localization of the s -p chloride c (s 12 2) z e m and a s … · 2019. 5. 6. · 136 ian dew,...

In: Zebrafish ISBN: 978-1-63117-558-9

Editors: Charles A. Lessman and Ethan A. Carver © 2014 Nova Science Publishers, Inc.

Chapter 7

LOCALIZATION OF THE SODIUM-POTASSIUM-

CHLORIDE COTRANSPORTER

(SLC12A2) DURING ZEBRAFISH

EMBRYOGENESIS AND MYOGENESIS

AND A SCREEN FOR ADDITIONAL ANTIBODIES

TO STUDY ZEBRAFISH MYOGENESIS

Ian Dew1, Linda M. Sircy

1, Lauren Milleville

1,

Michael R. Taylor2, Charles A. Lessman

3 and Ethan A. Carver

*1

1Department of Biological and Environmental Sciences,

University of Tennessee at Chattanooga, Chattanooga, TN, US 2Department of Chemical Biology and Therapeutics,

St. Jude Children‘s Research Hospital, Memphis, TN, US 3Department of Biological Sciences, The University of Memphis, Memphis, TN, US

ABSTRACT

The objective of the current study was to screen a series of commercially avaliable

antibodies in order to determine if they would bind specifically to target proteins in

zebrafish musculature for use in fluorscence confocal microscopy. Many of these

antibodies were not developed specifically for use in zebrafish; but because of the near

universality of many basic muscle proteins among species these antibodies could

potentially bind to homologus proteins in zebrafish. Of these, T4, for the protein Slc12a,

has not been well described. We reviewed antibody expression in the muscle and

evaluated the protein localization of Slc12a in zebrafish development. T4 was visualized

within the skeletal muscle, where it may play an important role in ion regulation during

muscle activity. Overall, the use of these antibodies will allow researchers access to tools

* Corresponding Author: Ethan A. Carver Ph.D. Department of Biological and Environmental Sciences, The

University of Tennessee at Chattanooga, 615 McCallie Avenue, 215 Holt Hall, Chattanooga, TN 37363, US,

Phone: (423) 425-4315, Email:[email protected]

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Ian Dew, Linda M. Sircy, Lauren Milleville et al. 136

known to work within the zebrafish and enable more studies using this organism as a

model system for muscle development.

Keywords: Zebrafish, antibodies, sarcomere, muscle development

INTRODUCTION

Zebrafish as a Model Organism

The zebrafish (Danio rerio) has established itself as a major vertebrate organism for the

study of developmental processes. Danio rerio Zebrafish are a tropical freshwater fish

belonging to the minnow family (Cyprinidae) and is native to India. There are many

characteristics of D. reriozebrafish that contribute to its use as a model organism. D. rerio

Zebrafish exhibit optical transparency during development, rapid temporal development, the

existence of mutant strains, and an ease and low cost associated with colony management. In

addition, zebrafish also exhibit external fertilization, which can be artificially induced by the

experimenter to produce a large number of progeny for laboratory use (Nüsslein-Volhard &

Dahm, 2002). Because this species is capable of breeding year round in controlled conditions,

continuous studies utilizing this organism can occur (Gilbert, 2006). The use of zebrafish

provides the researcher a model organism with the observational and manipulative ease

associated with invertebrate models as well as all of the developmental features common to

vertebrates (Liesche & Currie, 2007). These advantages allow the zebrafish to be utilized as a

model for a number of different areas. This chapter will focus on the use of Danio rerio

zebrafish as a model to study striated muscle formation.

Early Development and Structure of Musculature in Zebrafish

The skeletal musculature of zebrafish is a derivative of the mesoderm, which arises from

the somites during the segmentation stage of development. Muscle tissue functions to

coordinate movement, and can support and surround the skeleton and organ systems. In

zebrafish, the majority of skeletal muscle forms from the somites and is involved in

movement of the trunk and fins as well as the pharyngeal muscles used in respiration and

feeding. As the somite forms and differentiates, it will contain three distinct regions known as

the dermatome, the myotome and the sclerotome. In general, the sclerotome will form skeletal

structures such as the vertebrae. The dermatome and the myotome are originally found

together in a dorsal structure known as the dermamyotome, however this structure soon

separates and the dermatome helps form the dermis and the myotome contributes to a large

amount of the trunk skeletal muscle (Macintosh, 2006).

