K-Cadherin) Function in the Development Of

CADHERIN-6 (K-CADHERIN) FUNCTION IN THE DEVELOPMENT OF

ZEBRAFISH (Danio rerio) PHOTORECEPTORS

A Thesis

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Thomas Mbimba

August, 2007

CADHERIN-6 (K-CADHERIN) FUNCTION IN THE DEVELOPMENT

OF ZEBRAFISH (Danio rerio) PHOTORECEPTORS

Thomas Mbimba

Thesis

Approved: Accepted:

______Advisor: Department Chair Dr. Qin Liu Dr. Bruce Cushing

______Committee Member Dean of the College Dr. Richard Londraville Dr. Ronald F. Levant

______Committee Member Dean of the Graduate School Dr. Dan Ely Dr. George R. Newkome

______Date

ACKNOWLEDGMENTS

I would like to thank everyone in the Biology Department who helped in any way

possible in the completion of this project. I particularly appreciate all the work done by my advisor Dr. Qin Liu, and I thank my committee members for their guidance and time in the completion of the manuscript.. My thanks to everyone one in the Biology

Department for making my stay possible and comfortable. And finally, I dedicate this work to my family, parents and siblings in thanks for their support.

iii

TABLE OF CONTENTS Page

LIST OF FIGURES…………………………………………………….……….….v

CHAPTER

I. INTRODUCTION………………………………………………….…………….1

II. LITERATURE OVERVIEW……………………………………………………3

III. MATERIALS AND METHODS…………………………………...... 16

IV. RESULTS……………………………………………………………...... 22

V. DISCUSSION……………………..………………………………………….….32

VI. CONCLUSION………………………………………………………………….36

REFERENCES………………………………………………………………….…...37

APPENDIX: ANIMAL CARE AND USE PROTOCOL APPROVAL…...... 42

iv LIST OF FIGURES

Figure Page

1. Phylogenetic representation of cadherin sub-families………………………….5

2. Domain organization of representative members of the cadherin superfamily……………………………………………………...………………6

3. Representation of classic cadherin molecules at the cell surface………………..7

4. Cadherin calcium binding…………………………………………...... 9

5. Cadherin mechanism for calcium mediated assembly ……………...... 9

6. Expression of crx marker in control and cdh6 morphants ……………………...23

7. Expression of otx5 marker in control and cdh6 morphants.………...... 24

8. Expression of rho marker in control and cdh6 morphants .……………………..26

9. Expression of gnat1 marker in control and cdh6 morphants ……………………27

10. Expression of zpr1 marker in control and cdh6 morphants …..…………………28

11. Expression of gnat2 marker in control and cdh6 morphants ……………………29

12. Expression of uvo marker in control and cdh6 morphants ….……...... 30

13. Expression of irbp marker in control and cdh6 morphants.………...... 31

CHAPTER I

INTRODUCTION

During embryogenesis, tissue differentiation and organ development require cells expressing specific molecules that cue migration, aggregation and differentiation. One group of molecules involved in such processes is cadherin molecules, which are defined as calcium dependant adhesion molecules. They were first identified in the early 1980s by Dr. Takeichi of the Riken Kobe Institute in Japan. Since the discovery of the first cadherin molecule, numerous cadherins molecules have been isolated and studied from both vertebrate (human, frogs, rats, mice, and more recently zebrafish) and invertebrate such as fruit flies (Drosophilia).

Cadherin molecules play an important role in tissue differentiation and organ

development. In adults, cadherins help in maintaining tissue integrity, and also play a

role in tissue repair (Liu et al., 2002). Additionally, numerous studies have shown that

cadherins are involved in cancer development and progression (Babb et al., 2001). Based

on these diverse and important physiological roles of cadherin molecules, one can argue

that the study of such proteins is crucial for understanding animal development and

function.

There are different approaches to study gene function in tissue and organ

development. One approach is to downregulate or to eliminate the cadherin expression in

1 the organism, followed by assessing the effect of blocking this cadherin function on development of the target tissue or organ (loss-of-function approach).

This study is intended to investigate the role of cadherin-6 in the development of zebrafish pineal gland and retinal photoreceptors. Cadherin expression in the central nervous system (CNS) has been investigated in various vertebrate animals, but there is little information on cadherin function in retinal photoreceptor differentiation, and scarcer knowledge on cadherin function in the pineal gland development. To achieve this goal, I will use zebrafish as my model organism to study cadherin-6 function in the formation of photoreceptors in these structures. Zebrafish have been used as a model organism for studying vertebrate development due mainly to their many experimental advantages including transparency of the embryos, routine production of large numbers of eggs by females, fast embryonic growth, and their demonstrated utility as genetic model.

