IDENTIFICATION OF TWO NOVEL CVC DOMAIN-

CONTAINING HOMEOBOX GENES

Jens Looser

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Molecular and Medical Genetics

University of Toronto

O Copyright by Jens Looser 1995

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IDENTIFICATION OF TWO NOVEL CVC DOMAIN-

CONTAINING HOMEOBOX GENES.

Master of Science, 1995,

Jens Looser,

Graduate Department of Molecular and Medical Genetics,

University of Toronto.

ABSTRACT

The retinal C M 0 gene encodes a protein with both a homeodomain of the paired-

like type and a novel evolutionarily conserved protein domain of unknown function, the

CVC domain. In this thesis, 1 report the isolation of CHXIO- 2, a human gene related to

CHXIO. The CHXIO-1 cDNA was isolated from a screen for CHXIO homologues

expressed in adult human retina. CHXIO-I is expressed at low levels in the inner nuclear

and ganglion ce11 layers of the adult retina Chxl71, a murine CHXIO-1 homologue, differs

from CHXIO-1 in sequence and abundance. Genomic Southem analysis showed that

although Chxl7i is the closest mouse homologue of CHXIO-1, a more closely related

human gene may exist. This work demonstrates that multiple CVC domain-containing

homeobox genes are expressed in the mammalian retina. Like C M O , they are likely to

contribute to retinal development and maintenance.

ACKNO WLEDGMENTS

Sitting at my cornputer (a Mac, not some lousy IBM!) now after everything is over 1

try to remember everyone that helped me in completing this monumental piece. Suddenly

my brain feels as einpty as it has been for the last three months while 1 worked on the

following pages. Therefore, in case 1 forget your name in the following lines please forgive

me.

1 guess 1 wiii take the obvious ones fïrst. Thanks to d l past and present inernbers of

the McInnes Lab (in dphabetical order: Anna Soltyk, Bryan Snow, Carol Freund, Dingfang

Bu, Elizabeth Garami, Geoff Clarke, Ivy Liu, Jakub Noviik, Joe Zhou, Lin Liu, Lynda

Ploder, Midhat Osman, Roger Bascom) for their various contributions to iny work, the

cnimby remainders of my social life or rny booze-independent headaches. Special thanks to

Geoff Clarke for Our crash-and-bum science talks and the fact that he actually succeeded (at

times) in making me think.

Thanks to the University of Toronto, the Research lnstitute at the Hospital for Sick

Children and the Medical Research Council of Canada for the scholarships 1 received.

Thanks to everyone in the Hospital and the Department, especially Janet Rossant,

Johanna Rommens, Lee Montgomery, Lynn Mar and Vivien Measday, for uncountable

favors and advice on various aspects of life.

A particular thanks to Rod Mclnnes (old buddy, old pal) for taking the

(retrospectively seen not so big, eh!) risk of accepting some foreigner who burst into his

off~ce two summers ago. Thanks for teaching me aspects of d t i n g and speakinç that 1 did

not cal1 my own before being chained to one of your benches. 1 also appreciated your

support of my future plans even though they did not quite match yours.

Thanks to everyone who had to deal with my whining over the last two years,

especially during the last crack down on the thesis. Special thanks to Viçky Aneliunas (for

teaçhing me proper table manners), Pat Derkis (for taking my mind of work), Lian de

Lotbinière (for actuaily admiring what 1 am doing) and Christina Zeidler (for k ing herself).

Last and most importantly 1 would like to thank my family and fnends over in good

old Germûny. Your love, understanding and support enabled me to come and stay in

Canada. As a token of my love 1 want to dedicate this thesis to my parents for everything

they have ever done for me.