As an organ, muscle contains bundles of muscle cells, also known as fascicles, as well as

connective tissue, blood vessels, and nerves. Within each muscle fascicle, there reside many

muscle cells, also known as muscle fibers. Skeletal muscle fibers are elongated cells that run

the length of the muscle and contain multiple peripheral nuclei that lie immediately below the

sarcolemma, or plasma membrane. The muscle fiber‘s semi fluid cytoplasm, sarcoplasm, is

filled with mitochondria and hundreds of myofibrils. Myofibrils are banded, rod-like elements

Localization of the Sodium-Potassium-Chloride Cotransporter … 137

that contain the fiber‘s contractile machinery. Each myofibril is essentially a repetitive series

of sarcomeres. Sarcomeres are comprised of overlapping thick and thin filaments made of

myosin and actin proteins respectively that are visualized as muscle striations (Figure 1;

Stanfield, 2007). Actin is a muscle protein that forms filaments with a double-helical

structure. Associated with actin filaments are the proteins tropomyosin and troponin in

addition to nebulin, which aids in strengthening muscle structure (Macintosh, 2006). Myosin

is made of two globular heads and two -helix tails wrapped around one another. Hundreds

of individual myosin proteins link together in order to form one myosin filament (Macintosh,

2006).

Figure 1. Model of a sarcomere. This model names and places the major proteins within the contractile

element. The sarcomere is a repeated structure along a myofibril.

Together the thin actin filaments and the thick myosin filaments form the alternating dark

and light bands associated with muscle tissue and visualized as muscle striations. The myosin

filaments form the dark, anisotropic A-bands, and the actin filaments form the light, isotropic

I-bands. The actin and myosin filament partially overlap one another in resting muscle, but

there is a region, the H-zone, in which the two do not overlap. Within this region there is a

dark line that runs perpendicular to the actin and myosin filaments. This dark region is known

as the M-line and contains filaments of myomesin, which run parallel to the myosin filaments

and create a space between the myosin thick filaments (Macintosh, 2006). Titin filaments also

make up part of the muscle structure. These are bound to -actinin in the Z-disks and

myomesin in the M-line and act as stabilizers for the myosin thick filaments (Macintosh,

2006). Collectively, these make up a number of the proteins and structures within a sarcomere

(Figure 1). These form the repetitive units that make up a bulk of the myofiber, and then

collect together as part of the muscle fasicle.

A sac-like membranous network called the sarcoplasmic reticulum surrounds each of the

individual myofibrils. The sarcoplasmic reticulum and associated transverse (t) tubules help

transmit signals from the sarcolemma to the myofibrils, allowing the muscle cell to uniformly


respond to a neural input (Stanfield, 2007). Initial signals for muscle contraction begin with

the nerve cell as an action potential; subsequent myofibril contraction is then initiated as a

result of the release of Ca2+ ions from the sarcoplasmic reticulum. This sudden release of

Ca2+ ions changes the cell‘s membrane potential. Other ion gradients across the

plasmalemma become activated during muscle activity based on the release and reuptake of

Ca2+ ions with the fiber. These other ions, such as Na+, K

+ and Cl

-, maintain normal

transmembrane gradients and counteracts changes in ion gradients due to muscle activity.

Collectively, these proteins make up the contractile elements of skeletal muscle and are

present in vertebrate musculature. We use the zebrafish as a model to better understand the

interrelationships between these proteins and how they interact during development and

myogenesis to create the functional elements of the muscle.

In this chapter, we describe the identification of antibodies that cross-react with various

proteins within the muscle and sarcomere of zebrafish. Some of these antibodies have been

previously published (Brand et al. 1996; Barresi et al. 2001; Bessarab et al. 2008; Câmara-

Pereira et al. 2009; Codina et al. 2010; Hinitis et al. 2009 Hinits et al. 2012; Grosskurth et al.

2008;

Knight et al. 2008; Ochi & Westerfield, 2009; Piotrowski & Nüsslein-Volhard, C. 2000;

Tallafuss & Eisen, 2008; Tee et al. 2009; Walters et al. 2009).

