Investigations into the roles of thyroid hormone and

retinoic acid on opsin expression in juvenile rainbow trout

by

Tarek Suliman

B.Sc., Simon Fraser University, 2010

Thesis Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

in the

Department of Biological Sciences

Faculty of Science

Tarek Suliman 2019

SIMON FRASER UNIVERSITY

Summer 2019

ii

Approval

Name: Tarek Suliman

Degree: Master of Science

Title: Investigations into the roles of thyroid hormone and retinoic acid on opsin expression in juvenile rainbow trout

Examining Committee: Chair: Vicki Marlatt Assistant Professor

Iñigo Novales Flamarique Senior Supervisor Professor

Norbert Haunderland Supervisor Professor

Gerhard Gries External Examiner Professor

Date Defended/Approved: March 27, 2019

iii

Ethics Statement

iv

Abstract

Thyroid hormone (TH) and retinoic acid (RA) are powerful modulators of photoreceptor

differentiation during vertebrate retinal development. In the embryos and young juveniles

of salmonid fishes and rodents, TH induces switches in opsin expression within

individual cones, a phenomenon that also occurs in adult rodents following prolonged

(12 week) hypothyroidism. The ability of TH to modulate opsin expression in the

differentiated retina of fish, and the role of RA in inducing opsin switches, if any, is

unknown. Here I investigate the action of TH and RA on single cone opsin expression

and the absorbance of visual pigments in juvenile rainbow trout. Prolonged TH exposure

increased the wavelength of maximum absorbance (λmax) of the rod, and the medium

(M, green) and long (L, red) wavelength visual pigments, and affected single cone opsin

expression in the alevin. RA did not induce any opsin switches nor change the visual

pigment absorbance of photoreceptors.

Keywords: photoreceptor; retina; immunohistochemistry in-situ hybridization; microspectrophotometry

v

Dedication

I’d like to dedicate this thesis to my parents, to my

family, and to all the people who accompanied me

through the fires, floods, weapons, drugs, wild animals

and wild people.

vi

Acknowledgements

I thank Dr. Inigo Novales Flamarique for his continued patience and guidance

during the completion of this work and throughout my academic career. I’d also like to

thank Charlotte Lawson and Steve Arnold (Lower Fraser River trout hatchery,

Abbotsford, British Columbia, Canada) for the rainbow trout.

The microspectrophotometry data used in this thesis was obtained by my

supervisor, Dr. Novales Flamarique.

This thesis uses material previously published in the article Suliman and Novales

Flamarique, 2014, whose final version was drafted by my supervisor.

Suliman T, Novales Flamarique I. (2014). Visual pigments and opsin expression

in the juveniles of three species of fish (rainbow trout, zebrafish, and killifish) following

prolonged exposure to thyroid hormone or retinoic acid. Journal of Comparative

Neurology 522: 98-117.

vii

Table of Contents

Approval .......................................................................................................................... ii Ethics Statement ............................................................................................................ iii Abstract .......................................................................................................................... iv Dedication ....................................................................................................................... v Acknowledgements ........................................................................................................ vi Table of Contents .......................................................................................................... vii List of Tables .................................................................................................................. ix List of Figures................................................................................................................. ix

Chapter 1. Introduction ............................................................................................. 1 1.1. Introduction ............................................................................................................. 1 1.2. Photoreceptors, visual pigments, and opsin expression .......................................... 1 1.3. Thyroid hormone and the expression of thyroid hormone receptors ........................ 2 1.4. Role of retinoic acid in photoreceptor differentiation ............................................... 3 1.5. Mechanism of nuclear receptor action .................................................................... 3 1.6. Applications ............................................................................................................ 4 1.7. The life cycle of rainbow trout ................................................................................. 4 1.8. Aims of the research ............................................................................................... 5

Chapter 2. Investigation into the role of thyroid hormone and retinoic acid in the expression and spectral properties of visual pigments in rainbow trout alevins and smolts ........................................................... 7

2.1. Methods ................................................................................................................. 8 Animals ..................................................................................................... 8 2.1.1. In vivo thyroid hormone and retinoic acid treatments ................................. 8 2.1.2. In-situ hybridization and immunohistochemistry ......................................... 9 2.1.3. Riboprobes and antibodies ...................................................................... 10 2.1.4. Microspectrophotometry .......................................................................... 10 2.1.5.

2.2. Results ................................................................................................................. 11 SWS1 and SWS2 opsin expression in alevin single cone 2.2.1.

photoreceptors ........................................................................................ 11 SWS1 and SWS2 opsin expression in smolt single cone 2.2.2.

photoreceptors ........................................................................................ 13 Spectral properties of alevin and smolt cone photoreceptors ................... 16 2.2.3. TR alpha expression in alevin and smolt cone photoreceptors ................ 20 2.2.4.

2.3. Discussion ............................................................................................................ 21 Effects of thyroid hormone and retinoic acid on the opsin switch in 2.3.1.

differentiated single cones of rainbow trout .............................................. 21 Effect of retinoic acid treatment on opsin expression in rainbow 2.3.2.

trout ......................................................................................................... 22 Effects of thyroid hormone and retinoic acid on visual pigment 2.3.3.

absorbance .............................................................................................. 23 Unknown mechanisms of chromophore shifts in the vertebrate 2.3.4.

retina ....................................................................................................... 25

viii

Chapter 3. Further Discussion ................................................................................ 27 3.1. TH alters the spectral properties of rainbow trout visual pigments by

regulating opsin expression .................................................................................. 27 3.2. Opsin switching in single cones may be an adaptation to the changing

photic environment encountered during out-migration of rainbow trout ................. 27 3.3. Relevance to disorders of the human retina .......................................................... 28 3.4. Future Research ................................................................................................... 28

References ................................................................................................................ 29

ix

List of Tables

Table 2.1. Rainbow trout cone densities and double-to-single cone ratios .............. 13

Table 2.2. Spectral properties of rainbow trout visual pigments .............................. 17

List of Figures

Figure 2.1. Micrographs of tangential sections from the central retina of alevin rainbow trout following in-situ hybridization with the SWS1 and SWS2 riboprobes. .................................................................................. 12

Figure 2.2. Micrographs of tangential sections from the retina of smolt rainbow trout following in-situ hybridization with the SWS1 and SWS2 riboprobes. .................................................................................. 14

Figure 2.3. Micrographs of radial sections from the retina of smolt rainbow trout following in-situ hybridization with the SWS1 and SWS2 riboprobes. ............................................................................................. 15

Figure 2.4 Representative visual pigment absorbance spectra from isolated cone photoreceptors in rainbow trout (each trace is the mean of records from 4-7 cells from 3 fish). ......................................................... 18

Figure 2.5 Representative visual pigment absorbance spectra from isolated rod photoreceptors in rainbow trout (each trace is the mean of records from 7 cells from 3 fish). ............................................................ 19

Figure 2.6. Micrographs of tangential sections from rainbow trout retina following TRα protein immunohistochemistry. ........................................ 20

1

Chapter 1. Introduction

1.1. Introduction

Over the course of their life cycles, migratory Pacific salmon travel between their

natal freshwater lakes and streams to the marine ocean environments where they feed

and live as adults. As newly hatched salmon embryos grow to the juvenile stage, they

undergo physiological changes that prepare the fish for life in the open ocean. These

include new color patterning of the skin, increased tolerance to sea water, and shifting of

the spectral sensitivity of the retina. In this thesis, I explore whether thyroid hormone

plays a role in altering the spectral properties of the retina by modulating gene

expression during the growth of embryos and young juveniles. This research has

implications for understanding color vision, hormone signalling, and cell plasticity in

vertebrates.

