Implementation of Danio rerio (zebrafish) to Investigate Genes and mirnas Related to Epilepsy
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1 Implementation of Danio rerio (zebrafish) to Investigate Genes and mirnas Related to Epilepsy Claudia Maurer-Morelli Zebrafish Laboratory Dep. of Medical Genetics -FCM/UNICAMP
2 Danio rerio Popular named as zebrafish. Tropical freshwater fish. Five uniform, pigmented, horizontal blue stripes on the side of the body.
3 Fonte: WHY ARE ZEBRAFISH USED IN RESEARCH?
4 Searching PUBMED Zebrafish 1969 is worldwide accepted not 1994 only 2004 for genetic 2014 studies but also for modeling human diseases and drug discovery papers papers 03/28/15
5 The Zebrafish Size Larvae at 7 days post-fertilization (dpf)*. *Scale given in cm. Adult * Fully grown adults are around 3 to 5 cm long and 1 cm wide. The small size of the zebrafish is very convenient for saving costs and space in the laboratory as compared to rodents.
6 Maintenance Zebrafish are considered hard fish high capacity to acclimate to novel environments. Plates in Incubator (Embryos and Larvae) They can be kept at fairly high density in a small tank Rack System Conventional Glass Aquarium
7 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.
8 Comparable Features - Genome As a vertebrate, zebrafish share many similarities with commonly used laboratory species and are closer in the phylogenetic tree to humans than invertebrates models - Developmental traits - Conserved organization of common organs and tissues - Physiology - Behavior. Wheeler and Brandli, 2009.
9 Advantages in Research Females are able to spawn at intervals of 2 3 days, laying down about 200 eggs/time External fertilization and development UNBORN BABIES ON BOARD! LabZeb
10 Transparency Advantages in Research 4 cell 1h One day embryo three day embryos newly hatched 3dpf
11 Advantages in Research Rapid development and generation times (3-4 months) (
12 Zebrafish Sequence Project
13 ~ 75% genes are related to human genes Fonte: 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.
14 Reverse Genetics It is an approach to study gene function. Site-directed mutagenesis, gene knockout, gene knockdown FROM GENE TO PHENOTYPE
15 Genetic Tools Morpholino (MO) CRISPR Cas9
16 Zebrafish Community The massive amount of information obtained on zebrafish studies is recorded into databases
17 FROM ZEBRAFISH TO HUMAN
18 Cardiovascular Heart diseases Hematopoietic Diseases The zebrafish embryonic heart resembles the heart of the human embryo at three weeks gestation. Kidney Zebrafish model Skeleton Behavior Cancer Neurological Diseases Annu. Rev. Genomics Hum. Genet : HEART FAILURE ARRHYTHMIAS CONGENITAL HEART DISEASE
19 Zebrafish model Cardiovascular Heart Diseases Hematopoietic Diseases Kidney Skeleton Behavior The zebrafish is particularly amenable to the study of hematopoiesis. Blood circulation begins by approximately 24 hours post fertilization (hpf), and the number and morphology of circulating cells are visible under a microscope. Cancer Neurological Diseases Annu. Rev. Genomics Hum. Genet :311 40
20 Cardiovascular Heart disorders Hematopoietic Disorders Kidney It has the three major modular elements of all kidneys: the glomerulus tubules, and ducts. Zebrafish model Skeleton Behavior POLYCYSTIC KIDNEY DISEASE Cancer Neurological Diseases Annu. Rev. Genomics Hum. Genet :311 40
21 Cardiovascular Heart disorders OSTEOPOROSIS Hematopoietic Disorders OSTEOGENESIS IMPERFECTA CRANIOFACIAL SYNDROMES Kidney Zebrafish model Skeleton Behavior Behavior Cancer Annu. Rev. Genomics Hum. Genet :311 40
22 Cardiovascular Heart disorders Hematopoietic Disorders Kidney CIRCADIAN RHYTHMICITY - Adult zebrafish exhibit circadian rhythmicity ADDICTION ethanol exposure Zebrafish model Skeleton Behavior Cancer Brain
23 Cardiovascular Heart disorders Hematopoietic Disorders Kidney Zebrafish model Skeleton Behavior Cancer Neurological Diseases White RM et al., Cell Stem Cell Transparency in adult body allows for detailed visualization of cellular activity, circulation, metastasis and many other phenomena.