CHAPTER II

LITERATURE REVIEW

Cell Adhesion Molecules and Cadherin Molecules

Cell adhesion molecules (CAM) are surface molecules that mediate cell-cell

adhesion (Ivanov, 2001). They contribute to formation of specialized junctional

complexes to maintain multicellular structures. They are also integral parts of dynamic

cellular, development, and pathological processes (Siu et al., 2004). During development

many CAM show spatial and temporal regulations and some have been demonstrated to play important physiological roles during tissue and organ formation. Based on properties

such as homophilic adhesion, calcium dependency, association with ion channels and others properties, CAMs can be arranged into fives families: Immuglobulins, Integrins,

Cadherins, Selectins, and others.

The Cadherin Superfamily

Based on protein domain composition, genomic structure, and phylogenetic

analysis of protein sequences, cadherins can be put into six sub-families: classical (type I,

atypical/ type II), desmocollins, desmogleins, protocadherins, and other cadherin-related

molecules such as Flamingo cadherins (Nollet et al., 2000) (Figs.1 and 2). The classical

type I cadherins are the major component of cell-cell adhesive junctions, were the first to

be discovered, and are most studied and understood.

3 In general, cadherin molecules possess functional sites for adhesion recognition, calcium binding, membrane integration, cytoskeletal interactions and posttranslational modification such as glycosylation, phosphorylation and proteolysis. A typical type I or type II cadherin molecule has three major domains; a large extracellular domain, a small transmembrane domain, and a cytoplasmic domain (Fig. 2). The extracellular domain

(EC) in type I and II cadherins consists of five homologous repeats, with the most N- terminal repeat (ECI) as the binding site for the same kind of cadherin molecules from neighboring cells (Takeichi, 1991). The transmembrane domain is a small hydrophobic unit in bedded in the cell membrane. The intracellular domain is linked to actin filaments via α- and β-catenins and is known to be involved in the Wnt/APC (Wnt as a growth factor; and APC, adenomatosis polyposis coli) signaling pathway (Tsukita et al., 1992)

(Figs. 2 and 3).

Figure 1. Phylogenetic representation of cadherin sub-families and their representative members on the basis of domain EC1 (from: Nollet et al., 2000).

Legend Cadherin repeat Cadherin cytoplasmic domain Laminin A G domain Tyrosine kinase Non-chordate classic cadherin domain p120 binding site EGF-like domain B-catenin binding site Flamingo box His-Ala-Val motif single pass transmembrane domain Multiple pass transmembrane domain

Figure 2. Domain organization of representative members of the cadherin superfamily (from Truong et al., 2002)

Classical Cadherins

Classical cadherins are the best-known and most studied subgroup among the

cadherin molecules. Based on their overall sequence similarities and the conservation of

several motifs and aromatic amino acids residues, Suzuki proposed that the classical

cadherins be divided into two sub-families, type I and type II (Suzuki, 1996). The type I classical sub-family includes E-, N-, P-, and R-cadherins also called cadherin-1, -2, -3,

and -4, respectively. The type II sub-family includes cadherin-6, -7, -8, -10, and -11. The

type I cadherins typically have broad distributions that are segregated by embryonic germ

layer or tissue types (Nishimura et al., 1999). In contrast, type II cadherins exhibit more

fine-grained, and often overlapping, patterns of expression, notably within the developing

nervous system (Patel et al., 2005). One more major difference between the type I and

type II cadherins is the presence of HAV tripeptide in the EC1 region of the type I which

is substituted by a QIA tripeptide in the type II cadherins.

Figure 3. Schematic representation of classic cadherin molecules at the cell surface (From: Alattia et al., 1999). 7

Calcium Binding

Cellular adhesiveness has been long attributed to the presence of calcium, as shown in both the in vivo and in vitro in the absence of calcium (Kartenbeck et al., 1982).

The crystal structure of E-cadherin (cadherin-1) fragment (Figs. 4a, 5b, and 6) showed that calcium is central in dimer formation. The binding of calcium involves the residues that are located in the linker region or its vicinity, at the domain 1 and 2 interface (Alattia et al., 1999) (Figs. 4a, 4b). Experiments on calcium binding identified two forms of interaction between the calcium binding sites (Nagar et al., 1996). First, intramolecular interactions (based on the structure of E-cadherin extracellular domains 1 and 2, Ecad12) involving residues Glu11, Glu69, Asp103, and Asp136, link the three calcium ions bound to the Ecadh12 monomer (Fig. 4a). Second, intermolecular interaction involving residues

Asp100, Gln101, Asn102 and Asn143 which coordinate calcium ions on one monomer while forming intermolecular hydrogen bonds with the opposite monomer, either directly or via water molecules (Fig. 4b). Monomeric and dimeric cadherins show similar apparent calcium dissociation constants of 23 µM when measured at different protein concentrations suggesting no difference in their affinity for calcium (Alattia et al., 1999).