Here, we use these antibodies to focus on muscle structure and development and illustrate

their localization within muscle fibers. We also include a more comprehensive study of the

zebrafish Slc12a2 antibody (i.e., T4) during embryogenesis and muscle development. This

provides beneficial information on muscle development and begins to study the role that

Slc12a2 has in ion transport within this tissue.

METHODS

Zebrafish Husbandry and Egg Collection

The approved IACUC protocols to be used in this study include; protocols #0306EAC-

01, #0806EAC-02, #0306EAC-03, and are based upon and modified from prior zebrafish

work (Westerfield, 1994; Brand et al. 2002; Nüsslein-Volhard & Dahm, 2002). In this

experiment, wild-type zebrafish were raised in 10-gallon tanks. These tanks were maintained

at 28C and the water was filtered to remove fish waste and debris and to slow algal growth.

In each tank, between 10 to 20 male and female zebrafish were housed. The water supply is

slightly acidic, so in order to maintain the proper pH of 7 to 7.5, sodium bicarbonate was

added to the tank water.

To collect embryos, a plastic container with a layer of glass marbles was placed atop an

inverted glass bowl within each tank. During mating, the fertilized eggs would fall to safety

within the marbles.

Tanks were checked daily to ensure cleanliness, and proper temperature and pH were

maintained. If the tanks had experienced significant algal growth, the fish were carefully

removed and placed temporarily in holding tanks so that the mating tanks could be properly

cleaned. If needed, the tanks were topped-off with fresh tank water.


The mating tanks were checked daily for embryos. If embryos were present, they were

carefully removed using a plastic pipette and placed in a small container with special egg

water and a drop of anti-fungal methylene blue solution (Westerfield, 1994). The embryos

were either raised to maturity or incubated until the desired developmental stage was reached.

The water used for storing eggs is a diluted salt water solution of 1.5 milliliters of stock

solution (140 g Instant Ocean Sea Salts [Aquarium Systems, Inc.] in one liter of distilled

water) in one liter of reverse osmosis water.

Adult fish were fed dry flake food (Tetramin) daily. Newly hatched fry were fed

paramecium once or twice a day. If not used for this experiment, the young zebrafish were

moved into nursery tanks after twenty-one days, and were fed brine shrimp, paramecium and

flake food before being moved to adult maintanence tanks.

Histological Fixation of Embryos

When embryos reached the desired developmental stage (Kimmel, 1995), histological

fixation was used to preserve the embryos for future use.

Euthanized embryos of the desired developmental stage were placed into 1.5 mL

Eppendorf tubes and 1 mL of 4% Paraformaldehyde (PFA) was added to the tube. After

incubation at room temperature for 15 minutes, the PFA was removed and the samples were

then washed with 1 mL of Phosphate Buffered Saline (PBS) to dilute any residual PFA

remaining in the tube with the embryos. After dilution with the PBS, this solution, too, was

removed and 1 mL of 100% methanol was added to the tube, and the embryos were storred at

-20 until the embryos were needed.

Whole-Mount Antibody Staining The immunohistochemistry protocol used in this study was a modified version of the

protocol described in Nüsslein-Volhard (2002).

Day 1

Three to six embryos of the desired developmental time point were removed from their

respective Eppendorf tube and placed into a viewing dish along with a few milliliters of 100%

methanol. If the chorion was still intact it was carefully removed by viewing the embryo

under a dissecting microscope and using a set of tweezers to slowly peel away the chorion.

Once the embryos were properly prepared, they were placed back into an Eppendorf tube

and taken through a methanol-PBS gradient of descending concentrations of methanol. Each

wash was of a 5 minute duration and consisted of a 100% methanol wash, 75% methanol to

25% PBS, 50% methanol to 50% PBS, and 25% methanol to 75% PBS. The embryos were

then washed twice for ten minutes each wash in 1 mL 1X PBS solution. After the two 10

minute washes the embryos were washed four times in 1 mL PBS 1% Triton (PBST) solution

for 10 minutes each wash. The embryos were then immersed in 0.5 mL of primary antibody

solution and incubated overnight in 4C refrigeration.