1.2. Photoreceptors, visual pigments, and opsin expression

The retina is a multilayered tissue that transduces light into an image-forming

neuronal signal. Photons are captured in the photoreceptor layer, which consists of two

types of specialized neurons: rods and cones. Cone photoreceptors operate during day

light (photic conditions) and enable color vision whereas rod photoreceptors operate in

low-light (scotopic) conditions and are not part of color discrimination pathways.

Morphologically, the majority of cones in the retinas of non-mammalian vertebrates are

either single or double, the latter type consisting of two cones apposed together and

sharing a double membrane partition (Cheng et al, 2006). Rods and cones contain visual

pigments that are expressed in a modified cilium called the outer segment. The spectral

2

type of cone is dictated by the predominant visual pigment that it houses and can be

primarily sensitive to ultraviolet (UV), short-wavelength (S, or blue), middle wavelength

(M, or green) or long wavelength (L, or red) light (Yokoyama, 2000). Most

photoreceptors contain a single visual pigment, but it is possible for a cone to express

multiple visual pigments within the same cell, and for the type of visual pigment in a cone

to change during an animal's life time (Alexander et al., 1994; Cheng et al, 2006, 2007;

Cheng and Novales Flamarique, 2007a; Novales Flamarique, 2005). A visual pigment is

a molecular complex consisting of a protein (opsin) and a chromophore (a vitamin A

derivative). This complex absorbs light to begin the process of phototransduction.

1.3. Thyroid hormone and the expression of thyroid hormone receptors

The nuclear receptor ligands, thyroid hormone and retinoic acid, play crucial

roles during development of photoreceptors in the retinas of several vertebrates (Cvelk

and Wang, 2009; Swaroop et al., 2010; Stevens et al., 2011; Fischer et al., 2011; Weiss

et al., 2012). In salmonid fishes (Cheng et al., 2009; Gan and Novales Flamarique,

2010) and house mice (Roberts et al., 2006; Lu et al., 2009, Glashke et al., 2010) thyroid

hormone controls opsin expression and induces an opsin switch in the embryo or in the

young juvenile. The switch coincides with the transient expression of specific thyroid

hormone receptors (TRs) in the cone photoreceptor cells undergoing the switch: TRβ2 in

rodents and, likely, TRα in salmonid fishes (Roberts et al., 2005; Applebury et al., 2007;

Pessôa et al., 2008; Gan and Novales Flamarique, 2010; Ng et al., 2011). Until recently,

it was thought that the regulation of opsin expression by thyroid hormone was restricted

to the pre-adult stages of these animals, prior to a potential chromatin rearrangement

that would permanently silence transcription (Applebury et al., 2007; Glashke et al.,

2010). A recent study has shown, however, that prolonged (12 week) alteration to the

thyroid hormone metabolism of adult rodents modulates their opsin expression

(Glaschke et al., 2011). Whether such modulation occurs in other vertebrates that

undergo opsin switches is unknown.

3

1.4. Role of retinoic acid in photoreceptor differentiation

Retinoic acid is also known to affect photoreceptor differentiation and opsin

expression in a variety of vertebrates including zebrafish (Hyatt et al., 1996a,b;

Prabhudesai et al., 2005; Stevens et al., 2011), medaka, and mouse (Alfano et al.,

2011). In the retina of embryonic chicken (Mey et al., 1997; Hoover et al., 2001) and

mouse (McCaffery et al., 1992), retinoic acid is present when post-mitotic retinal

progenitor cells differentiate into photoreceptors (Carter-Dawson and Lavail, 1979;

Hoover et al., 1998; Trimarchi et al., 2008) and, later on, when retinoid receptors (RXRγ,

RORα and β) (Hoover et al., 1998; Roberts et al., 2005; Srinivas et al., 2006), along with

TRβ2 (Sjöberg et al., 1992; Ng et al., 2009) and chicken ovalbumin promoter

transcription factors (COUP-TFI and II) (Satoh et al., 2009) determine opsin expression,

at least in the mouse. In addition, retinoic acid promotes the expression of RXRγ, TRβ2,

COUP-TFI, and human L/M opsins in Weri-Rb-1 retinoblastoma cells (Li et al., 2003; Liu

et al., 2007), and activates the mouse Nrl promoter in Y79 retinoblastoma cells and in

cultures of photoreceptors from newborn rat and pig (Khanna et al., 2006). The latter

result provides a mechanism for the involvement of retinoic acid in regulating early

photoreceptor differentiation since Nrl, in combination with other transcription factors like

CRX and NR2e3, commits photoreceptor precursors to the rod fate (Swaroop et al.,

2010). In carp retina, retinoic acid induces the formation of spinules in horizontal cell

dendrites, acting as a potential modulator of visual information beyond phototransduction

(Dirks et al., 2004).

1.5. Mechanism of nuclear receptor action

Thyroid hormone and retinoic acid regulate gene expression by binding to their

nuclear receptors (Glass, 1996; Oetting and Yen, 2007). These receptors activate

transcription by interacting with specific DNA sequences termed response elements. In

the absence of ligand, the receptors couple with co-repressor proteins to silence

transcription from promoters of positively regulated genes to which they bind. In the

presence of ligand, however, a conformational change occurs in the receptors, which

release the co-repressors and bind co-activator proteins promoting transcription (Oetting

and Yen, 2007). Thyroid hormone and retinoic acid receptors (RARs) couple,

4

individually, to DNA as homodimers or, more commonly, as heterodimers in combination

with retinoid X receptors, RXRs (Glass, 1996; Eckey et al., 2003). Retinoid X receptors

bind, preferentially, 9-cis retinoic acid whereas RARs bind 9-cis or all-trans retinoic acid

(Glass, 1996). All-trans retinoic acid appears to be the predominant retinoid in the

developing retina (Hoover et al., 2001) and can bind RXR homodimers to induce 10% or

more of the maximal activation produced by 9-cis retinoic acid (Allenby et al., 1993). As

well, all-trans retinoic acid can bind the retinoid orphan receptor RORβ, depressing its

activity (Stehlin-Gaon et al., 2003).

