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1.
J Invest Dermatol ; 144(7): 1430-1439, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38752940

RESUMEN

Cutaneous wounds are common injuries that affect millions of people around the world. In vulnerable populations such as the elderly and those with diabetes, defects in wound healing can lead to the development of chronic open wounds. Although mammalian models are commonly used to study cutaneous wound healing, the challenges of in vivo imaging in mammals have hampered detailed observation of cell coordination and cell signaling during wound healing. The zebrafish is becoming increasingly popular for studying cutaneous wound healing owing to its genetic accessibility, suitability for experimental manipulation, and the ability to perform live, in vivo imaging with cellular or even subcellular resolution. In this paper, we review some of the techniques that have been developed for eliciting cutaneous wounds in the zebrafish, including an economical method we recently developed using a rotary tool that generates consistent and reproducible full-thickness wounds. Combined with the thousands of transgenic lines and experimental assays available in zebrafish, the ability to generate reproducible cutaneous wounds makes it possible to study key cellular and molecular events during wound healing using this powerful experimental model organism.


Asunto(s)
Modelos Animales de Enfermedad , Piel , Cicatrización de Heridas , Pez Cebra , Animales , Cicatrización de Heridas/fisiología , Piel/lesiones , Piel/patología , Animales Modificados Genéticamente , Humanos
2.
J Invest Dermatol ; 144(4): 888-897.e6, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37979772

RESUMEN

Cutaneous wounds are common afflictions that follow a stereotypical healing process involving hemostasis, inflammation, proliferation, and remodeling phases. In the elderly and those suffering from vascular or metabolic diseases, poor healing after cutaneous injuries can lead to open chronic wounds susceptible to infection. The discovery of new therapeutic strategies to improve this defective wound healing requires a better understanding of the cellular behaviors and molecular mechanisms that drive the different phases of wound healing and how these are altered with age or disease. The zebrafish provides an ideal model for visualization and experimental manipulation of the cellular and molecular events during wound healing in the context of an intact, living vertebrate. To facilitate studies of cutaneous wound healing in zebrafish, we have developed an inexpensive, simple, and effective method for generating reproducible cutaneous injuries in adult zebrafish using a rotary tool. We demonstrate that our injury system can be used in combination with high-resolution live imaging to monitor skin re-epithelialization, immune cell recruitment and activation, and vessel regrowth in the same animal over time. This injury system provides a valuable experimental platform to study key cellular and molecular events during wound healing in vivo with unprecedented resolution.


Asunto(s)
Piel , Pez Cebra , Animales , Adulto , Humanos , Anciano , Piel/diagnóstico por imagen , Piel/lesiones , Cicatrización de Heridas/fisiología , Repitelización , Inflamación
3.
Methods Mol Biol ; 2677: 113-125, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37464238

RESUMEN

Live imaging of adult tissue stem cell niches provides key insights into the dynamic behavior of stem cells, their differentiating progeny, and their neighboring support cells, but few niches are amenable to this approach. Here, we discuss a technique for long-term live imaging of the Drosophila testis stem cell niche. Culturing whole testes ex vivo for up to 18 h allows for tracking of cell-type-specific behaviors under normal and various chemically or genetically modified conditions. Fixing and staining tissues after live imaging allows for the molecular confirmation of cell identity and behavior. By using live imaging in intact niches, we can better uncover the cellular and molecular mechanisms that regulate stem cell function in vivo.


Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Masculino , Testículo , Nicho de Células Madre/fisiología , Células Madre , Drosophila melanogaster
4.
Elife ; 112022 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-35468055

RESUMEN

Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. Here we find that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. We propose that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation.


Asunto(s)
Quistes , Proteínas de Drosophila , Animales , Transdiferenciación Celular , Quistes/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Masculino , Receptores de Péptidos de Invertebrados/genética , Receptores de Péptidos de Invertebrados/metabolismo , Células Madre/fisiología , Testículo/metabolismo , Microambiente Tumoral
5.
Angiogenesis ; 24(2): 251-269, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33449300