Day 2

After incubation with the primary antibody, the embryos were removed from

refrigeration and the Eppendorf tube was incubated 15 minutes at room temperature. The

primary antibody was removed and placed back into its original tube for reuse. The embryos

were then washed four times with 1 mL of 1X PBS for 30 minutes each. This series of washes

dilutes any residual primary antibody. After the series of washes was completed the embryos

were incubated over night in AlexaFluor 488 secondary antibody at 4C.

Day 3

The Eppendorf tube was removed from incubation and the secondary antibody was

removed and placed back into the original container for reuse. The embryos were then

washed four times using 1 mL PBS for 30 minutes each wash. After the final wash the

embryos were mounted onto slides and counter-stained using Anti-Fade Dapi-Fluoromount-G

(Southern Biotech).

Antibodies Used in Screening

The following antibodies were used to probe various muscle proteins within the skeletal

musculature of zebra fish embryos:

A4.1025

The A4.1025 antibody, developed by Helen M. Blau at Stanford University, was obtained

from the Developmental Studies Hybridoma Bank developed under the auspices of the

NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA

52242 (Webster et al. 1998).

CT3

The CT3 antibody, developed by J. Lin, was obtained from the Developmental Studies

Hybridoma Bank developed under the auspices of the NICHD and maintained by The

University of Iowa, Department of Biology, Iowa City, IA 52242.

EB165

The EB165 antibody, developed by Everett Bandman at University of California at Davis,

was obtained from the Developmental Studies Hybridoma Bank developed under the auspices

of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City,

IA 52242 (Cerny & Bandman, E. 1986). EB165 binds to fast myosin heavy chain isoforms,

and was used at a 1:200 dilution and has been successful in zebrafish (Ochi & Westerfield,

2009; Tee et al. 2009; Walters et al. 2009).

F59

The F59 antibody, developed by Stockdale, was obtained from the Developmental

Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The

University of Iowa, Department of Biology, Iowa City, IA 52242 (Miller & Stockdale, 1986).


F59 binds to myosin heavy chain isoforms, and was used at a 1:200 dilution during

experiments.

MF20

The MF20 antibody, developed by Donald Fischman at Cornell, was obtained from the

Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and

maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. MF20

should bind to all sarcomere myosin and light meromyosin of skeletal muscle (Bader, Masak

& Fischman, 1982).

T11

The T11 monoclonal antibody was purchased from Sigma (T9030) (Wang, 1985).

T11 is a primary antibody that binds to titin filaments, which are present in sarcomeres of

striated muscles. This antibody was used successfully at a concentration of 1:1000.

T4

T4 is a polyclonal mouse anti-human NKCC1 antibody (1:2000; gift from Dr. Donald

Thomason) and was developed by Christian Lytle at University of California Riverside. It

was also obtained from the Developmental Studies Hybridoma Bank developed under the

auspices of the NICHD and maintained by The University of Iowa, Department of Biology,

Iowa City, IA 52242 (Lytle et al. 1995).

4D9

The 4D9 antibody, developed by Corey Goodman at the University of Californa at

Berkeley, was obtained from the Developmental Studies Hybridoma Bank developed under

the auspices of the NICHD and maintained by The University of Iowa, Department of

Biology, Iowa City, IA 52242 (Patel et al. 1989).

Secondary antibody: Alexa Fluor-488 conjugated Goat anti-mouse IgG (1:100;

Molecular Probes). The secondary antibody alone did not specifically bind to the embryo

proper in any measurable amount, however, some nonspecific binding did occur within the

yolk compartment. (data not shown).

Confocal Microscopy

The immunohistochemically stained zebrafish embryos were mounted and oriented on

slides for proper visualization of skeletal muscle using an Olympus FluoView FV1000

microscope. Embryos were studied under epi-fluorescent and confocal microscopy. The epi-

fluorescent setting was used to properly view whole embryos before using switching to the

laser confocal settings of the microscope. Confocal microscopy allowed for viewing the

skeletal musculature of the embryos at the cellular level, and analysis of binding of the

different antibodies to their respective muscle proteins. Images were captured and analyzed

using Fluoview software (Olympus America, Inc.) and saved in jpeg format. Then, images

were compiled using Photoshop CC5 (Adobe) and are shown with anterior to the right

throughout.


