Zebrafish tools for screening of therapeutic-relevant molecules affecting tissue mineralization | - CCMAR -
 

Zebrafish tools for screening of therapeutic-relevant molecules affecting tissue mineralization

Segunda, 25 Novembro, 2019 a Quarta, 27 Novembro, 2019
 
Course Description 

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

 

Objectives

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.
Format 

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. 

Pre-requisites 

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)

Agenda 

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)

 

Intended Audience 
Enthusiastic and motivated biologists, biomedical researchers and students who want to gain expertise in the use of zebrafish as a screening model for osteogenic compounds. Number of participants limited to a maximum of 15.
Scientific Organisation 
Instructors 

M. Leonor Cancela

Centre of Marine Sciences (CCMAR) & Department of Biomedical Sciences and Medicine (DCBM) - UALG, Faro, Portugal

Vincent Laizé

Centre of Marine Sciences (CCMAR) - UALG, Faro, Portugal
Vincent Laizé has a PhD in molecular Biophysics. He has joined the BIOSKEL lab at the CCMAR to study the mechanisms underlying bone formation and mineralization using fish in vitro and in vivo systems. He has participated in the development of the first cell lines of fish origin capable of in vitro biomineralization and is currently involved in the bioprospection of osteoactive compounds in several marine resources toward the development of pharmaceutical and nutraceutical solutions for human bone disorders.

Paulo J. Gavaia

Centre of Marine Sciences (CCMAR) - UALG, Faro, Portugal
Paulo Gavaia obtained his PhD in Biological Sciences. During this time, he acquired expertize in the use of marine fish and zebrafish in the fields of skeletal biology using cellular and molecular biology as tools to identify molecular regulators of calcification with biomedical applications. He joined the CCMAR as a staff Researcher of the BIOSKEL in 2007 and established a zebrafish facility to give support to the I&D activities centered in Comparative, Adaptive and Functional Skeletal Biology and in biomedical and pharmaceutic applications.

Anabela Bensimon-Brito

Max Planck Institute for Heart and Lung Research (MPI-HLR), Bad Nauheim, Germany
Anabela Bensimon-Brito has a joint PhD in Biological and Biomedical Sciences by UGent/UAlg where she characterized the vertebral column development in zebrafish. Since then, she worked on fin regeneration, leading to the optimization of imaging techniques for in vivo characterization of bone. Presently Anabela is a Postdoctoral Researcher working in Didier Stainier’s Lab, where she developed a new model for cardiac valve regeneration. She is also leading a project adapting in vivo imaging techniques to the adult zebrafish, including Echocardiography, MRI and Micro-CT.

João Cardeira

Max Planck Institute for Heart and Lung Research (MPI-HLR), Bad Nauheim, Germany
João Cardeira has a PhD in Mechanisms of Disease and Regenerative Medicine. During this period his research centered on the mechanisms underlying bone regeneration, using the regenerating zebrafish caudal fin as a studying model. He focused on how bone is patterned during regeneration, on how different bone cells play important roles during this morphogenetic process, while also developing quantitative tools to address bone regeneration. Recently, in 2018, he joined the Max-Planck-Institute for Heart and Lung Research, where he is developing work in the field of zebrafish cardiac regeneration.

Marco Tarasco

Centre of Marine Sciences (CCMAR) - UALG, Faro, Portugal
Marco Tarasco, PhD student in BIOSKEL Lab, is currently studying the effects of environmental pollutants on bone development and regeneration to gain insights into the molecular mechanism underlying osteotoxicity. He has been particularly interested in the development of new tools to better understand the effect of compounds on bone homeostasis and during his master thesis he has developed and optimized the zebrafish operculum as a system to screen osteoactive compounds.

Gil Martins

Centre of Marine Sciences (CCMAR) - UALG, Faro, Portugal
Gil Martins has a MSc. In Aquaculture and Fisheries with specialization in zebrafish nutrition. Since 2014, he has been working as a zebrafish facility manager at CCMAR and collaborated in numerous research projects on the field of skeletal biology and fish reproduction. In the last years, Gil work has been focus on the optimization of zebrafish rearing conditions and determination of zebrafish nutritional requirements.

Vânia P. Roberto

Centre of Marine Sciences (CCMAR) - UALG, Faro, Portugal
Vânia P. Roberto has a PhD in Biomedical Sciences. Her work has been focused on the epigenetic mechanisms underlying gene expression regulation in bone development and human diseases. Using cell-based systems and animal models she could characterize the function of 3 microRNAs in skeletogenesis. Currently, she is a member of the BIOSKEL Lab working on the beneficial impact of vitamin K1 in the skeleton and its potential effects at an epigenetic level.

Joana T. Rosa

Centre of Marine Sciences (CCMAR) - UALG, Faro, Portugal
Joana T. Rosa has a PhD in Molecular Biology. During her scientific career, she has been using fish models to study a wide range of questions related to the development, structure, and evolution of the skeleton. She has characterized new markers for skeletal development, developed new tools for bone and cartilage studies and validated the functionality of several mammalian tools in fish. Currently, she is working at BIOSKEL Lab on bioprospection toward the discovery of innovative compounds for human bone applications.
Accommodation 

Accommodation: Booking and payment must be arranged directly by the Trainee or Home Institution. A list of suggested hotels is provided below:

Venue 

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

Location 
ATT type 
Formação Avançada
Presentation Language 
English
Fee (starting from...) 
€ 150.00
Fee Covers 
  • All course materials
  • Coffe breaks
  • Lunches
Keywords 
Danio rerio, Operculum development, Caudal fin regeneration, Osteogenic activity, Drug screening