RESUMEN

Endothelial cells display an extraordinary plasticity both during development and throughout adult life. During early development, endothelial cells assume arterial, venous, or lymphatic identity, while selected endothelial cells undergo additional fate changes to become hematopoietic progenitor, cardiac valve, and other cell types. Adult endothelial cells are some of the longest-lived cells in the body and their participation as stable components of the vascular wall is critical for the proper function of both the circulatory and lymphatic systems, yet these cells also display a remarkable capacity to undergo changes in their differentiated identity during injury, disease, and even normal physiological changes in the vasculature. Here, we discuss how endothelial cells become specified during development as arterial, venous, or lymphatic endothelial cells or convert into hematopoietic stem and progenitor cells or cardiac valve cells. We compare findings from in vitro and in vivo studies with a focus on the zebrafish as a valuable model for exploring the signaling pathways and environmental cues that drive these transitions. We also discuss how endothelial plasticity can aid in revascularization and repair of tissue after damage- but may have detrimental consequences under disease conditions. By better understanding endothelial plasticity and the mechanisms underlying endothelial fate transitions, we can begin to explore new therapeutic avenues.


Asunto(s)
Diferenciación Celular , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Heridas y Lesiones/metabolismo , Pez Cebra/metabolismo , Animales , Arterias/metabolismo , Células Madre Hematopoyéticas , Humanos , Vasos Linfáticos/metabolismo , Venas/metabolismo , Heridas y Lesiones/terapia
6.
Cell Rep ; 24(13): 3466-3476.e8, 2018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30257208

RESUMEN

Homeostasis in adult tissues depends on the precise regulation of stem cells and their surrounding microenvironments, or niches. Here, we show that the cell cycle inhibitor and tumor suppressor Retinoblastoma (RB) is a critical regulator of niche cells in the Drosophila testis. The testis contains a single niche, composed of somatic hub cells, that signals to adjacent germline and somatic stem cells. Hub cells are normally quiescent, but knockdown of the RB homolog Rbf in these cells causes them to proliferate and convert to somatic stem cells. Over time, mutant hub cell clusters enlarge and split apart, forming ectopic hubs surrounded by active stem cells. Furthermore, we show that Rbf's ability to restrict niche number depends on the transcription factors E2F and Escargot and the adhesion molecule E-cadherin. Together this work reveals how precise modulation of niche cells, not only the stem cells they support, can drive regeneration and disease.


Asunto(s)
Autorrenovación de las Células , Proteínas de Drosophila/metabolismo , Proteína de Retinoblastoma/metabolismo , Nicho de Células Madre , Testículo/metabolismo , Factores de Transcripción/metabolismo , Animales , Cadherinas/metabolismo , Proliferación Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Masculino , Proteína de Retinoblastoma/genética , Testículo/citología , Factores de Transcripción/genética
7.
Methods Mol Biol ; 1463: 63-74, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27734347

RESUMEN

Live imaging of adult tissue stem cell niches provides key insights into the dynamic behavior of stem cells, their differentiating progeny, and their neighboring support cells, but few niches are amenable to this approach. Here we discuss a technique for long-term live imaging of the Drosophila testis stem cell niche. Culturing whole testes ex vivo for up to 12.5 h allows for tracking of cell-type specific behaviors under normal and various chemically or genetically modified conditions. Fixing and staining tissues after live imaging allows for the molecular confirmation of cell identity and behavior. Utilization of live imaging in intact niches will facilitate further understanding of the cellular and molecular mechanisms that regulate stem cell function in vivo.


Asunto(s)
Drosophila/citología , Testículo/ultraestructura , Imagen de Lapso de Tiempo/métodos , Animales , Rastreo Celular , Masculino , Microscopía Confocal/métodos , Técnicas de Cultivo de Órganos , Coloración y Etiquetado , Nicho de Células Madre , Testículo/citología , Fijación del Tejido
8.
PLoS Genet ; 10(11): e1004713, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25375180

RESUMEN

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). Here, we show a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.


Asunto(s)
Quinasas Janus/genética , Proteínas del Tejido Nervioso/biosíntesis , Receptores Inmunológicos/biosíntesis , Factores de Transcripción STAT/genética , Células Madre/metabolismo , Testículo/metabolismo , Animales , Diferenciación Celular/genética , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/crecimiento & desarrollo , Células Germinativas/metabolismo , Humanos , Quinasas Janus/biosíntesis , Masculino , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Factores de Transcripción STAT/biosíntesis , Transducción de Señal , Nicho de Células Madre/genética , Células Madre/citología , Testículo/crecimiento & desarrollo , Proteínas Roundabout
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