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