The same dissociation constant is relatively close to the average value determined for the entire extracellular region of E-cadherin (30µM) (Koch et al., 1997).

Figure 4a & 4b. (a) Calcium binding sites on single E-cadherin molecules, (b) Atomic detail of the calcium-mediated interaction in the E-cadherin dimmer (from: Alattia et al., 1999).

Figure 5: Schematic drawing of a proposed mechanism for calcium-mediated assembly of cadherin molecules (from Alattia et al., 1999)

Cadherin-6 (K-cadherin)

Cadherin-6 is a member of the subfamily type II cadherins. Cadherin-6, like other

type II cadherins, has the HAV motif substituted by a QIA tripeptide in the EC1

(Shimoyama et al., 1999).

During development, expression of cadherin6 is found in various regions of the

CNS in both zebrafish and mice (Inoue et al., 1997, 1998; Liu et al., 2006), and in the

peripheral nervous system (PNS) in zebrafish, mice and Xenopus (Inoue et al., 1997;

David, and Weldich, 2000; Liu et al., 2006). Using in situ hybridization methods

(detecting mRNA), Liu et al., (2006) studied cadherin6 mRNA (cdh6) expression in

developing zebrafish and reported that cdh6 was expressed in specific regions of the

embryonic zebrafish nervous system, and cdh6 expression in the developing nervous system was both spatially and temporally regulated.

As early as 18 hours post fertilization (hpf) when the dorsal forebrain

(telencephalon and diencephalon) becomes distinguishable, cdh6 was detected in the dorsal telencephalon and discrete region of the diencephalon. Specifically cdh6 was strongly expressed by the dorsal diencephalic region destined to become the pineal gland.

As development proceeded cdh6 continued to be expressed by the developing pineal gland through the first 3 days of development (Liu et al., 2006). cdh6 was also expressed

by the neural retina including the retinal ganglion cells, amacrine cells, cells adjacent to

the photoreceptors, and by the optic nerve during critical periods of their development.

The Development of Zebrafish Pineal Gland

The pineal gland (epiphysis) is an unpaired brain structure, located above the third ventricle in zebrafish. The pineal gland has a fully autonomous circadian clock: cultured zebrafish pineal gland held in the darkness by Cahill (1996), continue to produce melatonin in a rhythmic manner. The pineal gland is responsible for rhythmic production and secretion of melatonin which causes physiological changes in lower vertebrates (e.g. fish). Experiments showed that the pineal gland reacts to light in the same way does the eye, and it contains similar cell types as the eye, such as ganglion cells, cones and rods in the pineal gland (Robinson et al., 1993).

In zebrafish and other fish, the daily rhythm in melatonin production and secretion is modulated by serotonin-N-acetyltransferase (AANAT) (Gothilf et al., 20002).

The rhythmic oscillation of the zebrafish AANAT (zfAANAT) is in turn controlled by the clock gene, zperiod2 (zper2) (Ziv et al., 2005). Fish in general have two AANAT genes, AANAT-1 and -2, which are expressed respectively in the retina and pineal gland.

Zebrafish pineal gland development can be analyzed by expression of several photoreceptor specific markers including zpr-1 (for subset of cones), crx and otx5 (most pineal cells), gnat1 (for cones), uv opsin (cones), gnat2 (for rods) and rod opsin (rods)

(Shen and Raymond, 2003). There is no published study of cadherin function on the pineal gland development.

The Development of Zebrafish Eyes

Studies have found that the vertebrate retina is remarkably conserved during the vertebrate evolution. Similarities in the major cell classes of the retina and their organization can be traced as far back as the divergence of mammals from teleost fish

(Malicki et al., 2002). Such similarities make the use of zebrafish adequate for the study of the development of the vertebrate retina.

The development of the major sensory organs of the head, including the eye, is induced from the interactions of the neural tube with a series of epidermal thickenings called cranial ectodermal placodes. The development of the eye starts with the evagination of the optic vesicle from the brain, which contacts the overlying ectoderm and induces a lens placode. Lens cells are produced by the differentiation of the overlying ectoderm as the optic vesicle folds in on itself; thereafter the lens placode becomes the lens vesicle.

As the lens is internalized, the optic vesicle becomes the neural and pigmented retina.