1.6. Applications

During the last two decades, a major research effort has concentrated on

deciphering the action of thyroid hormone, retinoic acid, and their receptors in gene

regulatory networks that control photoreceptor development (Swaroop et al., 2010).

More recently, there has been interest in exploiting these agents to develop therapies

against congenital disorders of the retina and retinal injuries (Ooto et al., 2004; Mirabella

et al., 2005; Lin et al., 2012). In lower vertebrates, the roles of these ligands and their

receptors in establishing novel photoreceptors at metamorphosis (Hoke et al., 2006) has

received some attention (Mader and Cameron, 2006) but the field remains largely

unexplored. These endeavours, which have clinical (Lin et al., 2012) and industrial (e.g.,

in fish production, Power et al., 2001) applications require an understanding of

photoreceptor genesis and plasticity in the differentiated retina.

1.7. The life cycle of rainbow trout

The fertilized rainbow trout egg develops under the gravel for several months

before hatching as a yolk-sac alevin. Following yolk-sac absorption, the emerging fish,

termed an alevin, will progressively turn into another morph called parr (Groot and

Margolis, 1991). This stage is characterized by vertical marks along the fish's body, and,

during this time, ultraviolet (UV) cones will start transforming into blue cones and some

single (corner) cones will be lost from the ventral retina. During migration to the ocean,

the fresh-water dwelling parr becomes a salt-water tolerant smolt that eventually resides

5

in coastal waters. At this point in time, the majority of UV cones have transformed into

blue cones and corner cones have disappeared from most of the ventral retina (Cheng

et al., 2006, 2007; Cheng and Novales Flamarique, 2007). In some species, like the

sockeye salmon, Oncorhynchus nerka, the smolt will stay for several years in lakes

before migrating to the ocean. In nonanadromous salmonids like the rainbow trout,

Oncorhynchus mykiss, all life stages remain in freshwater.

1.8. Aims of the research

The similarities in opsin switches between rodents (Ng et al., 2001; Roberts et

al., 2006; Glaschke et al., 2010) and salmonid fishes (Cheng et al., 2007; Gan and

Novales Flamarique, 2010) led me to investigate whether opsin expression could be

modulated by thyroid hormone in the differentiated retina of rainbow trout (a salmonid

fish), as was recently demonstrated in the retina of adult rodents (Glaschke et al., 2011).

Retinoic acid could also influence opsin expression by modulating the activity of TR/RXR

heterodimers (Deeb and Liu, 2005). This ligand is known to promote the differentiation of

rods while inhibiting cone differentiation in the retinas of zebrafish and rodents

(McCaffery et al., 1992; Hyatt et al. 1996a,b; Kelley et al., 1999). In human and rat

retinal cell cultures treated with both retinoic acid and thyroid hormone, retinoic acid

induced a dose-dependent differentiation of progenitor cells into rods whereas thyroid

hormone promoted the differentiation of progenitor cells into cones (Kelley et al., 1994,

1995). Despite this evidence suggesting a dynamic equilibrium between both ligands in

determining photoreceptor differentiation and/or opsin expression, nothing is known

about the effects of retinoic acid on opsin expression in the differentiated retina.

In this study, I evaluated the effects of prolonged exposure to thyroid hormone or

retinoic acid on single cone opsin expression and visual pigment absorbance in the

differentiated retinas of juvenile rainbow trout. The goals were twofold: 1) to determine

whether thyroid hormone could act as a modulator of opsin expression or visual pigment

absorbance following the natural switch that occurs in the single cones of rainbow trout,

from SWS1 (ultraviolet, UV) to SWS2 (short wavelength, S) opsin (Cheng and Novales

Flamarique, 2007a), and 2) to determine whether retinoic acid had any effect on opsin

expression and visual pigment absorbance prior to and following the single cone opsin

6

switch. This study contributes to an increasing research effort aimed at understanding

retinal plasticity in the differentiated vertebrate retina.

7

Chapter 2. Investigation into the role of thyroid hormone and retinoic acid in the expression and spectral properties of visual pigments in rainbow trout alevins and smolts

2.1. Introduction

The rainbow trout, like other salmonid fishes, possesses cone photoreceptors that are

arranged in a repeated pattern throughout the retina termed the square mosaic (Cheng

et al., 2006; Cheng and Novales Flamarique, 2007). Two major morphological types of

cones comprise the mosaic: single cones, which have circular cross section, and double

cones, which are composed of two cells apposed together forming an elliptical cross

section at the level of the inner segment (Cheng and Novales Flamarique, 2007). Double

cones form the sides of the square mosaic unit while single cones are located at the

centre of the square and, when present, at the corners. Double cones are M/L cones,

housing a middle wavelength (M) visual pigment in one member and a long wavelength

(L) visual pigment in the other member. Single cones house a UV visual pigment during

early (embryonic) retinal development and then progressively transform to short

wavelength sensitive (S) cones as the animal grows to the smolt stage, at which point in

time the single cones are overwhelmingly S cones. The changes in opsin expression

and visual pigment properties that the cone mosaic undergoes following prolonged

exposure to thyroid hormone or retinoic acid in the larger juvenile are unknown. Given

the roles of these nuclear receptor ligands in regulating opsin expression in the retinas of

several rodents and fishes (Kelley et al., 1994; Hyatt et al., 1996a; Roberts et al., 2006;

Cheng et al., 2009; Gan and Novales Flamarique, 2010; Glashke et al., 2010; Glashke

et al., 2011; Stevens et al., 2011; Mitchell et al., 2015a,b; Stenkamp et al., 2014),

8

treatment with either ligand was expected to change opsin expression or visual pigment

properties in the various spectral cone types of the juvenile rainbow trout retina.

2.2. Methods

Animals 2.2.1.

All experimentation was approved by the Animal Care Committee of Simon

Fraser University, in compliance with guidelines set by the Canadian Council of Animal

Care. Wild stock rainbow trout (Oncorhynchus mykiss) were obtained from the Lower

Fraser Trout hatchery (Abbotsford, British Columbia, Canada). These were either small

juveniles that had recently absorbed their yolk sac, termed alevins (mean weight ± S.D.

= 0.41 ± 0.045 g, mean length ± S.D. = 4.0 ± 0.18 cm, n= 40), or larger, 2 year old,

juveniles, termed smolts (mean weight ± S.D. = 255 ± 31 g, mean length ± S.D. = 28.8 ±

1.77 cm, n= 30).

