zebrafish-keratocyte-explants-to-study-collective-cell-migration

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

establishment-of-a-clinic-based-biorepository

Establishment of a Clinic-based Biorepository

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

Midwestern University

Members of the family Halobacteriaceae in the domain Archaea are obligate extreme halophiles. They occupy a variety of hypersaline environments, and their cellular biochemistry functions in a nearly saturated salty milieu. Despite extensive study, a detailed analysis of their growth kinetics is missing. To remedy this, Arrhenius plots for 14 type species of the family were generated. These organisms had maximum growth temperatures ranging from 49 to 58 degrees C. Nine of the organisms exhibited a single temperature optimum, while five grew optimally at more than one temperature. Generation times at these optimal temperatures ranged from 1.5 h (Haloterrigena turkmenica) to 3.0 h (Haloarcula vallismortis and Halorubrum saccharovorum). All shared an inflection point at 31 +/- 4 degrees C, and the temperature characteristics for 12 of the 14 type species were nearly parallel. The other two species (Natronomonas pharaonis and Natronorubrum bangense) had significantly different temperature characteristics, suggesting that the physiology of these strains is different. In addition, these data show that the type species for the family Halobacteriaceae share similar growth kinetics and are capable of much faster growth at higher temperatures than those previously reported.

Growth kinetics of extremely halophilic archaea (family halobacteriaceae) as revealed by arrhenius plots.

The control of collective cell migration of zebrafish keratocyte sheets in explant culture is of interest for cell migration and epithelial wound healing and depends on the gene expression profile. In a zebrafish genome array, ∼17.5% of the probe sets were differentially expressed greater than two-fold (p≤0.003) between 1 and 7 days of explant culture. Among the differentially expressed genes were a variety of wound healing-related genes and many of the biomarkers for epithelial-mesenchymal transition (EMT), including a switch from keratin and E-cadherin to vimentin and N-cadherin expression and several EMT-related transcription factors were found to be differentially expressed. Supporting evidence for EMT is seen in both morphological change and rearrangement of the actin cytoskeleton and in expression of cadherins during explant culture with a visible disassembly of the cell sheet. TGFβ1 and TNFα expression were analyzed by qPCR at various time points and peak differential expression of both cytokines occurred at 3 days, indicating that the EMT process is ongoing under conditions routinely used in the study of fish keratocyte motility. These data establish that an EMT process is occurring during zebrafish keratocyte explant culture and support the use of this system as a wound healing model.

Zebrafish keratocyte explant cultures as a wound healing model system: differential gene expression & morphological changes support epithelial-mesenchymal transition.

Fish keratocytes are an established model in single cell motility but little is known about their collective migration. Initially, sheets migrate from the scale at ~145 μm/h but over the course of 24h the rate of leading edge advance decreases to ~23 μm/h. During this period, leader cells retain their ability to migrate rapidly when released from the sheet and follower cell area increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert within minutes with changes in cell-cell adhesions. Leader cells migrate as single cells when they detach from the leading edge and single cells appear to become leader cells if they rejoin the sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell-cell interactions determine the role of keratocytes within the migrating sheet.

Collective cell migration of primary zebrafish keratocytes.

The BRM and BRG1 tumor suppressor genes are mutually exclusive ATPase subunits of the SWI/SNF chromatin remodeling complex. The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses these subunits, SW13+, and one that expresses neither subunit, SW13-. Loss of BRM expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, most previously used HDAC inhibitors are toxic and broad-spectrum, providing little insight into the mechanism of the switch between subtypes. In this work, we explore the mechanisms of HDAC inhibition in promoting subtype switching and further characterize the oncogenic potential of the two epigenetically distinct SW13 subtypes.

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition.

Histone deacetylase (HDAC) inhibitors are powerful epigenetic regulators that have enormous therapeutic potential and have pleiotropic effects at the cellular and systemic levels. To date, HDAC inhibitors are used clinically for a wide variety of disorders ranging from hematopoietic malignancies to psychiatric disorders, are known to have anti-inflammatory properties, and are in clinical trials for several other diseases. In addition to influencing gene expression, HDAC enzymes also function as part of large, multisubunit complexes which have many nonhistone targets, alter signaling at the cellular and systemic levels, and result in divergent and cell-type specific effects. Thus, the effects of HDAC inhibitor treatment are too intricate to completely understand with current knowledge but the ability of HDAC inhibitors to modulate the immune system presents intriguing therapeutic possibilities. This review will explore the complexity of HDAC inhibitor treatment at the cellular and systemic levels and suggest strategies for effective use of HDAC inhibitors in biomedical research, focusing on the ability of HDAC inhibitors to modulate the immune system. The possibility of combining the documented anticancer effects and newly emerging immunomodulatory effects of HDAC inhibitors represents a promising new combinatorial therapeutic approach for HDAC inhibitor treatments.

Kathryn J. Leyva

Department of Microbiology & Immunology,
Arizona College of Osteopathic Medicine,
Department of Microbiology & Immunology, Arizona College of Osteopathic Medicine

Midwestern University

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Establishment of a Clinic-based Biorepository

Kathryn J. Leyva

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology & Immunology,Arizona College of Osteopathic Medicine,Department of Microbiology & Immunology, Arizona College of Osteopathic Medicine

Midwestern University

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Establishment of a Clinic-based Biorepository

Department of Microbiology & Immunology,
Arizona College of Osteopathic Medicine,
Department of Microbiology & Immunology, Arizona College of Osteopathic Medicine