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Implementation of Danio rerio (zebrafish) to Investigate Genes
and miRNAs Related to Epilepsy
Claudia Maurer-MorelliZebrafish Laboratory
Dep. of Medical Genetics-FCM/UNICAMP
Danio rerio
• Popular named as zebrafish.
• Tropical freshwater fish.
• Five uniform, pigmented, horizontal blue stripes on the side of the body.
http://aquagroup.weebly.com/photos--videos.html
WHY ARE ZEBRAFISH USED IN RESEARCH?
Fonte: http://www.youtube.com/watch?v=YO-LUC29zmk
www.ncbi.nlm.nih.gov/pubmed
Searching PUBMED
1969 1994 2004 2014
23663 papers 17088 papers 03/28/15
Zebrafish is worldwide accepted not only for genetic studies but also for modeling human diseases and drug discovery.
Larvae at 7 days post-fertilization (dpf)*.
Adult *
The Zebrafish Size
Fully grown adults are around 3 to 5 cm long and 1 cm wide.
*Scale given in cm.
The small size of the zebrafish is very convenient for saving costs and space in the laboratory as compared to rodents.
Maintenance
• Zebrafish are considered hard fish
– high capacity to acclimate to novel environments.
• They can be kept at fairly high density in a small tank
Rack System
Plates in Incubator(Embryos and Larvae)
Conventional Glass Aquarium
www.fylone-medical.com
Maintenance
• Water temperature: 28°C
• Feeding: flake food 2x/day + artemia
• Light/Dark cycle: 14h/10h
Zebrafish exhibit a robust circadian pattern of day-time activity and night-time rest, a state which is an important
similarity with sleep in mammals.
Comparable Features
Wheeler and Brandli, 2009.
As a vertebrate, zebrafish share many similarities with commonly used laboratory species and are closer in the phylogenetic tree to humans than invertebrates models
- Genome
- Developmental traits
- Conserved organization of common organs and tissues
- Physiology
- Behavior.
Advantages in Research• Females are able to spawn at intervals of 2–3 days, laying down
about 200 eggs/time
• External fertilization and development
http://protocols.davidson.edu
LabZeb
UNBORN BABIES
ON BOARD!
Advantages in Research
4 cell – 1h One day embryo
three day embryos newly hatched 3dpf
Transparency
www.cas.vanderbilt.edu
Advantages in Research
Rapid development and generation times (3-4 months)
• (
Zebrafish Sequence Project
Fonte: http://www.youtube.com/watch?v=YO-LUC29zmk~ 75% genes are related to human genes
Large synteny with humans (conserved gene order)
Genome partially duplicate
The information from zebrafish genome sequencing has advanced functional analyses of genes of interest and its role in determining the phenotype by genetic reverse.
Reverse Genetics
• It is an approach to study gene function.
– Site-directed mutagenesis, gene knockout, gene knockdown
FROM GENE TO PHENOTYPE
www.eusem.com
Morpholino (MO)
Genetic Tools
CRISPR–Cas9
www.cmrb.eu
Zebrafish Community
The massive amount of information obtained on zebrafish studies is recorded into databases
FROM ZEBRAFISH TO HUMAN
http://www.youtube.com/watch?v=YO-LUC29zmk
Zebrafishmodel
Heart
Hematopoietic Diseases
Kidney
Skeleton
Behavior
Cancer
Neurological Diseases
The zebrafish embryonic heart resembles the heart of the human embryo at three weeks gestation.
Cardiovascular diseases
Annu. Rev. Genomics Hum. Genet. 2002. 3:311–40
HEART FAILUREARRHYTHMIASCONGENITAL HEART DISEASE
Zebrafishmodel
Heart
Hematopoietic Diseases
Kidney
Skeleton
Behavior
Cancer
Neurological Diseases
Cardiovascular Diseases
The zebrafish is particularly amenable to the study ofhematopoiesis.
Blood circulation begins by approximately 24 hours post fertilization (hpf), and the number and morphology of circulating cells are visible under a microscope.
Annu. Rev. Genomics Hum. Genet. 2002. 3:311–40
Zebrafishmodel
Heart
Hematopoietic Disorders
Kidney
Skeleton
Behavior
Cancer
Neurological Diseases
Cardiovascular disorders
It has the three major modular elements of all kidneys: the glomerulustubules, and ducts.
POLYCYSTIC KIDNEY DISEASE
Annu. Rev. Genomics Hum. Genet. 2002. 3:311–40
Zebrafishmodel
Heart
Hematopoietic Disorders
Kidney
Skeleton
Behavior
Behavior
Cancer
Cardiovascular disorders • OSTEOPOROSIS
• OSTEOGENESIS IMPERFECTA• CRANIOFACIAL SYNDROMES
Annu. Rev. Genomics Hum. Genet. 2002. 3:311–40
Zebrafishmodel
Heart
Hematopoietic Disorders
Kidney
Skeleton
Brain
Behavior
Cancer
Cardiovascular disorders
CIRCADIAN RHYTHMICITY - Adult zebrafishexhibit circadian rhythmicity
ADDICTION – ethanol exposure
Zebrafishmodel
Heart
Hematopoietic Disorders
Kidney
Skeleton
Behavior
Cancer
Neurological Diseases
Cardiovascular disorders
Transparency in adult body allows for detailed visualization of cellular activity, circulation, metastasis and many other phenomena.