RESULTS

Most of the embryos tested in these experiments were between 24 and 48 hours post

fertilization (hpf). During this period of development the somites are rapidly specializing into

skeletal musculature of the trunk and tail of the young zebrafish. Nearly all of the features of

adult musculature are present by the 48-hpf stage of development. The muscle fibers consist

of myofibrils composed of a repetitive series of sarcomeres that contain the fiber‘s contractile

elements. The sarcomeres are primarily composed of myosin, actin, and titin proteins as well

as a number of other associated proteins involved in muscle contraction (Figure 1; Stanfield

& Germann, 2007).

Antibodies Showing Specificity for Muscle

T4

Figure 2. T4 localization within the early developing zebrafish. NKCC1 localization along during early

development is visualized. Small bars denote 20 um lengths. A) T4 localization at 1.5 hours post

fertilization with an objective magnification of 20X, with a brightfield background to visualize the cells

and yolk components. B) T4 localization at 1.5 hours post fertilization with an objective magnification

of 20X. This is the same field capture as seen in Figure 2A without brightfield. C) T4 localization at 3

hours post fertilization with a total magnification of 10X. The entire blastula is visible with localization

seen within blastomeres and as illustrated with yellow arrow, discrete regions of the chorion. D) T4

localization at 3 hours post fertilization with a total magnification of 10X. The entire blastula is visible

with a brightfield background to visualize the cells and yolk components. E) T4 localization at 3 hours

post fertilization with a total magnification of 10X. Fluorescence capture only without brightfield, as

seen in Figure 2D. F) T4 localization at 3 hours post fertilization with a total magnification of 40X. The

fluorescent localization is seen within blastomeres.


T4 is a polyclonal mouse anti-human Na+-K

+-2Cl

- co-transporter type 1 (NKCC1)

antibody, also known as Sodium-potassium-chloride cotransporter (Slc12a2). Overall, the T4

antibody has not been well documented in studies. With this in mind, we used whole-mount

immunohistochemistry utilizing the T4 antibody at various time points in zebrafish

development (Figures 2-6). During development, the T4 antibody stained for zebrafish

Slc12a2 at very early stages. Slc12a2 localization was confined to the blastomeres and not

present with the yolk (Figure 2A and B). This localization continues within the blastomeres,

during the blastula period, again within cells and not the yolk (Figure 2C-F). Slc12a2 protein

expression is also seen within the chorion as a punctate series of localizations. (Figure 2C-

yellow arrows). The blastomeres retain similar patterns of localization throughout

development (Figure 2B and F). By 16 hpf, Slc12a2 still localized within all cells of the

embryo proper; however, some fluorescence is seen within the yolk ( Figure 3). This

nonspecific binding in the yolk is seen with the secondary antibody alone (data not shown).

Figure 3. T4 localization along the entire tailbud region at 16 hours post fertilization is visualized.

Small bars denote 20 um lengths.

As muscle begins to form, we found localization of Slc12a2 within muscle myofibrils

(Figure 4). Slc12a2 has been associated with muscle tissue in rats (Won et al. 1999;

Kristensen, Hansen, & Juel, 2006), however, it has not been well visualized in zebrafish.

The T4 antibody bound within the skeletal myotubes in a very repetitive pattern (Figure

4). Under both low (Figure 4A) and high power magnification (Figure 4B), the Slc12a2

protein is visualized along the length of the myofibrils. This expression pattern was seen

within skeletal muscles during the course of development and does not vary (Figure 4 C and

D, and data not shown). T-tubules are narrow channels that encircle the myofibrils at regular

intervals and lie perpendicular to the long axis of the muscle fiber. These tubules run through

muscle fiber plasma membranes and act as part of excitation-contraction coupling

(MacIntosh, 2006). They function to conduct impulses from the surface to the interior of the

muscle fiber, initiating the release of Ca2+

from the lateral sacs of the SR (MacIntosh, 2006).

The repetitive patterns seen by these localizations lend to postulating that the T4 antibody


associates with the T-tubules themselves, or are in close association with them on the

sarcolemmal membrane.

