Zebrafish: Extended Definition

Gerardo Sandoval

English 363-02
 Zebrafish

Zebrafish (Danio rerio) are a freshwater fish known for their horizontal stripes along its

body and for their use in biological research. Most research conducted using zebrafish has been

in developmental genetics (studying how genes influence an organism’s growth) and only

recently has there been a push for broader applications in the sciences (i.e. neuroscience). The

zebrafish belongs to the cyprinid family which include familiar fish such as the pufferfish, carp,

and goldfish (Volhard & Dahm, 2002). The cyprinid family are classified as ray-finned fish.

Zebrafish can grow to be three to five centimeters in length and live on average three years.

Adulthood is reached at three to four months (Hill, 2019). In captivity, zebrafish should be kept

in optimal environments. Temperature and pH levels should remain at 28 degrees Celsius and pH

of 7 (Hill, 2019). These conditions increase lifespan and growth to five years and four

centimeters respectively. Males tend to have a red hue to their body, whereas females have a

silver shine along with a larger belly to distinguish them.

History

Beginning in the 1970s, researchers began to look at other organisms other than

invertebrates (i.e. Aplysia) for genetics study. In the1980s, zebrafish gained popularization as an

optimal vertebrate model for developmental genetics research (Volhard & Dahm, 2002). Dr.

George Streisinger, a professor at the University of Oregon, is considered to be the father of

zebrafish research for being the first to employ this animal model (Volhard & Dahm, 2002). His

research focused on genetic mutations in the nervous system. The zebrafish’s external

development (i.e. developing in an egg) proved to be advantageous for developmental genetics

allowing for visualization. His 1981 paper discussed the capability of in vitro fertilization and

development of homozygous lines (pure bred) (Volhard & Dahm, 2002). His development of the
first strain of zebrafish has led to the generation of various other strains to aid in the progression

of science and the literature.

Development

Figure 1. A schematic illustrating zebrafish

development. Adapted from D’Costa &

Shephard (2009).

Zebrafish are oviparous, meaning they lay eggs (Hill, 2019; Volhard & Dahm, 2002).

Females release on average 100 eggs every three days, while males release spermatozoa for

fertilization to occur. Once fertilized, development can be observed given their translucency.

Stages of development are determined by hours post-fertilization. Figure one shows an overview

of zebrafish’s development (Hill, 2019; Volhard & Dahm, 2002).

Within the first hour, fertilization reaches the zygote period (Volhard & Dahm, 2002).

After the initial hour up to two and half hours, the cleavage period begins. This is the period at

which the first mitosis, or splitting of the cell occurs (Hill, 2019; Volhard & Dahm, 2002). The

cells continue to divide and the cells are named based on the number of divisions that they have

undergone. The blastula period occurs from 2.5 to 5.25 hours post-fertilization. The embryo

becomes oblong and epiboly movement occurs (the outer layer forms outside the embryo). After
5.25 hour to 10 hours, the Gastrula period initiates (Volhard & Dahm, 2002). There are vital cell

movements that result in the end of epiboly, and formation of head and tail structures. From there

on and up until day three, the embryo begins to take on the form of a larvae (juvenile fish).

By day three, the embryo enters the hatching period. The embryo is now classified as

larvae (Hill, 2019; Volhard & Dahm, 2002). At this same period, the larvae develop the jaw, gill

arches, and pectoral fins. From four days to seven days-post-fertilization, the larvae grow larger

and are able to swim to surface level (Hill, 2019). At this time period, they begin to feed on live

food. Metamorphosis spans 14 to 30 days-post-fertilization (Hill, 2019; Volhard & Dahm, 2002).

During metamorphosis, the larvae develop the adult body shape, swim bladder, and

pigmentation. These juvenile zebrafish mature at three to four months as previously stated.

