Zebrafish

Zebrafish are small tropical freshwater fish native to South Asia and first used as model organism in 1930s (Creaser, 1934). They are commonly found in rivers and ponds of India, but can now be found in pet shops. They get their name from the horizontal blue stripes that adorn their bodies. They are social animals, preferring to live in large groups called shoals.

The spark that ignited the broad utilization of zebrafish occurred in the 90s with the "Big Screen". This screen was conducted by Christiane Nüsslein-Volhard in Tübingen (DE) and Wolfgang Driever in Boston (USA) and led to the the publication of 37 articles in a special issue of Development. In this screen, over 4000 ENU-mutant lines were generated and analysed in terms of development/behavior. In 2013, the sequencing of the zebrafish genome (Howe et al., 2013) revealed the mutations responsible for the mutant phenotypes. Zebrafish genome contains more than 25000 protein-coding genes - a higher number than that of humans due to the teleost-specific whole-genome duplication.

Although mice and humans are closely related from an evolutionary perspective being both mammals, zebrafish have many advantages that give them the upper fin over their mammalian rivals:

• High level of fertility (can produce up to 300 eggs at a time)
• Rapid development
• Transparent embryos
• External development/easy genetic manipulation
• Easy and cheap maintenance (compared to mice) - they can be housed together = less space
• High throughtput screening
• Vertebrate model
• 70% of human genes have at least one zebrafish orthologue
• 80% of proteins known to be associated with human diseases have a zebrafish counterpart
• Established behavioural paradigms

Whole genome duplication (WGD)

One of the differences between mammals and teleost fish is the duplication event which characterise their genome. The WGD is a process by which the whole genome of an organism is doubled. In vertebrates, two (three in teleost) WGD events have occurred. The result is the presence of different copies of the same gene which undergo:

1. subfunctionalization - duplicate genes with same expression pattern or function of ancestral genes with two duplicated genes;
2. neofunctionalization - one of the duplicated genes acquire a new function;
3. gene loss

Ontogenesis of the zebrafish central nervous system

The development of zebrafish central nervous system start at 6 hours post fertilization. At 24 hours post fertilization, the three main areas of the brain (forebrain, midbrain and hindbrain) have already formed. The forebrain comprises the two olfactory bulbs and the telencephalon which, in contrast to mammals, develops differently. In mammals, the neural tube moves internally following an evagination process, forming two telencephalic emispheres and two internal ventricles. In teleost fish, the neural tube moves externally (eversion model) forming two massive telencephalic emispheres and a single ventricle.

Mammals vs teleost telencephalon

Despite the different telencephalic development, the brain areas and neural circuits are very well conserved between mammals and teleost fish.

The main brain areas can be resumed as follows:

Zebrafish is a powerful model to study human disorders as 80% of genes known to be associated with human diseases have a zebrafish counterpart and neural circuits involved in the onset of such disorders are very well conserved.

Zebrafish are really useful to be employed both in forward (high reproductive rate providing many individuals for high-throughput screening) and reverse genetic (easy genetic manipulation due to the external embryonic development) studies, with the aim to identify possible therapeutic targets.

• Forward genetics (induce mutation > select pehotype of interest > identify gene/s involved)
  

Identify unknown gene/s potentially involved in causing behavioural traits characterising human psychiatric diseases (e.g., impulsivity, habituation deficit, anxiety or addiction beahaviours).

• Reverse genetics (select gene of interest > induce mutation/alter expression of the genes > analyse behavioural and molecular effects)
  

Induce mutations that alter the expression of gene/s identified but GWAS studies but whose casuality is not established or the mechanism of action are not known.

Examples of human diseases successfully modeled in zebrafish include:

1. Duchenne muscular dystrophy
2. Human melanoma
3. Epilepsy
4. Osteoporosis
5. Amyotrophic lateral sclerosis (ALS)
6. Inflammation
7. Atherosclerosis
8. Heart failure
9. Type 2 diabetes mellitus
10. Sensorineural hearing loss
11. Enteric nervous system disease
12. Cancer

Furthermore, despite non-human animal models do not show complex diseases such as psychiatric disease, it is possible to study traits that characterise such disorders in order to identify genes and molecular pathways involved. Examples of established zebrafish behaviours to study psychiatric disease phenotypes are:

1. Impulsivity
2. Stress and anxiety
3. Pre-pulse inhibition
4. Shoaling as model for social behaviours
5. Learning and memory

Last but not least, zebrafish larvae are a powerful model to perform high-throughput drug screens to identify novel compounds with similar effects as validated psychoactive drugs.