In vivo thyroid hormone and retinoic acid treatments 2.2.2.

Rainbow trout alevins and smolts were divided into 7 experimental groups, as

follows: (1) global control, (2) thyroid hormone treatment, (3) control for thyroid hormone

treatment, (4) retinoic acid treatment (single dose), (5) control for retinoic acid treatment

(single dose), (6) retinoic acid treatment (twice weekly dose), and (7) control for retinoic

acid treatment (twice weekly dose). The fish were held in separate, but identical, self

contained, tanks with water kept at 7 ºC, and oxygenated using bubbling air stones.

They experienced a 12 h dark: 12 h light regime during the duration of the study.

Global control fish were left untreated for the duration of the experiment. Thyroid

hormone-treated fish were exposed exogenously to L-thyroxine (T4, Sigma) by

dissolving the hormone into 0.1 M NaOH and adding it to the water in the holding tank to

a final concentration of 0.386 * 10-6 M. This treatment was performed every day for two

weeks in the case of alevins, and for 12 weeks in the case of smolts. Retinoic acid-

treated fish were immersed for 30 minutes in water containing all-trans retinoic acid

(Sigma Aldrich) dissolved in 0.1 M dimethyl sulfoxide (DMSO) to a final concentration of

9

10-6 M. This immersion procedure occurred either once, at the beginning of the

experiment, and the fish were left untreated for 6 weeks, or twice every week for a

period of 6 weeks. The retinoic acid immersion took place in separate tanks; the fish

were returned to their holding tanks after each exposure. Control fish for the various

treatments underwent the same handling procedure but exposure was restricted to the

vehicle solution. The exposures occurred in the afternoon, during the photopic part of the

circadian cycle. The treatments and durations were adapted from previous studies (Hyatt

et al., 1992; Prabhudesai et al., 2005; Cheng et al., 2009; Gan and Novales Flamarique,

2010) in which either of these ligands induced changes in opsin expression or

photoreceptor development.

In-situ hybridization and immunohistochemistry 2.2.3.

Fish were sacrificed in the light-adapted state. The left eyeball was removed, the

iris and lens discarded, and the remaining eyecup marked for ventral and nasal

orientation and immersed in cryo-fixative (4% paraformaldehyde in 0.08 M PBS, pH =

7.4). After 24 hr fixation at 4ºC, the retina was extracted from the eyecup, flattened by

making small peripheral incisions, and oriented over a grid with the optic nerve head at

the centre of the grid. This placement procedure ensured the analysis of similar pieces

of retina within and between treatments.

Each retina was then cut into 4 (alevin) or 20 (smolt) pieces. I analyzed all pieces

from the alevin retinas, and four pieces (two dorsal and two ventral) from the smolt

retina. The choice of retinal locations was guided by previous findings of photoreceptor

distributions, opsin expression and/or visual pigments present at the various locations

(Novales Flamarique and Hárosi, 2000; Cheng and Novales Flamarique, 2007a). Retinal

pieces were washed in 0.08 M PBS, cryoprotected in sucrose solution, and embedded in

O.C.T. frozen blocks for in-situ hybridization or immunohistochemistry, as detailed in

previous studies (Cheng et al., 2006; Gan and Novales Flamarique, 2010; Novales

Flamarique, 2011).

Sections (7-10 μm thick) obtained from the blocks were collected serially and

deposited on alternating slides for parallel or joint processing with rainbow trout-specific

10

SWS1 and SWS2 riboprobes. The sections were then incubated with anti-DIG Fab

fragments conjugated to alkaline phosphatase and the label visualized with NBT/BCIP.

To detect the presence of TRα protein, rainbow trout retina sections were incubated with

a rabbit anti-TRα1 antibody (Affinity BioReagents; 1:100 dilution in PBST), followed by a

goat anti-rabbit IgG conjugated to alkaline phosphatase, and the label visualized with

NBT/BCIP (Gan and Novales Flamarique, 2010).

For each retinal location analyzed, the density of spectrally-defined single cone

types and that of double cones was counted over an area ranging from 5760 to 23040

μm2 using a grid system on the computer monitor. From these measures, the ratio of

double to single cones was computed. Digital images of sections were acquired with an

E-600 Nikon microscope equipped with a DXM-100 digital camera and DIC or

fluorescence optics.

Riboprobes and antibodies 2.2.4.

The SWS1 and SWS2 riboprobes used have been described previously (Cheng

et al., 2006). In salmonid fishes, these riboprobes specifically label the UV and S single

cones, respectively (Cheng et al., 2006; Cheng and Novales Flamarique, 2007a,b).

The TRα1 antibody recognizes part of the rainbow trout TRα transactivation motif

(amino acids 212-218; GenBank accession number AAD30058), a conserved region of

the TRα ligand binding domain (Jones et al., 2002). The specificity of this antibody for

the TRα isoform has been confirmed previously (Mader and Cameron, 2006). In rainbow

trout alevins, this antibody labels a sub-population of single cones at the time of the

SWS1-to-SWS2 opsin switch (Gan and Novales Flamarique, 2010).

Microspectrophotometry 2.2.5.

Individual fish from the various treatments were dark adapted overnight.

Following this adaptation period, the fish was killed, one eye enucleated, and the retina

removed under infrared illumination. Small pieces of retina were teased apart and

prepared for viewing with the dichroic microspectrophotometer (DMSP) as per previous

studies (Hárosi, 1987; Novales Flamarique and Hárosi, 2000, 2002). The DMSP is a

11

computer-controlled, wavelength-scanning, single-beam photometer that simultaneously

records average and polarized transmitted light fluxes through microscopic samples

(Hárosi, 1987; Novales Flamarique and Hárosi, 2002). The DMSP was equipped with

ultrafluar (Zeiss) objectives: 32/0.4 for the condenser and 100/1.20 for the objective.

With the aid of reference measurements recorded through cell-free areas, individual

photoreceptor outer segments were illuminated sideways with a measuring beam of

rectangular cross section of ca. 2 x 0.6 µm. Absolute absorbance spectra were

computed in 2 nm increments from the obtained transmittances (each spectrum

consisted of an average of 8 scans). The solid spectra (fits) were derived from

experimental data by Fourier filtering (Hárosi, 1987).