Figure 4. T4 localization within zebrafish sarcomere. Small bars denote 20 um lengths. A) T4

localization at 1 day post fertilization with an objective magnification of 40X. B) T4 localization at 1

day post fertilization with a total magnification of 300X. C) T4 localization at 2 day post fertilization

with an objective magnification of 40X. D) T4 localization at 2 day post fertilization with an objective

magnification of 40X.

Slc12a2 has been shown to mediate electroneutral transport of Na+ and K

+ associated

with 2Cl–ions across membranes in a number of organs (Payne et al. 1995; Xu et al. 1994;

Abbas & Whitfield, 2009). At different time points in zebrafish development, we looked at

localization of Slc12a2 through T4 antibody staining outside of truck musculature.

Localization of Slc12a2 was observed in the developing lens at different stages of

development (Figure 5A and B). Also, different regions of skeletal muscle illustrate the


presence of Slc12a2, as seen in cranial skeletal muscle (Figure 5C and D). Otic expression in

zebrafish was seen as well (data not shown), and mice homozygous for a knockout of the

NKCC1 gene are profoundly deaf and have other growth and neurological defects (Delpire et

al. 1999; Flagella et al. 1999). Slc12a2 localization is seen in the cardiac muscle as well

(Figure 6). Overall, the T4 antibody has been shown to work well with zebrafish and provide

a resource for experimentation to better understand the role of Slc12a2 in development and

functionality within different organ systems.

Figure 5. T4 localization within different zebrafish organs. Small bars denote 20 um lengths. A) T4

localization in the lens at 1 day post fertilization with an objective magnification of 40X. B) T4

localization in the lens at 3 day post fertilization with a total magnification of 60X. T4 localization is

more intense at this stage and better defined within the lens and not the surrounding cells. C) T4

localization in cranial musculature at 5 day post fertilization with an objective magnification of 4X. The

yellow box encompasses the region that is shown in Figure 5D. D) T4 localization at 2 day post

fertilization with a total magnification of 140X.


Figure 6. T4 localization within the zebrafish heart. Small bars denote 20 um lengths. A) T4 localization

in the heart at day 2 post fertilization with an objective magnification of 40X. The heart is seen with

both a brightfield background and fluorescence to visualize the heart, surrounding cells and yolk

components (upper region of the figure). B) T4 localization in the ear at day 2 post fertilization with a

total magnification of 40X. This is the same image as Figure 6A without brightfield illumination. T4

localization within the sarcomere structures (white arrows) are seen within the cardiac muscle tissue.

A4.1025

The A4 monoclonal antibody has been tested at concentrations of 1:200 and bound

specifically to its target proteins. This antibody is specific for the myosin heavy chain protein;

this antibody has been successfully used in previous experiments on zebrafish (Bessarab et al.

2008; Hinitis et al. 2009). These proteins are both the fast and slow twitch striated muscle

myosin heavy chain isoforms (Blagden et al, 1997, Scnapp et al. 2009). Specimens stained

with A4 at this concentration had clearly visible fluorescence within the sarcomeres due to the

antibody binding to the myosin bands (Figure 7). Myosin represents the dark banding of the

striations seen under a regular light microscope within skeletal and heart muscle. The bands

of fluorescence are very broad, with small intervening spaces in between, and have similar

fluorescing regions for both 1 and 2 days post fertilization (Figure 7). This localization

pattern is indicative of the width of the myosin protein within each individual sarcomere

(Figure 1).

MF20

MF20 is an anti-myosin antibody. MF20 has been used successfully in prior experiments

on zebrafish (Barresi et al. 2001). In experiments where MF20 was diluted to concentrations

of 1:20, the specimens that had been stained with the antibody displayed a general wash of

fluorescence, which inhibited the proper visualization of the skeletal muscle structure.

However, when the antibody is diluted to concentrations of 1:40 repetitive myosin

localization within the sarcomeres were clearly visible from the antibody binding properly


(Figure 8A and B). This expression pattern is seen as early as 1 day post fertilization (dpf)

and throughout development (data not shown). One thing to note with this antibody as

compared to the A4.1025 anti-myosin antibody or the F59 anti-myosin antibody, it is that the

MF20 localization domain is less broad. (Figures 7 and 8). This may be due to the specificity

and actual target region differences between the two antibodies.