24 Cardiovascular Heart disorders Hematopoietic Disorders Zebrafish model Kidney Skeleton Behavior Alzheimer s Disease Parkinson s Disease Huntington Disease Hereditary Spastic Paraplesia Cancer Neurological Diseases
25 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).
26 IS THE ZEBRAFISH A GOOD MODEL FOR EPILEPSY/SEIZURE STUDIES?
27 15mM of Pentilenotetrazol (PTZ) Larvae 7dpf Behavior Larvae 7dpf Electrographic Larvae 7dpf c-fos in situ hibridization
28 Behavioral Manisfestations Stage I: increase in swim activity Stage II: rapid whirlpoollike 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: pp
29 Neural Activity Modified from Baraban et al., mM PTZ induced spontaneous epileptiform discharges which were initially brief, small in amplitude and occurred at high frequency after min of exposure and a period of clonic activity.
30 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
31 Antiepileptic Drugs (AEDs) Epileptic discharges were suppressed by AED as valproate and diazepam Baraban SC. Chapter 15. In: Pitkanen A, Schwartz Kroin PA, Moshé SL, editors. Models of seizures and epilepsy. Elsevier Academic Press; Amsterdam: pp
32 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
33 Zebrafish Laboratory -LabZeb- Department of Medical Genetics University of Campinas Campinas, São Paulo, Brazil
34 Knockdown candidate genes for epilepsy Mutation studies in patients with epilepsy Gene and mirna Expression
35 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
36 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)
37 Temporal transcript profile of il1b gene Figure 1. Temporal transcript profile of il1b gene in zebrafish larvae brain after Pentylenetetrazole-evoked seizure.
38 cox2a and cox2b mrna expression Figure 2. cox2a and cox2b mrna expression in zebrafish larvae brain after Pentylenetetrazole-evoked seizure
39 Indomethacin treatment cox2b gene Figure 3. Evaluation of the effect of indomethacin exposure prior to Pentylenetetrazole-induced seizure on cox2b transcript levels.
40 Indomethacin treatment c-fos gene c-fos Figure 4. Evaluation of the effect of indomethacin exposure prior to Pentylenetetrazole-induced seizure on c-fos transcrip levels.
41 Indomethacin treatment - behavior Figure 5. Seizure onset latency behavior during Pentylenetetrazole exposure. Figure 6. Number of seizure behavior during Pentylenetetrazole exposure.
42 Conclusion Zebrafish: Inflammatory response is indeed, present in zebrafish after seizures; valuable alternative model for studying molecular mechanisms underlying inflammation and seizures; Ongoing study: MO to knockdown il1b and cox2 genes and its receptors with posterior phenotype Valuable for investigation assessment of anti-inflammatory compounds that could be potentially therapeutics for seizure suppression.
43 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)
44 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 offspring Easy maintenance compared to rodents Genetic similarity to humans
45 Concluding Thoughts Zebrafish are considered an alternative model in Use of genetic reverse techniques to research; investigate the role of genes and genetic have many features variants (mutations) that contribute in scientific research; allow easy genetic manipulations and phenotypic assessment
46 Concluding Thoughts Zebrafish are considered an alternative model in research; Because its morphological and physiological similarities to mammals have many features that contribute in scientific research; Provide opportunities to accelerate the allow easy genetic manipulations and phenotypic process of drug assessment discovery Modeling human diseases and drugs screening
47 Supported by
48 THANKS! Modifyed from
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