2.3. Results

SWS1 and SWS2 opsin expression in alevin single cone 2.3.1.photoreceptors

Regardless of whether the alevins were left untreated, or were treated

with the vehicle solution (0.1 M NaOH or DMSO), all single cones in the

main, non-peripheral growth zone area of the retina of control fish labelled with

the SWS1 riboprobe (Figure 2.1 A) and none labelled with the SWS2 riboprobe

(Figure 2.1 B). Following two weeks of thyroid hormone treatment, none of

the single cones labelled with the SWS1 riboprobe (Figure. 2.1 C) and all labelled

with the SWS2 riboprobe (Figure 2.1 D) confirming the opsin switch in

single cones demonstrated in previous studies (Cheng et al., 2006; Cheng

and Novales Flamarique, 2007a). Regardless of the frequency of treatment

with retinoic acid, all single cones labelled with the SWS1 riboprobe

(Figure 2.1 E) and none with the SWS2 riboprobe (Figure 2.1 F) after 6 weeks

of treatment. The same results were obtained after two weeks of treatment

(data not shown). Double cones did not label with any of the riboprobes,

as expected since these cones are M/L pairs that express RH2 opsin in

one member and LWS opsin in the other (Cheng and Novales Flamarique,

2007a). Cone densities were statistically the same between treatments (Table 2.1)

12

Figure 2.1. Micrographs of tangential sections from the central retina of alevin rainbow trout following in-situ hybridization with the SWS1 and SWS2 riboprobes.

Note: A,B: All the single cones in the control label with the SWS1 riboprobe (black arrows, A) and none label with the SWS2 riboprobe (black arrowheads, B). White arrowheads point to the partitioning membrane that separate the two members of a double cone, none of which label with either riboprobe. A white asterisk denotes the location of a corner cone; these cones face the partitions of neighbouring double cones. C,D: Following two weeks of thyroid hormone exposure, none of the single cones label with the SWS1 riboprobe (black arrowheads, C) but all label with the SWS2 riboprobe (white arrows, D). E,F: After 6 weeks of retinoic acid exposure, all single cones label with the SWS1 riboprobe (black arrows, E) and none label with the SWS2 riboprobe (black arrowheads, F). Scale bar = 10 μm in A applies to all panels.

13

Table 2.1. Rainbow trout cone densities and double-to-single cone ratios

Fish Treatment Dorsal density Ventral density Dorsal d/s Ventral d/s

alevin Control 19740 (1398) 27649 (1445) 0.99 (0.018) 1.02 (0.014)

Alevin thyroid hormone 19515 (1703) 26984 (2035) 1.01 (0.026) 1.03 (0.023)

Alevin retinoic acid 20012 (1524) 26908 (1773) 1.01 (0.035) 1.00 (0.018)

Smolt Control 6353 (573) 7140 (421) 1.02 (0.034) 2.08 (0.16)

Smolt thyroid hormone 6557 (721) 6989 (436) 1.04 (0.041) 2.01 (0.12)

Smolt retinoic acid 6249 (819) 7205 (547) 1.05 (0.055) 2.04 (0.085)

Statistics of cone densities and double to single cone ratio (d/s) from the mid-dorsal and mid-ventral retina (n=6). Densities are in thousands per mm2. Each mean has a standard deviation (in parenthesis) associated with it. The means were not significantly different between treatments at α = 0.05.

SWS1 and SWS2 opsin expression in smolt single cone 2.3.2.photoreceptors

In rainbow trout smolts, regardless of treatment, none of the singles

cones from non-peripheral retina labelled with the SWS1 riboprobe

(Figure 2.2 A,D,G; Figure. 2.3 A,C,E) and all labelled with the SWS2 riboprobe

(Figure. 2.2 B,C,E,F,H,I; Figure. 2.3 B,D,F). None of the double cones labelled

with either riboprobe, and cone densities were statistically the same between

treatments (Table 2.1). The dorsal retina had corner cones (Figure. 2.2 B,E,H)

whereas these were primarily absent from the ventral retina (Figure 2.2 C,F,I).

The loss of corner cones from the ventral retina of rainbow trout is a gradual

process that begins at the alevin stage and results in their restricted presence

to the dorsotemporal retina of the smolt (Figure 2.2) and of the reproductive adult

(Cheng et al., 2006; Cheng and Novales Flamarique, 2007a,b). In radial sections,

the label was restricted to the myoid region of the single cones (Figure 2.3 B,D,F),

as has been observed previously (Cheng et al., 2006; Cheng and

Novales Flamarique, 2007a).

14

Figure 2.2. Micrographs of tangential sections from the retina of smolt rainbow trout following in-situ hybridization with the SWS1 and SWS2 riboprobes.

Note: Whether the fish was a control (A-C), or had been treated with thyroid hormone for 12 weeks (D-F), or with retinoic acid for 6 weeks (G-I), none of the single cones labeled with the SWS1 riboprobe (A,D,G) but all labeled with the SWS2 riboprobe. This labeling pattern was present both in the dorsal (B,E,H) and ventral (C,F,I) retina. The dorsal retina was characterized by a full mosaic with corner cones whereas the ventral retina lacked the majority of corner cones (see asterisk in I for an unusual corner cone occurrence). Symbols and nomenclature as in Figure 1. Scale bar = 10 μm in A applies to all panels.

15

Figure 2.3. Micrographs of radial sections from the retina of smolt rainbow trout following in-situ hybridization with the SWS1 and SWS2 riboprobes.

Note: In the light-adapted retina, the single cones are displaced vitreally with respect to the double cones. As per the results in Figure 2, none of the single cones labeled with the SWS1 riboprobe (A,C,E) and all labeled with the SWS2 riboprobe (B,D,F), regardless of treatment. The label was concentrated in the myoid region of each cell, as expected from the perinuclear location of the Golgi apparatus. Symbols and nomenclature as in Figure 1. Scale bar = 10 μm in A applies to all panels.

16

Spectral properties of alevin and smolt cone photoreceptors 2.3.3.

Absorbance measurements from the various cone photoreceptors

of alevin controls showed that the single cones had a UV visual pigment, and

the double cones were M/L pairs, one expressing an RH2 opsin, the other an

LWS opsin (Figure 2.4 A; Table 2.2). After two weeks of thyroid

hormone treatment, however, the single cones had an S visual pigment,

and the wavelength of maximum absorbance (λmax) of the M and L photopigments

had significantly shifted toward longer wavelengths (Figure 2.4 B; Table 2.2).

The bandwidths at half maximum absorbance (HBW) of the M and L visual

pigments from thyroid hormone treated fish were also greater compared with

those of controls (Table 2.2). In contrast, retinoic acid exposure resulted in visual

pigments that had statistically identical spectral characteristics to those of controls

(Figure 2.4 C, Table 2.2).

Similar measurements from the cone photoreceptors of smolt controls

showed that the single cones had an S visual pigment and the double cones

were M/L pairs (Figure 2.4 D; Table 2.2). As in the alevin, thyroid hormone

significantly increased the λmax of the M and L visual pigments, but did not

change the spectral characteristics of the S visual pigment, compared to that

of controls (Figure 2.4 E; Table 2.2). The visual pigment characteristics of

retinoic acid treated fish were the same as those of controls (Figure 2.4 F; Table 2.2).