Figure 7. A4.1025 localization to myofibrils within the zebrafish. Myosin localization along the length

of the myofibril in broad domains are seen. Small bars denote 20 um lengths. A) A4.1025 localization at

1 day post fertilization with an objective magnification of 40X. B) A4.1025 localization at 2 day post

fertilization with an objective magnification of 60X.

F59

F59 is another anti-myosin heavy chain antibody. F59 has been used successfully in prior

experiments on zebrafish (Codina et al. 2010, Tallafuss & Eisen, 2008, Hinits et al. 2012). In

experiments where MF20 was diluted to concentrations of 1:200, myosin localization was

repeated as a broad domain within the sarcomeres (Figure 8C and D). This localization

pattern closely mimics the broad pattern seen using the A4.1025 antibody (Figure 7 and

Figure 8C and D).

T11

T11 is an anti-titin antibody that binds to titin filaments present in sarcomeres of striated

muscles. The T11 antibody was used successfully at a concentration of 1:1,000. It has also

been used in zebrafish (Câmara-Pereira et al. 2009). Bands of titin protein were clearly visible

at 2 dpf. (Figure 9A). When we increased the magnification to 180X, we noticed a finer

sublocalization of Titin within the sarcomere. The antibody staining revealed a repeating

doublet of expression within the sarcomere (Figure 9B, arrows). Contrary to myosin patterns,

titin proteins are located on either side of a myosin protein, and are present twice within a

sarcomere (Figure 1). As shown in Figure 9B, the arrowheads point to the doublet expression.


The white arrowheads bracket one domain, sarcomere, of titin localization, and then the

yellow arrowheads denote the next sarcomere. The larger space between the domains is where

myosin proteins reside (Figure 7 and 8 C and D). Experiments using antibodies to T11 in

conjunction with A4.1025 or F59, will resolve this structure. This finer resolution localization

at a higher magnification and may allow for a better understanding of sarcomere assembly.

Figure 8. MF20 and F59 localization to myofibrils within the zebrafish. Myosin localization along the

length of the myofibrils are seen. Notice MF20 has a relatively small region of fluorescence, while F59

has a much broader region of expression similar to A4.1025. Small bars denote 20 um lengths. A)

MF20 localization at day 2 post fertilization with an objective magnification of 40X. B) MF20

localization at 2 day post fertilization with an objective magnification of 60X. C) F59 localization at

day 2 post fertilization with an objective magnification of 40X. D) F59 localization at 2 day post

fertilization with an objective magnification of 60X.


Figure 9. T11 localization to myofibrils within the zebrafish. Titin localization along the length of the

myofibril is seen. Small bars denote 20 um lengths. A) T11 localization at 1 day post fertilization with

an objective magnification of 40X. B) T11 localization at 2 day post fertilization with a total

magnification of 180X. Yellow and white arrowheads denote 1 sarcomere unit with T11 localization on

either end of the sarcomere.

Antibodies That Showed No Specific Localization to Sarcomere Components

4D9

The 4D9 primary antibody was tested at a dilution of 1:300 and failed to bind specifically

to its protein. When viewed under confocal microscopy the stained embryos presented a

general wash of fluorescence that did not yield any focused localization within the zebrafish

either within muscle or within other regions of the zebrafish (Figure 10A). The engrailed gene

products are present within zebrafish and have been visualized within the head regions of

zebrafish (Brand et al. 1996; Knight et al. 2008; Piotrowski & Nüsslein-Volhard, C. 2000).

Currently, we are testing different dilutions of the 4D9 antibody to improve results.

CT3

CT3 was diluted to a concentration of 1:100 but failed to bind specifically to a protein.

Once again embryos stained with such an antibody failed to provide specimens that could be

viewed properly with the confocal microscope (Figure 10B). It has been visualized in

zebrafish heart (Grosskurth et al. 2008). However, we have not seen this localization with our

dilutions. We are currently testing other dilutions of this antibody.

EB165

The EB165 antibody failed to bind specifically within the embryo at a 1:100 or 1:200

dilution (Figure 10C). It has been reported to bind to fast myosin heavy chain isoforms


successful in zebrafish (Ochi & Westerfield, 2009; Walters et al. 2009; Tee et al. 2009).

Further experiments with other dilutions are underway.