The corona is formed by the induction of the overlying ectoderm by the lens vesicles

(Gilbert, 2007). Zebrafish retina is similarly organized and contains similar cell types as other vertebrate retina. There are three major cellular layers: the photoreceptor cell layer that contain rods and cones, the inner nuclear layer where horizontal cells, bipolar cells and amacrine cells are localized, and the ganglion cell layer. The cellular layers are separate by two fibrous layers: the outer plexiform layer where photoreceptor cells connect with cells in the inner nuclear layer (e.g. bipolar cells), and inner plexiform layer where ganglion cells interact with cells from the inner nuclear layer. Zebrafish retina contains several types of photoreceptors including rods, short single cones, long single

12 cones, double cones (reviewed by Malicki, 1999). Based on morphology and expression

of photoreceptors-specific markers, these photoreceptors can be distinguished from one

another. These markers include rod cell-specific genes rod opsin and transducing α- subunit (gnat1), cone cell-specific transducing α-subunit (gnat2) (Brockerhoff et al.,

2003), blue opsin (specific for long single cones), uv opsin (specific for short single cones), red opsin and zpr-1 (for double cones) (Larison and BreMiller, 1990; Raymond et al., 1993; Hisatomi et al., 1996). All photoreceptor cells express interphotoreceptor retinoid-binding protein gene (irbp) (Stenkamp et al., 1998). In the course of zebrafish retinal development, photoreceptors start to become postmitotic between 43 and 48 hours post fertilization (hpf), beginning in the ventral retina. A population of the earliest

generated photoreceptors begin to express visual pigments by 48 hpf (Robinson et al.,

1995), and by 72 hpf, photoreceptors throughout the retina express most of the photoreceptor-specific genes (Raymond et al., 1995; Stenkamp et al., 1998; Brockerhoff et al., 2003). In addition to these specific markers, several transcription factors such as

Crx and Pax6, have been demonstrated to regulate eye development in all vertebrate species and most invertebrates examined. These molecules exert their effect through downstream targets genes including cadherins, and their mutations are involved in an array of human diseases.

During development, cadherin molecules play an important role in adhesion and connection and differentiation of vertebrate retina cells. Both the classical type I cadherins (e.g. cadherin-2 and cadherin-4 also called N-cadherin and R-cadherin, respectively), and type II cadherins (e.g. cadherin-6, and cadherin-7) are expressed by mammal (Honjo et al., 2000) and zebrafish retina (Liu et al., 2003; Liu et al., 2006a, b). 13 Moreover, disruption of cadherin function (e.g cadherin-2 and cadherin-4) in chicken and

zebrafish results in several retinal defects (Matsunaga et al., 1988; Masai et al., 2003;

Malicki et al., 2003; Babb et al., 2005). There is no published study on the type II cadherins function on vertebrate eye development.

Morpholino Oligomers

Morpholino antisense oligomers (or oligo nucleotides, abbreviated as MOs) are sequence-specific binding polymers designed to block translation of selected mRNAs

(the sense strand) (Summerton and Weller, 1997). MOs are chemically modified DNA

ologonicleotides with similar base hybrization abilities as DNA but have a morphine

moiety instead of a ribose (Summerton and Weller, 1997). In addition, a

phosphorodiamidate linkage is used, resulting in a neutral charge backbone. This design

makes MOs modified and highly soluble polymers that are capable of hybridizing with

single-stranded nucleic acid sequences (e.g. mRNA). In addition, MOs have high

specificity with little cellular toxicity. Moreover, MOs are not subject to any known

endogenous enzymatic degradation activity, therefore their effects are long lasting

(Ekker, 2000). The binding of MOs to the targeted sequences causes deactivation of the

gene (via reduced translation of the target protein or resulting production of abnormally

spliced mRNA). The application of MOs in animal models (e.g. zebrafiish and Xenopus)

has helped unlock the secret to many genes in the vertebrate genome (Ekker, 2000). This

technique has also been used in therapeutic applications. Dr. Liu’s lab has successfully

used MO methods to study cadherin-2 and cadherin-4 function in zebrafish development

(Kerstetter et al., 2004; Badd et al., 2005)

Hypotheses

Since cdh6 is expressed in the zebrafish pineal gland and eye during critical periods of their formation, I hypothesize that blocking cadherin6 expression in zebrafish embryos likely results in disruption of the pineal and retinal development. The hypothesis was tested by examining differentiation of photoreceptors in the pineal gland and retina of zebrafish embryos injected with cdh6 specific MOs using some of the above mentioned markers.