The λmax of the rod visual pigments from thyroid hormone treated alevins

(Figure 2.5 A) or smolts (Figure 2.5 B) were significantly greater than those

of controls, and the HBWs were overall greater as well (Table 2.2). In contrast,

the λmax and HBW of rod visual pigments from retinoic acid treated fish were

statistically the same as those of controls (Figure 2.5 A,B; Table 2.2).

17

Table 2.2. Spectral properties of rainbow trout visual pigments

Stage Treatment Cone type λmax (±S.D.) HBW (±S.D.) N

Alevin Control UV 382 (6.28) 6014 (684) 9

Alevin Control M 500 (5.77) 4531 (396) 24

Alevin Control L 563 (6.12) 3688 (391) 25

Alevin Control Rod 508 (4.55) 4524 (318) 14

Alevin thyroid hormone S 435 (6.09) 4915 (508) 15

Alevin thyroid hormone M 545 (8.31)* 4647 (445) 24

Alevin thyroid hormone L 625 (11.4)* not available 20

Alevin thyroid hormone Rod 528 (7.34)* 4676 (335) 13

Alevin retinoic acid UV 379 (6.15) 6163 (702) 11

Alevin retinoic acid M 499 (6.68) 4533 (411) 17

Alevin retinoic acid L 566 (5.89) 3801 (437) 21

Alevin retinoic acid Rod 508 (5.63) 4512 (375) 16

Smolt Control S 434 (5.65) 5017 (568) 15

Smolt Control M 521 (5.19) 4304 (402) 34

Smolt Control L 564 (7.03) 3869 (387) 30

Smolt Control Rod 509 (3.58) 4410 (371) 35

Smolt thyroid hormone S 435 (5.09) 5090 (576) 13

Smolt thyroid hormone M 553 (7.12)* 4471 (485) 25

Smolt thyroid hormone L 628 (8.76)* not available 22

Smolt thyroid hormone Rod 527 (6.92)* 4904 (433) 27

Smolt retinoic acid S 432 (4.10) 5041 (492) 17

Smolt retinoic acid M 520 (5.74) 4295 (383) 38

Smolt retinoic acid L 565 (6.78) 3789 (321) 38

Smolt retinoic acid Rod 510 (4.87) 4518 (318) 31

Statistics of visual pigments in rainbow trout. Each computed mean has a standard deviation (in parenthesis) associated with it. Abbreviations: λmax is the wavelength of maximum absorbance, HBW is the bandwidth at half maximum absorbance, and n is the number of photoreceptors examined. The asterisks denote means that are significantly different at α = 0.05 from those of the other, corresponding, treatments. The HBW of the L visual pigment in thyroid hormone-treated fish could not be discerned due to a lack of data beyond 650 nm. UV, S, M, L and rod photoreceptors express SWS1, SWS2, RH2, LWS, and RH1 opsins, respectively.

18

Figure 2.4. Representative visual pigment absorbance spectra from isolated cone photoreceptors in rainbow trout (each trace is the mean of records from 4-7 cells from 3 fish).

Note: In both the alevin (A-C) and the smolt (D-F), thyroid hormone treatment (B,E) significantly increased the wavelength of maximum absorption (λmax) of the M and L visual pigments in comparison with those from control (A,D) and retinoic acid-treated (C,F) fish. The M and L visual pigments were found in double cones, one per double cone member. In the alevin, single cones of control and retinoic acid-treated fish had the same UV visual pigment, whereas those of thyroid hormone treated fish had an S visual pigment. In the smolt, single cones from all treatments had the same S visual pigment. See Table 1 for visual pigment statistics. The embedded photographs show representative fish from each stage and treatment. Scale bar = 2 cm in A (applies to panels A-C) and 14.5 cm in D (applies to panels D-F).

19

Figure 2.5. Representative visual pigment absorbance spectra from isolated rod photoreceptors in rainbow trout (each trace is the mean of records from 7 cells from 3 fish).

Note: The λmax of the rod visual pigment in thyroid hormone-treated alevin (A) and smolt (B) fish was significantly greater than that from control and retinoic acid-treated fish. See Table 1 for visual pigment statistics.

20

TR alpha expression in alevin and smolt cone 2.3.4.photoreceptors

As reported previously (Gan and Novales Flamarique, 2010), we found that TRα

was expressed by a subpopulation of corner cones in the alevin (Figure 2.6 A).

Expression of TRα was, however, absent from the cone photoreceptors of smolts,

regardless of treatment (Figure 2.6 B-D).

Figure 2.6. Micrographs of tangential sections from rainbow trout retina following TRα protein immunohistochemistry.

Note: A: Expression of TRα by a single, corner, cone (black arrow) in the retina of control alevin. B-D: TRα was not detected in cones of the smolt retina irrespective of treatment. Black arrowheads point to unlabelled single cones in control (B), thyroid hormone treated (C), and retinoic acid-treated (D) smolt. Other symbols and nomenclature as in Figure 1. Scale bar = 10 μm in A applies to all panels.

21

2.4. Discussion

Effects of thyroid hormone and retinoic acid on the opsin 2.4.1.switch in differentiated single cones of rainbow trout

Previous research had demonstrated a switch in opsin expression of rainbow

trout single cones from SWS1 to SWS2 in the differentiated retina of the alevin (Cheng

et al., 2006; Cheng and Novales Flamarique, 2007a). This switch could be induced

prematurely with thyroid hormone but only before the natural opsin switch had taken

place, a process that begins in the alevin and ends in the young smolt (Cheng et al.

2007, 2009). The single cone opsin switch proceeds from the ventral to the dorsal retina

in a pattern that correlates with a gradient of TRα expression in the single cone

population (Gan and Novales Flamarique, 2010). Two weeks of thyroid hormone

exposure was insufficient to modulate opsin expression in the single cones of the smolt,

none of which expressed TRα (Gan and Novales Flamarique, 2010). These findings

resembled previous literature on adult mouse where two weeks of hypothyroidism failed

to alter opsin expression (Applebury et al., 2007).