The Zebrafish Model

Why use zebrafish for research rather than other organisms? Zebrafish possess a

relatively simple nervous system and are translucent (Fetcho & Liu, 1998; Friedrich, Genoud, &

Wanner, 2013; Roberts, Bill, & Glanzman, 2013). The translucency of the fish allows for in vivo

optical imaging of neuronal circuits. These features, in combination with an extensive toolbox to

manipulate neural function, make them a useful model to understand memory formation at a

systems level (Best et al., 2008; Friedrich et al., 2013; Roberts et al., 2013). A non-invasive

approach can be implemented to monitor large populations of neural activity in the intact

organism(Ahrens et al., 2012; Dieris, Ahuja, Krishna, & Korsching, 2017) . Further, other

techniques such as Photoablation (disrupting neural networks with lasers), help functionally

annotate neuronal activity (Fetcho & Liu, 1998). Along with lesion experiments, behavior can

be further investigated by identifying certain cell types (i.e. using green fluorescent protein-
expressing larvae zebrafish) and lesioning brain regions to establish causal relationships (Liu &

Fetcho, 1999; Roeser & Baier, 2003).

Applications

Since Streisinger’s use of this vertebrate, the creation of mutants has allowed for

neuroscience to study genetics, development, and behavior. As previously mentioned, their

translucent bodies at such a young age allow for visualization. The combination of key biological

attributes (i.e., translucency, small brain size) and extensive tools (i.e. transgenic lines) to

investigate neural function make zebrafish a powerful animal system. Zebrafish can be easily

genetically modified to create transgenic lines that allow for imaging of specific cell types

through expression of specific neurons using proteins (Portugues, Severi, Wyart, & Ahrens,

2013). Typically, imaging of neural activity is conducted on zebrafish larvae that are 5-14 days

post-fertilization (dpf) because their relative translucency is favorable for brain imaging (Fetcho

& Liu, 1998; Miyasaka et al., 2014; Miyasaka et al., 2009). The GAL4/Upstream Activating

Sequence (GAL4/UAS) system allows for visualization of neural activity. This system provides

a mechanism for fluorescent proteins, to be targeted to specific neurons (Dieris et al., 2017).

Essentially, Fluorescent calcium indicators like GCamP, reflect neural activity as calcium flows

into the active neurons. For example, when a neuron is active it fluoresces more.
 Figure 2. GCaMP6-expressing zebrafish under confocal microscope. A. Auto

fluorescence of zebrafish. B. Olfactory Bulb region in zebrafish brain as indicted as the green

region within the purple ring. (Gerardo Sandoval).

Habituation in Zebrafish Larvae

Current research with zebrafish can take advantage of these technological advances to

examine learning and memory. Habituation is non-associative memory characterized as a

decreased response due to repeated sensory stimulation (Roberts et al., 2016; Thompson &

Spencer, 1966). This decrease in response strength is unrelated to sensory adaptation, fatigue, or

injury. This response reduction can last from minutes to hours depending on the organism and

training regimen (Thompson & Spencer, 1966). This form of learning is present in various

organisms ranging from invertebrates (i.e. Aplysia, Drosophila) to vertebrates (i.e. Mus

musculus, humans), (Friedrich et al., 2013; Glanzman, 2009). In zebrafish, habituation to visual

and auditory stimuli has been observed (Best et al., 2008; Braubach, Wood, Gadbois, Fine, &

Croll, 2009; Randlett et al., 2018; Roberts et al., 2013; Roberts et al., 2016; Wolman, Jain, Liss,

& Granato, 2011).

New technologies in neuroscience are arising that aim to generate neural circuit maps and

understand the physiological changes that occur in the vertebrate nervous system (Ahrens et al.,

2013; Friedrich et al., 2013; Panier et al., 2013). These technologies favor simple translucent

vertebrates, such as zebrafish, over murine systems and may allow for a whole-brain and

functionally useful understanding of memory. The myriad of optical tools available to measure

neural responses, control neural activity, and monitor synapses to elucidate habituation in larval

zebrafish make this organism an advantageous animal model.

 Acknowledgments

I would like to thank Arian for the constructive feedback and aiding in stating areas in my

prompt that need correct citation.

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Figure 1:

D'Costa, Allison & Shepherd, Iain. (2009). Zebrafish Development and Genetics: Introducing

Undergraduates to Developmental Biology and Genetics in a Large Introductory

Laboratory Class. Zebrafish. 6. 169-77. 10.1089/zeb.2008.0562.

 Post Write

In outlining my extended definition, I hoped to guide the reader using strategies that would

make the reading understandable. I used graphics to help facilitate the in-text descriptions.

Incorporating images helped illustrate zebrafish development and depict what a certain strain of

zebrafish looks like. I also chose to partition the definition into subheadings that allow for a

better visual appeal and flow of information. Each section serves its own purpose to help explain

the over-arching (broad) word.