CHAPTER III

MATERIAL AND METHODS

Animals

Wild-type adult zebrafish, Danio rerio, were provided by Dr. Liu from his

zebrafish colony (University of Akron, Biology Department, Akron, Ohio). The fish will be kept in 10-gallon tanks maintained at 25-26°C, with 12h/12h light/dark. The embryos were obtained from breeding of the adult zebrafish. Both the control and the experimental

embryos were placed in filtered fish tank water supplemented with methylene blue (to

reduce fungi growth) and PTU (1-phenyl-2-thiourea, 0.003%, to prevent pigment

formation). In accordance with animal regulation, all procedures are approved by the

University of Akron Committee of Use and Care of Animal in Research and according to the standard zebrafish handling procedures (Westerfiled, 2000).

MOs treatment

Cadherin-6 morpholino antisense oligonucleotides (cdh6MOs)

Two translation blocking cadherin-6 morpholino antisense oligonucleotide

(cdh6MOs) were used to knock down cadherin-6 protein levels. Their sequences are;

cdh6MO1: 5’-AAG AAG TAC AAT CCA AGT CCT CAT C -3’, cdh6MO2: 5’- TCC

GCT CTT AGG GTG TCT TAC AGG G- 3’. A cdh6MO1 specific control MO, (5-mis cdh6MO1, 5’-AAC AAG TAG AAT GCA ACT CCT CGA TC-3’) was used as a

16 control. The MOs were purchased from Gene Tools (Philomath, OR). The design of the

MOs was done according to Gene Tools targeting guidelines (Naservicius and Ekker,

2000). The cdh6MOs sequences were compared to the databases using BLAST, and no

significant similarities were found to any sequences other than the zebrafish cadherin-6.

MO injection

Newly laid eggs (1-4 cell stage) were placed in agarose gel plate, and injected

with MOs at 0.25 mM (for cdh6MO1, and 5-mis cdh6MO1) or 0.12 mM (for cdh6MO2)

in Daneau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES pH 7.6) using a Narishige microinjection system under a Leica dissection microscope and using phenol red (20%) as an injection indicator. An injected total volume of 1-2 nl (0.6-2.4 ng) was delivered per embryo. Because dividing cells

(blastomeres) and yolk are connected by large pores before the 8-cell stage in zebrafish embryos (Westerfield, 2000), DNA or RNA injection results in distribution of the injected material to all dividing cells. There were two groups of control embryos. Some embryos from the same batch of embryos used for the cdh6MOs injection were injected with the 5-mis-cdhMO; another group of embryos were not injected.

Both the injected and control embryos were placed in filtered fish tank water and allowed to develop to 50 hours post fertilization (hpf) to 80 hpf at 28.5°C. An overdose of Tricain (MS222, 0.5%) was used to euthanize the embryos. A solution of 4% paraformaladehyde in phosphate buffered saline (PBS, pH-7.4) was used to fix the embryos overnight at 4°C. The embryos will be washed in PBS and followed by rinsing

17 in increasing concentrations of methanol and finally stored in 100% methanol at -20°C

until use.

Whole-mount Immunocytochemistry

To determine protein distribution, standard whole mount immunocytochemistry

(ICC) (Liu et al., 1999a, 2001) was performed using a zpr-1 antibody that labels a subset

of cone photoreceptors (double cones) (Zebrafish Resource Center, Eugene, OR). The

process of ICC begins with rehydrating the embryos in decreasing concentrations of

methanol in PBS, followed by protease K digestion (this step allows the reagents to

penetrate into cells). The embryos were then fixed in 4% paraformaldehyde for 20 min. at room temperature. The embryos were washed in PBS before incubation in a blocking solution (1-1.5% normal horse serum in PBS with supplement DMSO) for 2 hours to block non-specific antibody binding. The embryos were incubated with the primary antibody (zpr-1, 1:1500, diluted in the blocking solution) at 4°C, overnight on a rotating shaker. To rinse off unbound antibody, the next morning the embryos were washed 4 times for 30 minutes each in PBST (PBS with Tween 20 to prevent sticking of the embryos to each other and to the container). The immunoperoxidase preparation consists of incubating the embryos with a biotinylated second antibody (anti-mouse IgG; Vector

Laboratory) which was also diluted in the blocking solution at room temperature for 6-7 hours on constant movement. After 4 washes in PBS, a solution of avidin-biotin complex

(Vector Laboratories) was used to incubate the embryos for 2 hours at room temperature.

After washing in PBST the reaction product was visualized using 0.01% hydrogen peroxide and 0.05% diaminobenzine as the chromogen.

Whole mount in situ hybridization

Six probes were used in this study: crx, otx5 those two probes label most cells in

the pineal gland and most photoreceptor precursor cells in the retina, gnat1 (labels

cones), gnat2 (for rods), rod opsin (for rods) and uv opsin (for a subset of cones). All the

probes, digoxigenin-labeled, were generated by Dr. Liu using a Roche’s in vitro probe

synthesis kit (Roche, Indianapolis, Indiana). The whole mount in situ hybridization comprises the following four steps.

