextracellular matrix i... more Integrins, transmembrane molecules that facilitate cell-to-cell and cell-toextracellular matrix interactions, are heterodimers that consist of an α-and β-subunit. The integrin α4 gene (itgα 4) is expressed in various type of cells and tissues. Its biochemical functions and physiological roles have been revealed using cultured cell assays. In contrast, the primary effect caused by itgα 4 deletion on vertebrate development is poorly understood, because knockout mice exhibit multiple defects that can lead to embryonic lethality in the uterus. Zebrafish are a convenient vertebrate model to investigate morphogenesis during embryogenesis, because of their external fertilization and subsequent development outside the female's body. Here, we generated a zebrafish mutant line named itgα 4 ko108 using the CRISPR/Cas9 genome editing system; the mutant genome harbored an approximately 2.0-kb deletion in the itgα 4 locus. A truncated transcript was detected in itgα 4 (+ / −) or (− / −) fish but not in (+ / +) fish. The mutant transcript was hypothesized to encode a truncated Itgα4 protein due to a premature stop codon. itgα 4 (− / −) embryos obtained from the mating of heterozygous parents exhibited no apparent phenotype during development at 24 hours post-fertilization (hpf). However, approximately half of them exhibited cephalic hemorrhage at 48 hpf. The incidence ratio was significantly higher than that in (+ / +) or (+ / −) embryos. Embryonic hemorrhage has also been reported previously in Itgα4 knockout mice. In contrast, embryonic lethality with the other defects reported in the knockout mice was not observed in our zebrafish model. Therefore, the mutant line itgα 4 ko108 should be a useful model to investigate a physiological function for Itgα 4 in the blood circulation system.
Integrins are transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matr... more Integrins are transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matrix (ECM) interactions. Integrin molecules are heterodimers that consist of α-and β-subunits. The integrin β1 gene is widely expressed in vivo and is the major β molecule in many tissues; however, tissue-specific roles of integrin β1 are still elusive. In this study, we investigated integrin β1 function in endothelial cells of zebrafish. An integrin β1b mutant zebrafish exhibited morphological abnormalities in blood vessel formation, cephalic hemorrhage and a decreased responsiveness to tactile stimulation during development. To determine the role of integrin β1b in vascular formation, we developed a Gal4/UAS-mediated conditional inactivation of integrin β1 by expressing the cytoplasmic region of integrin β1 that acts as a dominant-negative (DN) isoform. Expression of integrin β1 DN in endothelial cells induced blood vessel abnormalities as in integrin β1b mutants. These results show that endothelial cells require integrin activity for the formation and/or maintenance of blood vessels in zebrafish. Furthermore, our time-lapse recording visualized the breakpoint of cephalic vessels and the hemorrhage onset. Taken together, our tissue-specific inactivation of integrin β1 in zebrafish is powerful tools for functional analysis of integrin β1 in developing tissues.
Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, proce... more Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broadspectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2017
During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this pro... more During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this process remain elusive. Here, we addressed this issue by functionally analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. Loss-of-function studies of SALL4 showed that this transcription factor is required for the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also up-regulated integrin gene expression. The impaired cell migration observed in SALL4 knockdown cells was restored by overexpression of integrin α6 and β1. In addition, we clarified that integrin α6 and β1 formed a heterodimer. At the molecular level, loss of the SALL4-integrin α6β1 network lost focal adhesion dynamics, which impairs cell migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho activation was suppressed by integrin α6β1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4-integrin α6β1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated Highlights SALL4 up-regulates integrin α6β1 expression at the transcription level. The SALL4-integrin α6β1 network is required for a spindle-shaped morphology. The SALL4-integrin α6β1 network activates focal adhesion dynamics. The SALL4-integrin α6β1 network modulates Rho activation for cell migration.
CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic respo... more CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.
... Isaac, A., Rodriguez-Esteban, C., Ryan, A., Altabef, M., Tsukui, T., Patel, K., Tickle, C. an... more ... Isaac, A., Rodriguez-Esteban, C., Ryan, A., Altabef, M., Tsukui, T., Patel, K., Tickle, C. and Izpisua-Belmonte, JC, 1998. Tbx genes and limb identity in chick embryo development. Development 125, pp. 867875. Papaioannou, VE and Silver, LM, 1998. The T-box gene family. ...
Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise... more Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise to the membranous septa and valves. Here we show that Meltrin h/ADAM19, a novel metalloprotease-disintegrin, participates in the development of the endocardial cushion. Mice lacking Meltrin h exhibit ventricular septal defect (VSD) and immature valves, and most of the animals die soon after birth. During development of the endocardial cushion, epithelial-mesenchymal transformation (EMT) of endocardial epithelial cells generates most of the cushion mesenchymes that constitute the main components of the septa and valves. Meltrin h is expressed in both the epithelia and the mesenchymes of the endocardial cushion. In the absence of Meltrin h, the cushion is small or thin in the septum-forming region and show poor remodeling of cardiac jelly components; both of these characteristics suggest impaired growth and differentiation of the endocardial cushion. When embryonic fibroblasts are cultured sparsely, Meltrin h-lacking cells exhibit aberrant ectodomain shedding of type I Neuregulin, one of the ErbB ligands expressed in endocardial cells. These results suggest the necessity of proteolytic regulation of ErbB ligands by Meltrin h for proper heart development.
Meltrin /ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of... more Meltrin /ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern of meltrin  during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin  participates in the proteolytic processing of membrane-anchored NRGs. When NRG-1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin , which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-1. Furthermore, expression of protease-deficient mutants of meltrin  exerted dominant negative effects on the basal processing of NRG-1. These results indicate that meltrin  participates in the processing of NRG-1. Since meltrin  affected the processing of NRG-4 but not that of NRG-␣2, meltrin  was considered to have a preference for -type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin  participates in the intracellular processing of NRGs rather than the cleavage on the cell surface. Various intracellular signaling and adhesion molecules govern the cell-cell interactions during the development of multicellular organisms. The actions of these molecules are regulated not only by transcriptional and translational controls but also by post-translational modifications such as phosphorylations and proteolytic processings. Numerous membrane-anchored signaling molecules are subjected to proteolytic processing to release their extracellular domains. Such modifications may cause qualitative and irreversible changes in the functions of these molecules.
Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of post... more Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously. Principal Findings: We report here that Meltrin b, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor a mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin b–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin b–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 k...
Biochemical and biophysical research communications, 2005
Maturation of the neuromuscular junction is accompanied by molecular switching of acetylcholine r... more Maturation of the neuromuscular junction is accompanied by molecular switching of acetylcholine receptor (AChR) channels from embryonic types with gamma-subunits to adult ones with epsilon-subunits after birth. As a step toward understanding the molecular mechanisms of the gamma-to-epsilon switch, we addressed the question of whether embryonic- and adult-type AChRs constitute different endplates during the transitional period. From analyses with double- or triple-staining with anti-gamma- and/or anti-epsilon-antibodies together with alpha-bungarotoxin, which binds to alpha-subunits, we demonstrated that during neonatal stages in mice, adult-type AChRs are incorporated into individual endplates expressing embryonic-AChRs and replace these embryonic-AChRs gradually. The main period of AChR transition in the mouse diaphragm was between postnatal days 5 (P5) and P7, similar to the period described previously in which endplates shift from multi-axon to single-axon innervation. This findi...
Meltrin beta (ADAM19) is a metalloprotease-disintegrin expressed in the peripheral nervous system... more Meltrin beta (ADAM19) is a metalloprotease-disintegrin expressed in the peripheral nervous system and other organs during embryogenesis. We report here an alternatively spliced isoform, meltrin beta mini, that lacks the prodomain, metalloprotease and disintegrin domains. A comparison of the cDNA and genomic sequences suggested the existence of a new exon. This isoform was detected in murine dorsal root ganglion and neuronal cell lines by RT-PCR. Overexpression of meltrin beta mini but not meltrin beta induced neurite outgrowth in neuronal cells. These studies suggest that the novel meltrin beta isoform has a distinct function related to neurogenesis.
During muscle regeneration, extracellular signal-regulated kinase (ERK) promotes both proliferati... more During muscle regeneration, extracellular signal-regulated kinase (ERK) promotes both proliferation and migration. However, the relationship between proliferation and migration is poorly understood in this context. To elucidate this complex relationship on a physiological level, we established an intravital imaging system for measuring ERK activity, migration speed, and cell-cycle phases in mouse muscle satellite cells. We found that in vivo, ERK was maximally activated in satellite cells two days after injury, and this is then followed by increases in cell number and motility. With limited effects of immediate ERK activity on migration, we hypothesized that ERK increases migration speed in the later phase by promoting cell-cycle progression. Our cell-cycle analysis further revealed that in satellite cells, ERK activity is critical for the G1/S transition, and cells migrate more rapidly in the S/G2 phase three days after injury. Finally, migration speed of satellite cells was suppre...