More recently, a study using adult house mice and brown Norway rats showed

that a longer exposure time (minimum of 5-7 weeks) was necessary to alter circulating

levels of thyroid hormone in adult rodents and change their opsin expression, as

assessed after 12 weeks of treatment (Glaschke et al., 2011). Based on this study and

previous work claiming a reversal in spectral sensitivity, from S to UV, of rainbow trout

smolts subjected to 6 weeks of thyroid hormone treatment (Browman and Hawryshyn,

1994a), we surmised that a 12 week exposure would induce an opsin switch reversal in

the single cones from SWS2 to SWS1. This was not observed (Figures 2.2, 2.3) and

neither was the presence of TRα receptors in the single cones (Figure.2.6) suggesting a

permanent state of transcriptional silencing following the natural opsin switch. There are

two additional lines of evidence that support this conclusion. First, reproductive adults,

which undergo a surge in thyroid hormone during maturation and a reduction following

reproduction (Holloway et al., 1999) have single cones that express SWS2 opsin

transcript exclusively (Cheng and Novales Flamarique, 2007a,b). Second, following the

process of smoltification, smolts have reduced circulating levels of thyroid hormone

22

(Alexander et al., 1994), yet a reversal to SWS1 opsin expression in singles cones does

not occur (Figures 2.2, 2.3; Novales Flamarique, 2005; Cheng et al., 2007a). In rodents,

there are persistent levels of TRβ2 receptor in the adult retina and induced

hypothyroidism leads to a reversal in opsin expression from M/LWS to SWS1 (Glaschke

et al. 2011). There is therefore greater plasticity in opsin expression in the differentiated

retina of rodents compared to that in salmonid fishes.

Effect of retinoic acid treatment on opsin expression in 2.4.2.rainbow trout

Previous studies reporting spectral sensitivity changes in rainbow trout as a

function of TH (Browman and Hawryshyn, 1992, 1994a) or RA (Browman and

Hawryshyn, 1994b) suffer from confounding variables due to the use of fish that were

naturally undergoing the SWS1 to SWS2 opsin switch, inconsistent illumination of the

retina, and insufficient resolution of the histology (Beaudet et al. 1997; Novales

Flamarique, 2001; Cheng and Novales Flamarique, 2007a). More recent reports on the

loss and regeneration of corner cones following thyroid hormone treatment (Hawryshyn

et al., 2003; Allison et al., 2006) have been proven self-contradictory and unreliable

(Cheng and Novales Flamarique, 2007a,b; Cheng et al. 2009). The findings in this thesis

show that modulation of opsin expression by thyroid hormone can only be achieved prior

to the natural opsin switch (Figures 2.1, 2.2). This is inconsistent with the reported

change in spectral sensitivity of smolts, from S to UV, measured under a long

wavelength adapting background, following 6 weeks of thyroid hormone treatment

(Browman and Hawryhsyn, 1994a).

In contrast to the action of thyroid hormone at the alevin stage, I found no effect

of retinoic acid on opsin expression at the alevin or smolt stages (Figures 2.1, 2.2). This

result stands in contrast to the spectral sensitivity changes from UV to S, and from S to

UV, reported for rainbow trout parr and smolt, respectively, following a single 20 minute

exposure to retinoic acid and assessment after 6 weeks (Browman and Hawryshyn,

1994b). As per the thyroid hormone studies (Browman and Hawryshyn, 1992, 1994a),

the retinoic acid results on pre-smolt fish can be explained by differential illumination of

the retina and the use of fish that were at an intermediate stage between the alevin and

23

smolt, termed parr. The single cone opsin switch is well under way in parr fish (Cheng et

al., 2009) such that it is impossible to compare results between treatments or individuals

within a treatment, especially when these vary widely in size (8-32g; Browman and

Hawryshyn, 1992, 1994a,b).

The results reported for smolts (fish weighing ~ 60-120g; Browman and

Hawryshyn, 1994a,b) are puzzling in that I do not observe any changes in single cone

opsin expression or visual pigment absorbance to substantiate the reported changes in

spectral sensitivity. One possible explanation could be the use of smaller smolts by

Browman and Hawryshyn (1994a,b), compared to those in the present study, some of

which may have had a residual population of UV cones in the dorso-temporal retina. The

large variation in size of smolts used by Browman and Hawryshyn (1994a,b) could

explain their results (Novales Flamarique, 2001). Spectral sensitivity experiments on

smolts should be repeated using fish that have completed the UV to S single cone

transformation. Based on the present findings, I predict that neither thyroid hormone nor

retinoic acid will alter spectral sensitivity of smolts due to the action of single cones.

Effects of thyroid hormone and retinoic acid on visual 2.4.3.pigment absorbance

Regardless of the rainbow trout stage examined, thyroid hormone significantly

increased the λmax of the M, L and rod visual pigments (Table 2.2). Thyroid hormone did

not affect the spectral characteristics of the S visual pigment, suggesting different

mechanisms of visual pigment regulation between cone types in the differentiated retina.

In accordance with the opsin distributions observed using molecular markers, retinoic

acid did not affect the visual pigments in single cones (UV or S), nor those in double

cones (M or L), or the opsins in rods (Table 2.2).

The absorbance of a visual pigment is determined by the opsin expressed and

the chromophore type that the opsin is conjugated to. For a given opsin, conjugation to

the vitamin A1-derived chromophore, 11-cis retinaldehyde, results in a lower λmax than

conjugation to the vitamin A2-derived chromophore, 11-cis 3,4-dehydroretinaldehyde

(Isayama and Makino, 2012). To assess whether the production of new opsin types

could partly explain the absorbance of visual pigments following thyroid hormone

24

treatment, the λmax limits of vitamin A1-A2 conjugated opsin pairs were computed using

semi-empirical mathematical models (Hárosi, 1994). It was assumed that the visual

pigments of control fish represent the absorbance of 11-cis retinaldehyde conjugated

opsins even though some cones may have contained a mixture of chromophores

(Hárosi, 1994). This assumption did not alter the validity of the analysis nor the overall

conclusions.

Control alevin rainbow trout had visual pigments with mean λmax at 382 nm (UV),

500 nm (M), 563 nm (L), and 508 nm (rod) (Table 2.2). Assuming that these values

reflect the absorbance of 11-cis retinaldehyde conjugated opsins, the corresponding 11-

cis 3,4-dehydroretinaldehyde pairs are computed to be (Hárosi, 1994): 394 nm (UV), 525

nm (M), 632 nm (L), and 537 nm (rod). The change in mean photopigment λmax of single

cones following thyroid hormone treatment (435 nm; Table 2.2) was beyond the

predicted limit for a UV visual pigment, indicating a switch in opsin expression. Indeed,

the in-situ hybridization results demonstrated a switch in opsin from SWS1 to SWS2 in

the single cones (Figure 2.1). With the exception of the M visual pigment, the λmax of the

other photopigments following thyroid hormone treatment (Table 2.2) were within the

predicted ranges of 11-cis 3,4-dehydroretinaldehyde conjugated opsins. Thus, the

changes in λmax could be explained by visual pigment chromophore shifts from 11-cis

retinaldehyde to 11-cis 3,4-dehydroretinaldehyde. In contrast, the thyroid hormone-

induced shift in λmax of the M visual pigment was ~20 nm beyond the maximum predicted

for the corresponding 11-cis 3,4-dehydroretinaldehyde conjugated opsin, suggesting the

expression of another RH2 opsin. This result is in line with findings from a study on coho

salmon parr where thyroid hormone treatment for four weeks revealed changes in M

cone absorbance indicative of expression of two RH2 opsins (Temple et al., 2008).