1. Preparation of the embryos:

The embryos, processed as described above and stored in 100% methanol at 20ºC, were rinsed in fresh 100% methanol for 5 min at RT. The embryos were rehydrated in methanol series (MeOH/PBST-75%/25%, 50%/50%, 25%/75%), for 5 min. each at RT, then washed 4 times in 100% PBST for 5 min. each time. The embryos were then incubated in proteinase K (10 µg/ml, in PBST), followed by a brief rinse in PBST with a

5 min. wash in PBST. The embryos were then refixed in 4% paraformaldehyde for 20 min. at RT. To finish the first step the embryos were washed five times in PBST for 5 min. per wash.

2. Hybridization:

The embryos were transferred to a new 1.5 ml siliconized RNase free centrifuge tube. Prehybridize the embryos for 2 hours or longer at 56-60°C in a hybrization solution without a probe. The embryos were then placed in the hybridization buffer with the probe

(~ 700 µl each, probe concentration ≤ 1μg/ml), then hybridized overnight at 56-60°C.

The next morning, the embryos will be rinsed briefly in 2X SSC. Following this rinse, the

19 embryos were washed in 50% formamide /2X SSC (0.5ml) at 60°C for 1 hour. The

embryos were then washed twice in 2X SSCT (0.01% Tween, 1.0 ml) for 10 min. each at

60°C. Perform two stringent washes in 0.2 X SCCT (0.01% Tween, 1.0 ml) at 70oC for

30 min. each, followed by a serial washes (75%/25%, 50%/50%/, 25%/75%, 0%/100% of

0.2X SCCT/PBST) at RT, for 10 min. per wash.

3. Blocking non-specific staining using a blocking solution and incubating with an anti- dig-antibody:

The embryos were placed in a blocking solution (5% normal goat serum, 2 mg/ml

BSA, 1% DMSO in PBST) for 2 hours at room temperature to block non-specific binding of the antibody. This is followed by incubating the embryos with an alkaline phosphatase conjugated anti-dig-Ab (1:5000, from Roche) at 4°C overnight with constant agitation.

4. Antibody-Detection:

The next day wash the embryos were washed 8 times for 15 min. each in PBST.

The embryos were washed again 3 times for 10 min. each in an alkaline phosphate substrate buffer (100 mM NaCl, 100 mM Tris-Cl, pH 9.5, 50 mM MgCl). This solution is also called Genius buffer 3. The embryos were transferred to a 24-well tissue culture plate. The embryos were then incubated in alkaline phosphatase chromogen reaction mixture for 0.5-2 hours at RT in the dark. The reaction was stopped once the color reaction becomes satisfactory using Genius buffer 3, followed by PBST rinses, for 5 min.

each time in RT. The staining was fixed with 4% paraformaldehyde in PBS, either 20

min. at RT or overnight at 4°C. And finally the embryos were washed 2 times for 10 min.

each with PBST and mount under coverslip in 100% glycerol.

20 A compound microscope equipped with a SPOT digital camera system was used to observe and record morphological phenotypes and staining patterns of the markers of both the control and the experimental groups.

CHAPTER IV

RESULTS

Expression of photoreceptor-specific markers was greatly reduced in the cdh6 morphant eye, but not in the morphant pineal gland

Since cdh6 expression was observed in the developing pineal gland and retina during critical periods of their development (Liu et al., 2006), it is reasonable to assume that cadherin-6 is involved in the development of these structures. To determine the function of cadherin-6 in the differentiation of the pineal gland and retina, I first examined the expression of two transcription factors that are known to regulate photoreceptor differentiation in the vertebrate retina and/or pineal gland: crx (Furukawa et al., 1997; Blackshaw et al., 2001; Shen and Rayamond, 2004), and otx5 (Gamse et al.,

2002). Both crx (Fig. 1) and otx5 (Fig. 8) were strongly expressed by the pineal gland of both control embryos (panels A and B in each figure) and cdh6 morphants (panels D and

C in each figure) at 50 hpf. These two genes were strongly expressed by the retina of control embryos (panels A and B in each figure), but their expression was greatly reduced in the retina of cdh6 morphants (panels C and D in each figure). Moreover, there was no obvious difference in the size of the pineal gland between the morphants and control embryos, but the morphant heads and eyes were clearly smaller compared to those of control embryos.

Figure 7. Expression of crx in control (panels A and B) and cdh6 morphant pineal gland (P) and retina. Panels A and C are lateral views of the head region of whole mount embryos, with anterior to the left and dorsal up. Panels B and D are ventral views of whole-mount embryo heads with dorsal up.