Biochemical and Biophysical Research Communications
The Ras homologous (Rho) proteins are a family of small GTPases, which regulate the cytoskeleton ... more The Ras homologous (Rho) proteins are a family of small GTPases, which regulate the cytoskeleton and are related to stress fibers and focal adhesion. The Rho-associated protein kinases (ROCK) constitute part of the Rho effectors that regulate cell shape and movement via phosphorylation of the myosin light chain and actin depolymerizing factor/cofilin. ROCK members are widely expressed and play roles in various cell types during vertebrate development and morphogenesis; therefore, ROCK-knockout animals exhibit multiple defects mostly initiated at the embryonic stage. Analyzing the distinct roles of ROCK in cell shape and movement during the embryonic stages using live mammalian models is difficult. Here, we inhibited the Rho/ROCK pathway in zebrafish, which is a small fish that can be conveniently used as a developmental animal model in place of mammals. To inhibit the Rho/ROCK pathway, we designed a dominant-negative ROCK-2 (dnROCK-2) that lacked the kinase domain and was under the control of an upstream activation sequence (UAS). To evaluate the effects of expression of dnROCK-2, transgenic zebrafish lines were generated by mating strains expressing the construct with counterpart strains expressing the Gal4 activator in target tissues. In this study, we crossed the dnROCK-2-expressing line with two such Gal4-expressing lines; (1) SAGFF(LF)73A for expression in the whole body, and (2) Tg(fli1a: Gal4FF)ubs4 for endothelial cell-specific expression. The phenotypes of the fish obtained were observed by fluorescent stereomicroscopy or confocal microscopy. Overexpression of dnROCK-2 in the whole body resulted in an inhibition of development, notably in cephalic formation, at 1-day post-fertilization (dpf). Confocal microscopy revealed that Hensen's zone became unclear in the trunk muscle fibers expressing dnROCK-2. Endothelial cell-specific expression of dnROCK-2 caused abnormalities in cardiovascular formation at 2-dpf. These results suggest that dnROCK-2 can act as a dominant negative construct of the Rho/ROCK pathway to affect regulation of the cytoskeleton. This construct could be a convenient tool to investigate the function of ROCK members in other vertebrate cell types.
American Journal of Physiology-Heart and Circulatory Physiology
A disintegrin and metalloproteinase (ADAM)12 is considered to promote cardiac dysfunction based o... more A disintegrin and metalloproteinase (ADAM)12 is considered to promote cardiac dysfunction based on the finding that a small-molecule ADAM12 inhibitor, KB-R7785, ameliorated cardiac function in a transverse aortic constriction (TAC) model by inhibiting the proteolytic activation of heparin-binding-EGF signaling. However, this compound has poor selectivity for ADAM12, and the role of ADAM12 in cardiac dysfunction has not yet been investigated using genetic loss-of-function mice. We revealed that ADAM12 knockout mice showed significantly more advanced cardiac hypertrophy and higher mortality rates than wild-type mice 4 wk after TAC surgery. An ADAM12 deficiency resulted in significantly more expanded cardiac fibrosis accompanied by increased collagen-related gene expression in failing hearts. The results of a genome-wide transcriptional analysis suggested a strongly enhanced focal adhesion- and fibrosis-related signaling pathway in ADAM12 knockout hearts. The loss of ADAM12 increased t...
Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intraepithelial lymph... more Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intraepithelial lymphocytes (IELs), especially CD8αα+ IELs (CD8αα IELs). In the intestine, IL-15 is produced by intestinal epithelial cells (IECs), blood vascular endothelial cells (BECs) and hematopoietic cells. However, the precise role of intestinal IL-15 on IELs is still unknown. To address the question, we generated two kinds of IL-15 conditional knockout (IL-15cKO) mice: villin-Cre (Vil-Cre) and Tie2-Cre IL-15cKO mice. IEC-derived IL-15 was specifically deleted in Vil-Cre IL-15cKO mice, whereas IL-15 produced by BECs and hematopoietic cells is deleted in Tie2-Cre IL-15cKO mice. The cell number and frequency of CD8αα IELs and NK IELs were significantly reduced in Vil-Cre IL-15cKO mice. By contrast, CD8αα IELs were unchanged in Tie2-Cre IL-15cKO mice, indicating that IL-15 produced by BECs and hematopoietic cells is dispensable for CD8αα IELs. Expression of an anti-apoptotic factor, Bcl-2, was decreased,...
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Papers by Atsuko Sehara