White RM et al., Cell Stem Cell. 2008
Zebrafishmodel
Heart
Hematopoietic Disorders
Kidney
Skeleton
Behavior
Cancer
Neurological Diseases
Cardiovascular disorders
Alzheimer’s Disease
Parkinson’s Disease
Huntington Disease
Hereditary Spastic Paraplesia
Zebrafish CNS
• Zebrafish and mammalian encephalon share many structural properties
– fore-, mid- and hind-brain, including diencephalon, telencephalon and cerebellum
– main cellular types, such as neurons, microglia, oligodendrocytes, cerebellar Purkinje cells, motor neurons and astrocytes.
Gupta T., Mullins M.C. (2010).http://www.jove.com/details.php?id=1717
IS THE ZEBRAFISH A GOOD MODEL FOR EPILEPSY/SEIZURE STUDIES?
15mM of Pentilenotetrazol (PTZ)
Behavior
Larvae 7dpf
Electrographic
Larvae 7dpf
c-fos in situ hibridization
Larvae 7dpf
Behavioral Manisfestations
• Stage I: increase in swim activity
• Stage II: rapid whirlpool-like circling swim behavior
• Stage III: a series of brief clonus-like movements leading to a loss of posture
Baraban SC. Chapter 15. In: Pitkanen A, Schwartz Kroin PA, Moshé SL, editors. Models of seizures and epilepsy. Elsevier Academic Press; Amsterdam: 2006. pp. 89–198.
Neural Activity
Modified from Baraban et al., 2005
15mM PTZ induced spontaneous epileptiform discharges which were initially brief, small in amplitude and occurred at high frequency after 10 – 30 min of exposure and a period of clonic activity.
Gene Expression c-fos
It is well known that immediate early genes are over expressed in brain regions corresponding to sites of seizure initiation.
c-fos expression is up-regulated after seizure in a pattern very similar to those found in rodents
Antiepileptic Drugs (AEDs)
Baraban SC. Chapter 15. In: Pitkanen A, Schwartz Kroin PA, Moshé SL, editors. Models of seizures and epilepsy. Elsevier Academic Press; Amsterdam: 2006. pp. 89–198.
Epileptic discharges were suppressed by AED as valproate and diazepam
Screening of AED
1- electrographic evidence of epileptic-like activity;
2- seizure-like behaviors manifest as tonic-clonic motor movement
(Woodbury 1969)
Zebrafish are considered a powerful model for AED screening
Zebrafish Laboratory-LabZeb-
Department of Medical GeneticsUniversity of Campinas
Campinas, São Paulo, Brazil
Mutation studies in patients with epilepsy
Knockdown candidate genes for epilepsy
Gene and miRNA Expression
Epilepsy and Inflammation
• The relationship between epilepsy and inflammation is a relevant issue in clinical practice
• There is evidence that inflammatory mediators are up-regulated after seizures in both humans and rodents
Objectives
• To investigate temporal transcript profile of
il1b and cox2 genes after PTZ-induced seizure in 7dpf zebrafish brain
• To study the effect of indomethacin prior to PTZ exposure, on seizure behavior and neuronal activity (by mesuring c-fos transcript levels)
Temporal transcript profile of il1b gene
Figure 1. Temporal transcript profile of il1b gene in zebrafish larvae brain after Pentylenetetrazole-evoked seizure.
cox2a and cox2b mRNA expression
Figure 2. cox2a and cox2b mRNA expression in zebrafish larvae brain afterPentylenetetrazole-evoked seizure
Indomethacin treatment – cox2b gene
Figure 3. Evaluation of the effect of indomethacin exposure prior toPentylenetetrazole-induced seizure on cox2b transcript levels.
Indomethacin treatment – c-fos gene
Figure 4. Evaluation of the effect of indomethacin exposure prior toPentylenetetrazole-induced seizure on c-fos transcrip levels.
c-fos
Indomethacin treatment - behavior
Figure 5. Seizure onset latency behavior during Pentylenetetrazole exposure.
Figure 6. Number of seizure behavior during Pentylenetetrazole exposure.
Conclusion
• Zebrafish:
– Inflammatory response is indeed, present in zebrafish after seizures;
– valuable alternative model for studying molecular mechanisms underlying inflammation and seizures;
– Valuable for investigation of anti-inflammatory compounds that could be potentially therapeutics for seizure suppression.
Ongoing study: MO to knockdown il1b and cox2 genes and its receptors with posterior phenotype
assessment
Concluding Thoughts
Zebrafish
• are considered an alternative model in research
Mainly because of the widespread use of the unprotected embryo and larval form up to 5 days stage (Directive Animal Welfare 2010/63/EU)
Concluding Thoughts
Zebrafish
• are considered an alternative model in research;
• have many features that contribute in scientific research
Fertilization and development extra-utero Transparency
Number of offspringEasy maintenance compared to rodents
Genetic similarity to humans
Concluding Thoughts
Zebrafish
• are considered an alternative model in research;
• have many features that contribute in scientific research;
• allow easy genetic manipulations and phenotypic assessment
Use of genetic reverse techniques to investigate the role of genes and genetic
variants (mutations)
Concluding Thoughts
Zebrafish
• are considered an alternative model in research;
• have many features that contribute in scientific research;
• allow easy genetic manipulations and phenotypic assessment
• Modeling human diseases and drugs screening
Because its morphological and physiological similarities to mammals
Provide opportunities to accelerate the process of drug discovery
Supported by
THANKS!
Modifyed from http://www.keepcalm-o-matic.co.uk
Epilepsy in Adult Zebrafish