Figure 8. Expression of otx5 in control (panels A and B) and cdh6 morphant pineal gland (P) and retina. Panels A and C are lateral views of the head region of whole mount embryos, with anterior to the left and dorsal up. Panels B and D are ventral views of whole-mount embryo heads with dorsal up.

24 To further study cadherin-6 function in zebrafish retina and pineal photoreceptor

differentiation, I examined the control and cdh6 morphant embryos using several rod-

specific markers such as rod opsin (rho, Fig. 9) and gnat1 (Fig. 10), cone specific

markers such as zrp-1 (Fig. 11), gnat2 (Fig. 12) and uv opsin (uvo, Fig. 13) and a marker

that labels both rods and cones, irbp (Fig. 14). All three markers were expressed by the developing pineal gland (except the uvo which is expressed only in the retina) and throughout the outer region of the retina of control embryos at 72 hpf, but their expression was greatly reduced in the retina of cdh6 morphants. Moreover, the pineal gland size, as judged by labeled cells, was similar in size between control embryos and the morphants, while the morphant head and eyes were noticeably smaller than the

control group.

Figure 9. Expression of rho in control (panels A and B) and cdh6 morphant pineal gland (P) and retina. Panels A and C are dorsal views of the head region of whole mount embryos, with dorsal up. Panels B and D are ventral views of the head region of the whole mount embryos with ventral up

Figure 10. Expression of gnat1 in control (panels A and B) and cdh6 morphant pineal gland (P) and retina. Panels A and C are lateral views of the head region of the whole mount embryos, with anterior to the left and dorsal to the right. Panels B and D are ventral views of whole-mount embryo heads with dorsal up.

Figure 11. Expression of zpr1 in control (panels A and B) and cdh6 morphant pineal gland (P) and retina. Panels A and C are dorsal views of the head region of whole mount embryos, with dorsal up. Panels B and D are ventral views of the head region of the whole mount embryos with ventral up

Figure 12. Expression of gnat2 in control (panels A and B) and cdh6 morphant pineal gland (P) and retina. Panels A and C are lateral views of the head region of whole mount embryos, with anterior to the left and dorsal to the right. Panels B and D are ventral views of whole-mount embryo heads with dorsal up.

Figure 13. Expression of uvo in control (panel A) and cdh6 morphant retina (panel B). Panels A and B are ventral views of whole-mount embryo heads with dorsal up.

Figure 14. Expression of irbp in control (panels A and B) and cdh6 morphant pineal gland (P) and retina. Panels A and C are lateral views of the head region of whole mount embryos, with anterior to the left and dorsal to the right. Panels B and D are ventral views of whole-mount embryo heads with dorsal up.

CHAPTER VII

DISCUSSION

Although there are numerous studies of cadherin-6 expression in the vertebrates, there is little information on cadherin-6 function in differentiation of the vertebrate retina, and no published report on cadherin-6 function in development of the vertebrate pineal gland. Ruan and colleagues recently studied cadherin-6 function Xenopus eye development (Ruan et al., 2006). But their focus was mainly on the formation of the early eye cup, retinal lamination, and lens development, while differentiation of the photoreceptors was dealt with very briefly using only one photoreceptor marker (rod opsin). Due to similar expression and function of cadherins in vertebrate species, including humans, understanding cadherin-6 function in zebrafish likely provides us with

insights into its function in human development.

Cadherin-6 MOs are likely specific

MOs technology has become one of the most used and powerful methods to study

gene function in vertebrate species such as zebrafish and Xenopus (Ekker, 2000). Dr.

Liu’s laboratory has successfully employed this technique to study cadherin function in

developing zebrafish cardiovascular system (Bagatto et al., 2005), cranial ganglia and

lateral line system (Kerstetter et al., 2004; Wilson et al., 2007), and visual system (Babb

et al., 2005). The cadherin-6 MOs used in this study were likely specific due to the

following reasons.

32 First, the specificity of cdh6MO1 was recently demonstrated by Kubota and colleagues

(2007) in their study of cadherin-6 function in zebrafish kidney development. The authors

showed that the phenotype (small eye and head, short yolk extension, edema in thorax

region, and/or curved body) could be rescued by co-injection of cdh6MO1 with zebrafish

cadherin-6 mRNA. Second, the phenotype obtained by injection of cdh6MO1 and

cdh6MO2 was similar and dose dependant. Third, injection of the control MO (5-miss

matched cdh6MO1) resulted in embryos that were indistinguishable to uninjected wild

type embryos. Therefore, the photoreceptor phenotype observed in my study likely

resulted from disrupted cadherin-6 function.