Figure 10. Illustration of antibody localization to skeletal muscle. These proteins have given a general,

nonspecific binding to all cells within the developing zebrafish. Small bars denote 20 um lengths. A)

CT3 pattern of localization at day 2 post fertilization with an objective magnification of 40X. B) 4D9

pattern of localization at day 2 post fertilization with an objective magnification of 40X. C) EB165

pattern of localization at day 2 post fertilization with an objective magnification of 40X.

DISCUSSION

The objective of this project was to gain a more definitive view of zebrafish musculature

through the use of monoclonal antibodies that might bind to specific target proteins within the

zebrafish musculature. Examining these antibodies collectively and examining their activity is

useful for building a better picture of zebrafish muscle development.

Out of the antibodies that were tested, three were nonspecific and the others produced

specific staining patterns that could be visualized using confocal microscope. The nonspecific

antibodies produced a general background wash of fluorescence, which inhibited the proper

visualization of muscle structure at the cellular level. The MF20 monoclonal antibody binds

to sarcomere myosin in striated muscle and was tested successfully at concentrations of 1:40.

The use of the MF20 antibody on zebrafish specimens allowed for clear visualization of the

sarcomeres of the skeletal musculature. The A4 monoclonal antibody binds to both fast and

slow twitch striated muscle myosin heavy chain isoforms (Blagden et al, 1997, Scnapp et al.

2009). When zebrafish samples were visualized with this antibody, the sarcomeres were

visible from the A4 antibody binding to myosin bands. The T11 antibody binds to titin

filaments within the sarcomeres of striated muscle, and titin bands were easily visualized in

zebrafish samples that were stained with this antibody.

From this study it is clear that zebrafish musculature can be characterized by the use of

various monoclonal antibodies, which bind to specific proteins in the muscle tissue. The

successful antibodies work well, produce low amounts of background fluorescence, and the


results obtained from whole-mount antibody staining are highly reproducible. These

antibodies can be used in future studies examining the development of zebrafish musculature.

For example, the effects of certain mutations affecting muscle proteins can be compared to

wild-type musculature through a comparative staining. The expression at certain

developmental time periods of specific muscle proteins can also be examined using these

antibodies. Many issues involving the development of zebrafish skeletal muscle can be

addressed using the antibodies screened in this study.

CONCLUSION

Overall, we screened a series of commercially avaliable antibodies that localized to

zebrafish musculature using in fluorscence confocal microscopy. Many of these antibodies

have been published (Brand et al. 1996; Barresi et al. 2001; Bessarab et al. 2008; Câmara-

Pereira et al. 2009; Codina et al. 2010; Hinitis et al. 2009 Hinits et al. 2012; Grosskurth et al.

2008; Knight et al. 2008; Ochi & Westerfield, 2009; Piotrowski & Nüsslein-Volhard, C.

2000; Tallafuss & Eisen, 2008; Tee et al. 2009; Walters et al. 2009), but we verified and

expanded their localization within skeletal muscle. We illustrated differences in the

localization domains between different anti-myosin antibodies, and illustrated a fine detail

localization of the T11, anti-titin antibody. We focused on the protein localization of Slc12a

using the T4 antibody in early zebrafish development and later during muscle formation,

where it may play an important role in ion regulation during muscle activity. Overall, the use

of these antibodies will allow researcher access to tools known to work within the zebrafish

and enable more studies using this organism as a model system for muscle development.

ACKNOWLEDGEMENTS

This research material is based upon workwas supported by National Science Foundation

under grant Grant No. (DBI-0922941)., Research reported in this publication was supported

by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National

Institutes of Health under Award Number 1R15AR055798-01A1. The content is solely the

responsibility of the authors and does not necessarily represent the official views of the

National Institutes of Health. National Institutes of Health Grant #1R15AR055798-01A1

Other funding includes, UTC startup funds and UC Foundation Faculty Research Grant

#R04-1011-088 to EAC. A UTC provost award funds part of the research to LM. Portions of

this chapter are part of research conducted in partial fulfillment of Undergraduate

Departmental Honors projects (LS and LM). Open access publication costs of this chapter

were defrayed though the generosity of Dr. Jeff Elwell, Dean of the College of Arts and

Sciences at the University of Tennessee at Chattanooga.


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