ABSTRACT
Skin that is exposed to radiation has an impaired ability to heal wounds. This is especially true for whole-body irradiation, where even moderate nonlethal doses can result in wound-healing deficits. Our previous attempts to administer dermal cells locally to wounds to correct radiation-induced deficits were hampered by poor cell retention. Here we improve the outcome by using biodegradable fibrin microbeads (FMBs) to isolate a population of mesenchymal marrow-derived stromal cells (MSCs) from murine bone marrow by their specific binding to the fibrin matrix, culture them to high density in vitro, and deliver them as MSCs on FMBs at the wound site. MSCs are retained locally, proliferate in site, and assist wounds in gaining tensile strength in whole-body irradiated mice with or without additional skin-only exposure. MSC-FMBs were effective in two different mouse strains but were ineffective across a major histocompatability barrier. Remarkably, irradiated mice whose wounds were treated with MSC-FMBs showed enhanced hair regrowth, suggesting indirect effect on the correction of radiation-induced follicular damage. Further studies showed that additional wound-healing benefit could be gained by administration of granulocyte colony-stimulating factor and AMD3100. Collagen strips coated with haptides and MSCs were also highly effective in correcting radiation-induced wound-healing deficits.
Subject(s)
Fibrin/pharmacology , Mesenchymal Stem Cell Transplantation/methods , Radiation Injuries, Experimental/therapy , Skin Diseases/therapy , Wound Healing/physiology , Absorbable Implants , Animals , Cells, Cultured , Dermis/physiology , Dermis/radiation effects , Disease Models, Animal , Female , Fibrin/physiology , Germ-Free Life , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Microspheres , Radiation Injuries, Experimental/physiopathology , Skin Diseases/physiopathology , Tensile Strength/physiology , Whole-Body Irradiation/adverse effects , Wound Healing/radiation effectsABSTRACT
BACKGROUND: Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. RESULTS: Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number. CONCLUSION: Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network.
Subject(s)
Cytoplasmic Granules/ultrastructure , Gene Expression Regulation, Developmental , Germ Cells/ultrastructure , Zebrafish/embryology , Animals , Animals, Genetically Modified , DEAD-box RNA Helicases/genetics , Dyneins/genetics , Embryo, Nonmammalian/embryology , Genetic Markers , Green Fluorescent Proteins , In Situ Hybridization , Microtubules/ultrastructure , Zebrafish/anatomy & histology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Epithelial mesenchymal transition (EMT) is a complex process that involves changes in gene expression, cytoskeleton organization, cell adhesion, and extracellular matrix composition. Screening for genes mediating EMT and cancer metastasis, Waerner, Alacakaptan, and colleagues identified ILEI, a cytokine-like protein that plays an essential role in EMT, tumor growth, and late steps of metastasis.
Subject(s)
Epithelial Cells/cytology , Interleukins/biosynthesis , Mesoderm/cytology , Animals , Cell Line, Transformed , Cell Transformation, Neoplastic , Humans , Interleukins/metabolism , Mice , Mice, Nude , Neoplasm Metastasis , Neoplasm Transplantation , Neoplasms/etiology , Neoplasms/physiopathologyABSTRACT
The high-affinity copper transporter 1 (Ctr1) is a highly conserved transmembrane protein that mediates the internalization of copper ions from the extracellular medium. In this study, we have isolated the zebrafish ctr1 gene. The zebrafish ctr1 cDNA encodes a protein with 69% identity to the human orthologue and shows conservation of specific amino acid residues involved in copper transport. We find only a single ctr1 gene in the zebrafish genome which maps to linkage group 5. The genomic structure of the zebrafish gene shows that it consists of five exons and that exon-intron boundaries are absolutely conserved with the mammalian ctr1 genes. Expression in embryos was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Zebrafish ctr1 is maternally loaded, and transcripts can be detected throughout development and in adult fish. Distribution of ctr1 message appears ubiquitous during early stages becoming restricted to the brain and ventral tissues by 24 h post fertilization (hpf). Beginning at 3 days post fertilization (dpf), expression is found mainly in the developing intestine. Specific knockdown of ctr1 by antisense morpholino oligonucleotides (MOs) causes early larval lethality. Defects include cell death in tissues where ctr1 is most heavily expressed, a finding similar to that described for a mouse knockout of mCtr1. Despite the existence of at least one other copper transport mechanism in the fish, our studies show that zebrafish ctr1 is an essential gene for development.
