Zebrafish tools for screening of therapeutic-relevant molecules affecting tissue mineralization
Bone disorders affect millions of people worldwide and current treatments have a limited efficacy often accompanied by side effects. The discovery of novel bone anabolic compounds with either pharmaceutical or nutraceutical value is thus needed. The zebrafish have emerged as a model for human bone diseases but also as a system for the screening of compounds that would treat or prevent these diseases. Beyond the technical advantages over traditional models (e.g. rodents), the high similarity of targets, physiology, drug metabolism and pharmacology in comparison to humans further highlight the potential of zebrafish as a robust model for drug screenings. The BISOKEL lab (CCMAR, University of Algarve) has developed several zebrafish assays to evaluate the osteogenic potential of natural or synthetic compounds and molecules and would like to share, within the scope of a training school (TS), both the theoretical and practical knowledge required to go through all the steps of those procedures.
Register Here: https://www.ccmar.ualg.pt/webform/zebrafishscreening
This hands-on TS aims at providing advance training to both academia and company personnel on the use of specific zebrafish systems toward drug screening, in particular bone anabolic compounds. It will also foster knowledge exchange between attendees and contribute to the establishment of scientific collaborations on bioprospection using zebrafish. Specifically, this TS will transfer conceptual and practical known-how on the following topics:
- Basis of zebrafish biology and husbandry;
- Current knowledge on drug screenings;
- Routes of compounds exposure in zebrafish;
- Imaging of bone structures and morphometric analysis;
- Assessment of compound osteogenic and regeneration potential.
Zebrafish tools presented in this workshop are the basis of a first-line medium throughput screening methodology.
In the drug discovery pipeline, first line screenings represent a critical filter to reduce the amount of compounds to be further tested and are therefore time and money savers. Until recently, in vitro cell systems or in vivo rodent systems were commonly used, although several bottlenecks existed (e.g. a cell line cannot represent the complexity of a whole organism, rodents are slow growers, expensive to maintain and have an intra-uterine early development). However, in vivo zebrafish systems overcome those obstacles and have emerged as a promising alternative to the traditional systems.
This workshop will provide both conceptual and experimental know-how on zebrafish screening systems through lectures given by experts in the field. Attendees will also have the opportunity to acquire some technical expertise through hands-on sessions where they will learn to manipulate zebrafish tools toward the screening of osteoactives.
Basic formation in biology, biomedical sciences or biochemistry. Knowledge of basic molecular biology techniques is necessary to understand some concepts.
IMPORTANT: Participants are asked to bring their own computers to perform the image analysis and morphometric assessments. If possible ImageJ should be previously installed (available for free at https://imagej.net)
Monday 25th | Background and hands-on on zebrafish biology and drug screening
9:30 - Welcome Reception
The course will start with a brief welcome reception and an overview of the main infrastructures and areas of research in UALG.
9:45 - Lecture 1 | Basis of zebrafish biology and husbandry. (Paulo Gavaia, CCMAR & Gil Martins, CCMAR)
10:30 - Coffee-break
11:00 - Lecture 2 | Fundamentals, principles and importance of drug screenings; Introduction on zebrafish screening systems; Methods used to administrate compounds (e.g. waterborne, embryo microinjection, intraperitoneal injection and dietary supplementation). (Vincent Laizé, CCMAR & Jorge Dias, SPAROS Lda)
12:30 - Lunch
14:00 - Hands-on 1 | Manipulation and microinjection of zebrafish embryos and intraperitoneal injection of zebrafish adults. (Paulo Gavaia, CCMAR; João Cardeira, MPI-HLR; Marco Tarasco, CCMAR; Gil Martins, CCMAR)
16:00 - Coffee-break
16:30 - Hands-on 1 (continuation)
Tuesday 26th | Drug screening for osteogenic molecule using zebrafish based-systems
09:30 - Lecture 3 | Zebrafish toolbox: mutant lines modeling human bone disorders and transgenic lines/in vivo systems to assess osteogenic or osteotoxic activities. (M. Leonor Cancela, CCMAR-UALG)
10:30 - Coffee-break
11:00 - Lecture 4 | Zebrafish genetics: principles of transgenesis and mutagenesis. (Anabela Bensimon-Brito, MPI-HLR)
11:45 - Lecture 5 | Basis of zebrafish imaging, image analysis and data processing regarding the pro and anti-osteogenic potential of compounds. (Anabela Bensimon-Brito, MPI-HLR; João Cardeira, MPI-HLR)12:00 - Coffee-break
12:30 - Lunch
14:00 - Hands-on 2 - The operculum system
Hands-on 2.1. Exposure of zebrafish larvae to experimental compounds | Hands-on 2.2. Alizarin red staining of zebrafish larvae and visualization of calcified structures | Hands-on 2.3. Using transgenic fish expressing fluorescent markers of different bone proteins | Hands-on 2.4. Imaging of the opercular bone using a fluorescence stereomicroscope | Hands-on 2.5. Image analysis and morphometric assessment of zebrafish structures. (Marco Tarasco, CCMAR; Joana Rosa, CCMAR; João Cardeira, MPI-HLR; Anabela Bensimon-Brito, MPI-HLR; Vânia Roberto, CCMAR; Gil Martins, CCMAR)
16:30 - Coffee-break
17:00 Hands-on 2 (continuation)
19:30 Workshop dinner (optional)
Wednesday 27th | Drug screening and regeneration: the caudal fin based-system
10:00 - Lecture 6 | Basis of regeneration and regenerative medicine. (João Cardeira, MPI-HLR)
10:30 - Lecture 7 | Zebrafish caudal fin and scales as biomedical tools for the screening of pro-osteogenic, pro-resorptive and regenerative molecules. The pipeline of procedures used will be presented and discussed. (João Cardeira, MPI-HLR & Marco Tarasco, CCMAR)
11:00 - Coffee-break
11:30 - Lecture 8 | Basis of caudal fin imaging acquisition, image analysis and data processing regarding the osteogenic/regenerative potential of compounds. (João Cardeira, MPI-HLR & Marco Tarasco, CCMAR)
12:30 - Lunch
14:00 - Hands-on 3 | The caudal fin system
Hands-on 3.1. Caudal fin amputation and exposure to experimental compounds | Hands-on 3.2. Alizarin red staining of zebrafish cauda and visualization of new bone structures | Hands-on 3.3:Imaging of the newly formed lepidotrichia using a fluorescence stereomicroscope | Hands-on 3.4: Image analysis and morphometric assessment of new bone mineralization and patterning. (Marco Tarasco, CCMAR; João Cardeira, MPI-HLR; Anabela Bemsimon-Brito, MPI-HLR)
16:30 - Coffee-break
17:00 - Hands-on 3 (continuation)
M. Leonor Cancela
Paulo J. Gavaia
Vânia P. Roberto
Joana T. Rosa
Accommodation: Booking and payment must be arranged directly by the Trainee or Home Institution. A list of suggested hotels is provided below:
CCMAR - Centre of Marine Sciences
University of Algarve
Campus de Gambelas, 8005-139 Faro, Portugal
Tel: +351 289 800 057, +351 289 800 900 ext. 7057
Fax: +351 289 800 069
- All course materials
- Coffe breaks