Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.

Intestinal epithelial cell-derived IL-15 determines local maintenance and maturation of intra-epithelial lymphocytes in the intestine.

Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intra-epithelial 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 was 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, whereas Fas expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. Forced expression of Bcl-2 by a Bcl-2 transgene partially restored CD8αα IELs in Vil-Cre IL-15cKO mice, suggesting that some IL-15 signal other than Bcl-2 is required for maintenance of CD8αα IELs. Furthermore, granzyme B production was reduced, whereas PD-1 expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. These results collectively suggested that IEC-derived IL-15 is essential for homeostasis of IELs by promoting their survival and functional maturation.

A disintegrin and metalloproteinase 12 prevents heart failure by regulating cardiac hypertrophy and fibrosis.

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 the abundance of the integrinß1 subunit and transforming growth factor (TGF)-ß receptor types I and III, and this was followed by the phosphorylation of focal adhesion kinase, Akt, mammalian target of rapamycin, ERK, and Smad2/3 in the heart, which resulted in cardiac dysfunction. The present results revealed that the loss of ADAM12 enhanced focal adhesion and canonical TGF-ß signaling by regulating the abundance of the integrinß1 and TGF-ß receptors.NEW & NOTEWORTHY In contrast to a long-believed cardio-damaging role of a disintegrin and metalloproteinase (ADAM)12, cardiac hypertrophy was more severe, cardiac function was lower, and mortality was higher in ADAM12 knockout mice than in wild-type mice after transverse aortic constriction surgery. The loss of ADAM12 enhanced focal adhesion- and fibrosis-related signaling pathways in the heart, which may compromise cardiac function. These results provide insights for the development of novel therapeutics that target ADAM12 to treat heart failure.

Generation and evaluation of a transgenic zebrafish for tissue-specific expression of a dominant-negative Rho-associated protein kinase-2.

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.

In silico analysis-based identification of the target residue of integrin α6 for metastasis inhibition of basal-like breast cancer.

Metastasis causes death in breast cancer patients. To inhibit breast cancer metastasis, we focused on integrin α6, a membrane protein that contributes to cell migration and metastasis. According to in silico analysis, we identified Asp-358 as an integrin α6-specific vertebrate-conserved residue and consequently as a potential therapeutic target. Because Asp-358 is located on the surface of the ß propeller domain that interacts with other molecules for integrin α6 function, we hypothesized that a peptide with the sequence around Asp-358 competitively inhibits integrin α6 complex formation. We treated basal-like breast cancer cells with the peptide and observed reductions in cell migration and metastasis. The result of the immunoprecipitation assay showed that the peptide inhibited integrin α6 complex formation. Our immunofluorescence for phosphorylated paxillin, a marker of integrin-regulated focal adhesion, showed that the peptide reduced the number of focal adhesions. These results indicate that the peptide inhibits integrin α6 function. This study identified the functional residue of integrin α6 and designed the inhibitory peptide. For breast cancer patients, metastasis inhibition therapy may be developed in the future based on this study.

Mesenchymal stromal cells in bone marrow express adiponectin and are efficiently targeted by an adiponectin promoter-driven Cre transgene.

Stromal cells in bone marrow (BM) constitute a specific microenvironment supporting the development and maintenance of hematopoietic cells. Adiponectin is a cytokine secreted by adipocytes. Besides its anti-diabetic and anti-atherogenic roles, adiponectin reportedly regulates the development and function of hematopoietic cells in BM. However, it remains unclear whether mesenchymal stromal cells in BM express adiponectin. Here, we show that PDGFRß+VCAM-1+ stromal cells express adiponectin. Lineage tracing revealed that a majority of PDGFRß+VCAM-1+ cells were targeted by an adiponectin promoter-driven Cre (Adipoq-Cre) transgene. Additionally, the Adipoq-Cre transgene targets a minority of osteoblasts at a younger age but larger populations are targeted at an older age. Furthermore, the Adipoq-Cre transgene targets almost all CXCL12-abundant reticular (CAR) cells and most of the stromal cells targeted by the Adipoq-Cre transgene are CAR cells. Finally, deletion of interleukin-7 (IL-7) by the Adipoq-Cre transgene resulted in severe impairment of B lymphopoiesis in BM. These results demonstrate that PDGFRß+VCAM-1+ stromal cells in BM express adiponectin and are targeted by the Adipoq-Cre transgene, suggesting a broader specificity of the Adipoq-Cre transgene.

PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

Skeletal muscle satellite cells (SMSCs), the major stem cells responsible for the regeneration of skeletal muscle, are normally cell cycle arrested but differentiate to generate myocytes upon muscle damage, forming new myofibers along with self-renewing stem cells in preparation for subsequent injury. In this study, we investigated which factors stimulate the proliferation and differentiation of SMSCs and found that pyruvate, the end product of glycolysis, stimulates their differentiation. Pyruvate antagonizes the effects of hypoxia on preferential self-renewal of SMSCs through dephosphorylation or activation of pyruvate dehydrogenase (PDH), which mediates opening of the gateway from glycolysis to the tricarboxylic acid (TCA) cycle by producing acetyl coenzyme A from pyruvate. PDH kinase 1, highly expressed under hypoxia, is down-regulated under normoxic conditions, leading to an increase in dephosphorylated PDH. Conditional deletion of PDH in SMSCs affects cell divisions generating myocytes and subsequent myotube formation, inefficient skeletal muscle regeneration upon injury, and aggravated pathogenesis of a dystrophin-deficient mouse model of Duchenne muscular dystrophy. Thus, the flow from glycolysis to the TCA cycle mediated by PDH plays a pivotal role in the differentiation of SMSCs, which is critical for the progression of skeletal muscle regeneration.-Hori, S., Hiramuki, Y., Nishimura, D., Sato, F., Sehara-Fujisawa, A. PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

Integrin ß1 activity is required for cardiovascular formation in zebrafish.

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 ß-subunits. The integrin ß1 gene is widely expressed in vivo and is the major ß molecule in many tissues; however, tissue-specific roles of integrin ß1 are still elusive. In this study, we investigated integrin ß1 function in endothelial cells of zebrafish. An integrin ß1b 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 ß1b in vascular formation, we developed a Gal4/UAS-mediated conditional inactivation of integrin ß1 by expressing the cytoplasmic region of integrin ß1 that acts as a dominant-negative (DN) isoform. Expression of integrin ß1 DN in endothelial cells induced blood vessel abnormalities as in integrin ß1b 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 ß1 in zebrafish is powerful tools for functional analysis of integrin ß1 in developing tissues.

Anteroposterior molecular registries in ectoderm of the echinus rudiment.

BACKGROUND: Echinoderms and hemichordates are sister taxa that both have larvae with tripartite coeloms. Hemichordates inherit the coelom plan and ectoderm from larvae, whereas echinoderms form the adult rudiment comprising rearranged coeloms and a vestibule that then develops into adult oral ectoderm. Molecular networks that control patterns of the ectoderm and the central nervous system along the anteroposterior (AP) axis are highly conserved between hemichordates and chordates, respectively. In echinoderms, however, little is known about the AP registry in the ectoderm. RESULTS: We isolated ectodermal AP map genes from the sand dollar Peronella japonica and examined their expression. Comparative expression analyses showed that (1) P. japonica orthologs of hemichordate anterior markers are expressed in the larval apical plate, which degenerates during metamorphosis; (2) P. japonica orthologs of the medial markers are expressed in the ambulacral ectoderm of the rudiment; and (3) few P. japonica orthologs of the posterior markers are expressed in ectoderm. CONCLUSIONS: We suggest that echinoids only inherit the ambulacral ectoderm from a common ambulacrarian ancestor, which largely corresponds to the collar ectoderm in hemichordates. The ectodermal AP registry provides insights into the AP axis and evolutionary processes of echinoderms from a common ambulacrarian ancestor. Developmental Dynamics 247:1297-1307, 2018. © 2018 Wiley Periodicals, Inc.

The Sal-like 4 - integrin α6ß1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

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 ß1. In addition, we clarified that integrin α6 and ß1 formed a heterodimer. At the molecular level, loss of the SALL4 - integrin α6ß1 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 α6ß1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4 - integrin α6ß1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated that this gene network enhances cell migration in vivo. Our findings identify a potential new therapeutic target for the prevention of metastasis, and provide an improved understanding of cancer cell migration.

Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

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 broad-spectrum 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.

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages.

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta-actin promoter. The aNLS-EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time-lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.

ADAM12-deficient zebrafish exhibit retardation in body growth at the juvenile stage without developmental defects.

ADAM (a disintegrin and metalloprotease) constitutes a family of multi-domain proteins that are involved in development, homeostasis, and disease. ADAM12 plays important roles in myogenesis and adipogenesis in mice; however, the precise physiological mechanisms are not known, and the function of this gene in other vertebrates has not been examined. In this study, we used a simple model vertebrate, the zebrafish, to investigate the functions of ADAM12 during development. Zebrafish adam12 is conserved with those of mammals in the synteny and the amino-acid sequence. We examined adam12 expression in zebrafish embryos by whole mount in situ hybridization and the promoter activity of the adam12 upstream sequence. We found that adam12 is strongly expressed in the cardiovascular system, erythroid progenitors, brain, and jaw cartilage during zebrafish development, and adam12-knockout zebrafish exhibited reduced body size in the juvenile stage without apparent morphological defects. Taken together, these results suggest that adam12 plays a significant role in the regulation of body growth during juvenile stage in zebrafish, although the precise molecular mechanisms await further study.

Neuregulin-1/glial growth factor stimulates Schwann cell migration by inducing α5 ß1 integrin-ErbB2-focal adhesion kinase complex formation.