The use of photoreceptor-specific markers to analyze differentiation of zebrafish photoreceptors

Crx and otx5 are transcription factors known to regulate vertebrate photoreceptor

development (Furukawa et al., 1997; Games, et al., 2002; Shen and Raymond, 2004).

Both genes are expressed by most retinal cells and pineal gland cells in both younger (24-

50 hpf) and older (3-4 day) zebrafish, and have been used to assess differentiation of

early fish retina and pineal gland (Shen and Raymond, 2004; Liu et al., 2007). Zpr-1

labels strongly differentiating and mature double cones in zebrafish retina, and it has been

used as a double cone marker in numerous studies (Masai et al., 2003; Malicki et al.,

2003; Babb et al., 2005; Liu et al., 2007). gnat1 (for rods) and gnat2 (for cones) are

expressed by differentiating photoreceptors in both the retina and pineal gland starting

around 60 hpf in the retina, and 48 hpf in the pineal gland (Brockerhoff et al., 2003),

while irbp is expressed by both photoreceptors (both in the retina and pineal gland) and retinal pigmented epithelium of developing zebrafish (Stenkamp et al., 1998). uv opsin

33 and rod opsin are expressed by a subset of cones in the retina, and all rods in both the

retina and pineal gland, respectively (Raymond et al., 1995). All these markers (gnat1, gnat2, irbp, uv opsin and rod opsin) have been used to determine differentiation of zebrafish photoreceptors in the retina and/or pineal gland (Shen and Raymond, 2004; Liu et al., 2007).

Cadherin-6 is crucial for differentiation of zebrafish retinal photoreceptors

The great reduction in the number of retinal cells expressing the photoreceptor- specific markers in cdh6 morphants suggests that cadherin-6 plays an important role in differentiation of the retinal photoreceptors. This reduction was unlikely due to a general developmental delay, because morphants at 72 hpf expressed less of those markers than control embryos at younger stages (e.g. 60 hpf, data not shown). Cadherin-6 function in regulating the retinal photoreceptor development is likely an indirect one, because the photoreceptors do not express cadherin-6 (Liu et al., 2006). Other retinal cells, such as the retinal ganglion cells, amacrine cells, and cells located adjacent to the photoreceptors

(likely horizontal cells) express cadherin-6 during critical periods of their development

(Liu et al., 2006). Moreover, differentiation of these cells is greatly affected in cdh6 morphants (Liu and Wilson, unpublished observation). This is similar to findings in cadherin-4 morphants in which cadherin-4 is expressed by the ganglion cells and amacrine cells, but not in the photoreceptors, yet photoreceptor differentiation is severely disrupted in the cadherin-4 morphant. These results suggest that normal photoreceptor development depends on development of other retinal cells (Liu et al., 2007). The reduced eye size in cdh6 morphants is mainly due to decreased cell proliferation in the

34 cdh6 morphant eye (Liu and Wilson, unpublished observation), which is similar to the study in Xenopus (Ruan et al., 2006). In contrast, the small eye phenotype in cadherin-4 morphants is due mainly to increased cell death (Babb et al., 2005).

Cadherin-6 may not be required for the pineal gland development

cadherin-6 was strongly expressed in the zebrafish pineal gland during critical stages of its development (Liu et al., 2006), therefore it is reasonable to speculate that cadherin-6 is involved in the pineal gland formation. My results, showing that expression of all of the photoreceptor-specific markers was similar between the control embryos and cdh6 morphants, were surprising. It is possible that cadherin-6 function was not completely removed in the morphants, and a small amount of cadherin-6 protein may be sufficient for pineal photoreceptor differentiation. Cadherin-6 function may also be compensated for by that of other cadherins such as cadherin-2 and cadherin-4, which are expressed in developing zebrafish pineal gland (Liu unpublished observation). It is also possible that both processes (incomplete disruption of cadherin-6 function and compensation by other cadherins) contributed to the little or no perturbation in the pineal photoreceptor differentiation. My results provide further support to the idea that differentiation photoreceptor differentiation in the pineal gland is regulated by different mechanisms from those that control retinal photoreceptor differentiation (Games et al.,

2002).

CHAPTER VIII

CONCLUSION

Using MO technique, I was able to study the function of cadherin-6 in zebrafish photoreceptors development in both retina and pineal gland. My results showed that cadherin-6 affected retinal development more than the development of the pineal gland.

These results may be due to the fact that the two organs use different mechanisms for photoreceptors differentiation or in the case of the pineal gland other cadherins molecules such as cadherin-2 and cadherin-4 may have supported its normal development.

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APPENDIX

ANIMAL CARE AND USE PROTOCOL APPROVAL