In rainbow trout smolt controls, the mean λmax of the visual pigments, assumed to

be 11-cis retinaldehyde-dominated, were: 434 nm (S), 521 nm (M), 564 nm (L), and 509

nm (rod) (Table 2.2). The predicted 11-cis 3,4-dehydroretinaldehyde corresponding pairs

are: 441 nm (S), 557 nm (M), 634 nm (L), and 538 nm (rod). Thus, the mean absorbance

λmax of each photopigment following thyroid hormone treatment (Table 2.2) fell within the

upper limit set by the predicted 11-cis 3,4-dehydroretinaldehyde opsin conjugate.

Although no expression of new opsin types need be invoked to explain the changes in

25

λmax observed, it is likely that a shift in RH2 opsin expression in favour of the longer

wavelength isoform occurred in the smolt. This is suggested by the higher mean λmax of

the M visual pigment found in thyroid hormone-treated smolts (553 nm) compared to

alevins (545 nm) (Table 2.2).

Mechanisms of chromophore shifts in the vertebrate retina 2.4.4.

An in vitro study using retinal pigment epithelium cells extracted from coho

salmon showed that thyroid hormone exposure altered the production of didehydro

retinoids (Alexander et al., 2001). In zebrafish and amphibians, a cytochrome P450

enzyme of previously unknown function was shown to catalyze the conversion of vitamin

A1 retinoids to vitamin A2 retinoids in a TH-dependant manner (Enright et al. 2015).

Cyp27c1 is a 3,4-dehydrogenase that adds a double bond to the β-ionone ring of retinol

(vitamin A1) to yield 3,4-didehydroretinol (vitamin A2). Cyp27c1 is expressed in the

retinal pigment epithelia of zebrafish and the American bullfrog and is upregulated by TH

treatment (Enright et al. 2015). Catalysis of retinoids by cyp27c1 may be a possible

mechanism for the spectral tuning of chromophores in salmon.

First studied in zebrafish (Enright et al., 2015), cyp27c1 and its analogues have

been found in cichlids, birds, humans, and lampreys. The human analog, hP450 27C1,

is expressed as mRNA in the kidney, liver and skin, but the functional enzyme is only

detected in the keratinocytes of the skin and is not known to play a role in vision. In

humans the enzyme catalyzes the production of all-trans retinol (vitamin A1) to all-trans

3,4-dehydroretinol (vitamin A2) and may have aid in protecting the skin from UV damage

(Johnson et al., 2017; Kramlinger et al., 2016). Birds have an apocarotenoid metabolism

with a cyp27c1 analogue that allows for conversion between galloxanthin and 11’,12’-

dihydrogalloxanthin. The apocarotenoids, found in the oil droplet of the avian cone

photoreceptor, act as tunable spectral filters that contribute to the bird’s ability to

discriminate color (Toomey et al., 2016). Populations of cichlids that have been

separated by as little as 2000 generations have shown differences in expression of

cyp27c1 genes that reflect responses to their varied photic environments (Härer et al.,

2018). The lamprey’s spectral sensitivity red shifts as it matures and migrates to a new

photic environment. The change in the lamprey's spectral sensitivity is achieved through

26

the switching of visual pigment chromophores from 11-cis retinol to 3,4 didehydroretinol,

which occurs concomitantly with an increase in cyp27c1 expression in the retinal

pigment epithelium. Cyp27c1 mediating chromophore ratios as part of an evolutionarily

conserved spectral tuning mechanism may have Cambrian origins as it is observed in

lamprey as well as fish and humans (Morshedian et al., 2017).

27

Chapter 3. Further Discussion

3.1. TH alters the spectral properties of rainbow trout visual pigments by regulating opsin expression

My findings are that TH triggers a UV-to-blue opsin switch in alevins, but older

juveniles that have completed smoltification are not affected by prolonged exposure to

TH. I also found that RA did not induce a change in opsin expression in alevins or

smolts.

A UV-to-blue opsin switch was detected using in-situ hybridization and

corresponds to the shift from UV to S visual pigment observed using

microspectrophotometry, confirming previous findings that TH modulates opsin

expression in alevin rainbow trout.

TH treatment also altered the spectral properties of the M visual pigment beyond

what would be predicted by a chromophore change alone, suggesting a switch to a

novel RH2 opsin isoform.

3.2. Opsin switching in single cones may be an adaptation to the changing photic environment encountered during out-migration of rainbow trout

Rainbow trout have a pelagic existence in surface waters as young juveniles, and

then progress to life in deeper waters following metamorphosis (Evans and Fernald,

1990; Novales Flamarique, 2005). This is accompanied by the loss of UV visual

pigments, and their replacement by longer wavelength photopigments that are better

28

adapted to sense the light spectrum in deeper waters (Forsell et al., 2001; Cheng and

Novales Flamarique, 2004; Cheng et al., 2006). The switch from SWS1 to SWS2 opsin

that occurs in the single cones of rainbow trout is likely an adaptation that optimizes

feeding on zooplankton by alevins and on larger, opaque prey by smolts (Novales

Flamarique, 2013).

3.3. Relevance to disorders of the human retina

TH is required for the development of normal vision in humans. Infants with

congenital hypothyroidism, or who had mothers with hypothyroidism, have deficits in

contrast sensitivity (Mirabella et al., 2005). TRβ2 mutations in humans causes a

decrease in M and L cone function and an increase in S cone function, indicating that

TRβ2 signaling is required for normal cone cell differentiation (Weiss et al., 2012).

Endogenous TH exposure upregulates M and L opsin targets in human WERI cells (Liu

et al., 2007). Mammalian S opsin and salmonid UV opsin are derived from an ancestral

short wavelength sensitive opsin (SWS1) and are both regulated by TH signaling during

growth and development. The ability of TH to influence opsin expression in salmonid

fishes may generate new insight into nuclear receptor signaling and the gene regulatory

networks involved in photoreceptor retinopathies.

3.4. Future Research

Receptor subtype-specific agonists and antagonists could be used to determine

which TR subtype is responsible for changes in opsin expression.

There was a TH-induced shift detected in the M visual pigment of rainbow trout

photoreceptors. It is possible that the shift in visual pigment absorbance corresponds to

the expression of a novel medium-wavelength-sensitive opsin protein. If riboprobes were

generated that were specific to the putative M opsin, then it would be possible to confirm

the existence of a TH-induced opsin switch in rainbow trout double cones.

29

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