After peripheral nerve injury, Schwann cells gain a migratory phenotype and remodel their extracellular matrix to provide a supportive environment for axonal regeneration. The soluble neuregulin-1 isoform, that is, glial growth factor (GGF), is expressed in regenerating axons of injured peripheral nerves and regulates Schwann cell motility by activating the ErbB family of tyrosine kinase receptors, but how GGF/ErbB signaling contributes to Schwann cell motility remains unclear. Here, we show that GGF stimulates Schwann cell migration by inducing the formation of a protein complex containing the fibronectin receptor α5ß1 integrin, ErbB2, and focal adhesion kinase (FAK). ErbB2 co-localizes and co-immunoprecipitates with the focal complex members including α5ß1 integrin and FAK after GGF treatment. These effects of GGF appear to involve FAK activation, which occurs downstream of ErbB2 stimulation. RNAi-mediated down-regulation of α5 integrin expression in primary cultured Schwann cells resulted in significantly decreased interaction between FAK and ErbB2, as well as decreased GGF-induced migration. An increase in the α5ß1 integrin-ErbB2-FAK complex formation was observed in injured nerve Schwann cells, but not uninjured control. Taken together, these data suggest that GGF plays an important modulatory role in Schwann cell migration after nerve crush by inducing α5ß1 integrin-ErbB2-FAK complex formation.

Generation of a monoclonal antibody reactive to prefusion myocytes.

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion-incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell-cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.

Findings from recent studies by the Japan Aerospace Exploration Agency examining musculoskeletal atrophy in space and on Earth.

The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly.

Restored interlaced volumetric imaging increases image quality and scanning speed during intravital imaging in living mice.

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 imaging quality within a volume, however, is degraded by involuntary movements and trades off between the imaged volume, imaging speed and quality. To balance such trade-offs incurred by two-photon excitation microscopy during intravital imaging, we developed a unique combination of interlaced scanning and a simple image restoration algorithm based on biological signal sparsity and a graph Laplacian matrix. This method increases the scanning speed by a factor of four for a field size of 212 µm × 106 µm × 130 µm, and significantly improves the quality of four-dimensional dynamic volumetric data by preventing irregular artifacts due to the movement observed with conventional methods. Our data suggest this method is robust enough to be applied to multiple types of soft tissue.

Intravital imaging reveals cell cycle-dependent myogenic cell migration during muscle regeneration.

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 cell-derived myogenic cells. We found that in vivo, ERK is maximally activated in myogenic cells two days after injury, and this is then followed by increases in cell number and motility. With limited effects of ERK activity on migration on an acute timescale, we hypothesized that ERK increases migration speed in the later phase by promoting cell-cycle progression. Our cell-cycle analysis further revealed that in myogenic cells, ERK activity is critical for G1/S transition, and cells migrate more rapidly in S/G2 phase 3 days after injury. Finally, migration speed of myogenic cells was suppressed after CDK1/2-but not CDK1-inhibitor treatment, demonstrating a critical role of CDK2 in myogenic cell migration. Overall, our study demonstrates that in myogenic cells, the ERK-CDK2 axis promotes not only G1/S transition but also migration, thus providing a novel mechanism for efficient muscle regeneration.

ADAM12: a potential target for the treatment of chronic wounds.

Wound healing is a complex process involving multiple cellular events, including cell proliferation, migration, and tissue remodeling. A disintegrin and metalloprotease 12 (ADAM12) is a membrane-anchored metalloprotease, which has been implicated in activation-inactivation of growth factors that play an important role in wound healing, including heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and insulin growth factor (IGF) binding proteins. Here, we report that expression of ADAM12 is fivefold upregulated in the nonhealing edge of chronic ulcers compared to healthy skin, based on microarrays of biopsies taken from five patients and from healthy controls (p = 0.013). The increase in ADAM12 expression in chronic ulcers was confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Moreover, immunohistochemical analysis demonstrated a pronounced increase in the membranous and intracellular signal for ADAM12 in the epidermis of chronic wounds compared to healthy skin. These findings, coupled with our previous observations that lack of keratinocyte migration contributes to the pathogenesis of chronic ulcers, prompted us to evaluate how the absence of ADAM12 affects the migration of mouse keratinocytes. Skin explants from newborn ADAM12-/- or wild-type (WT) mice were used to quantify keratinocyte migration out of the explants over a period of 7 days. We found a statistically significant increase in the migration of ADAM12-/- keratinocytes compared to WT control (p = 0.0014) samples. Taken together, the upregulation of ADAM12 in chronic wounds and the increased migration of keratinocytes in the absence of ADAM12 suggest that ADAM12 is an important mediator of wound healing. We hypothesize that increased expression of ADAM12 in chronic wounds impairs wound healing through the inhibition of keratinocyte migration and that topical ADAM12 inhibitors may therefore prove useful for the treatment of chronic wounds.

Disruption of integrin α4 in zebrafish leads to cephalic hemorrhage during development.

Integrins, transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matrix interactions, are heterodimers that consist of an α- and ß-subunit. The integrin α4 gene (itgα4) 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 itgα4 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 itgα4 ko108 using the CRISPR/Cas9 genome editing system; the mutant genome harbored an approximately 2.0-kb deletion in the itgα4 locus. A truncated transcript was detected in itgα4 (+/-) or (-/-) fish but not in (+/+) fish. The mutant transcript was hypothesized to encode a truncated Itgα4 protein due to a premature stop codon. itgα4 (-/-) 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 itgα4 ko108 should be a useful model to investigate a physiological function for Itgα4 in the blood circulation system.