TABLE OF CONTENTS

Abstract

Acknowledgmen ts

Table of Contents

List of Figures

CHAPTER 1: INTRODUCTION

1. RETINA

1 . 1 Physiology of the adult mamrnalian retina

1.2 Development of the marnmalian retina

2. HOMEOBOX GENES

2.1 Discovery and Charactenzation of the homeobox

2.2 Phylogeny of the homeobox

2.3 The Paxfprd class of homeobox genes

2.4 Pax6/eyeiess; Master contd genes of eye development

3. ChxlO

3.1 Ch10 biology in the mammalian retina

3.2 Ch10 mutations and ocular retardution

3.3 Ch10 homologues in rnice and goldfish

4. RATIONALE FOR THIS PROJECT

CHAPTER 2: ORIGINAL RESEARCH

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

1. Attribution of data

2. Human retinal screen

3. OTX3, a novel orthdenticle homologue

4. Human LIM homeobox gene LH2

5 CHXIO-1, a novel CHXlO homologue

5.1 Cloning

5.2 Putative protein domains

5.3 Mapping to the chromosomes

5.4 Northem analysis on total RNA

5.5 Northern analysis on poly-(A)+ RNA

5.6 In situ hybridizations

6. Isolation of the murine CHXIO-1 orthologue. C h 1 71

6.1 Cloning

6.2 Putative protein domains

6.3 Northem analysis

6.4 Phylogenetic relationship to CHXIO-1

DISCUSSION

CHAPTER 3: FUTURE DIRECTIONS

LIST OF CITATIONS

LIST OF FIGURES

Figure 1 - 1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 1-5

Figure 1-6

Figure 2- 1

Figure 2-2

Figure 2-3

Figure 2-4

Figure 2-5

Figure 2-6

Figure 2-7

Figure 2-8

Figure 2-9

Figure 2- 10

Figure 2- 1 1

Figure 2- 12

Figure 2- 13

Figure 2-14

Figure 2- 15

Retinal layer structure

Murine eye development

ChxlO compared to Px/prd homeobox genes

C M 0 in situ hybridizations

ocular retardution phenotypes

previously known Chri0 homologues

Flow chart of the human retinal screen

Class L,2 and 3 plaque pure phages

OTX3 alignments

CHXlO-1 clone rnap and cDNA

chx 10- l/chx 10: aiignment and peptide structures

Mapping of CHXIO-1 io the chromosomes

CHXlO-1 Northern analysis

CHXIO-1 in situ hybridizations

Ch171 clone map and cDNA

c h 17 llchx 10- 1 : dignment and peptide structures

Chxl71 Nort hem analy sis

Evolutionary conservation of chx 10

Aiignment of al1 CVC homeobox genes

Mouse genomic Southem analysis

Human genomic Southem analysis

CHAPTER 1: INTRODUCTION

In this thesis, 1 present the results of a screen for homologues of the 'retinal'

homeobox gene ChxlO. In this chapter, 1 present a brief overview of: 1. selected aspects

of neuroretinal biology, 2. homeobox-containing genes in general and, in more detail, the

Pax/prd class, and 3. the biology of Ch10 and its importance for the retina.

1. RETINA

1.1 Physiology of the adult mammalian retina

Light absorption in vertebrates occurs in a thin sheet of neural tissue called the

neuroretina, which lines the back of the eye cup. Within this thin sheet of neural tissue,

visual image information is captured, transduced into a neuronal signal and sent to the

visual centers of the brain. Six major classes of neuronal cells (photoreceptors, horizontal,

amacrine, interplexiform, bipolar and ganglion) and two classes of glial cells (Müller and

astrocyte) are found in the retina (Figure 1-1 .). The neuronal ce11 nuclei are organized into

three well defined parallel nuclear layers, which are separated by two plexiform layers that

host the synaptic processes (Adler and Farber, 1986; Rowe, 199 1).

Retinal organization is the inverse of what one might intuitively suspect, in that the

photoreceptors are facing away from the light source and are covered by multiple retinal ce11

layers (Figure 1- 1). Light that enters the eye therefore has to travel through al1 three

nuclear layers before being captured by the photoreceptors. Distal to the photoreceptors

lies the retinal pigment epithelium (RPE), a neuroepithelial layer of single ce11 thickness.

The pigment granules in this layer help absorb scattered light and thereby enhance the

image quality. Additionally, the RPE serves to transport metabolites and nutrients to the

photoreceptors as well as to promote phagocytosis of shed discs from the outer segments

of the photoreceptor cells. The inner segments of the photoreceptor cells, which contain

the major metabolic machinery of the ce11 and the nucleus, make up the outer nuclear layer.

Vitreal Border

Figure 1-1

Schematic representation of the mammaiian retina with aii its ce11 types. Abbreviation of

ceU narnes: astrocyte (A), bipolar (B), cone (C), horizontal (H), interplexiform a), Müller

(M), rod (R). Taken from Adler and Farber (1986).

Photoreceptors extend their synaptic termini into the outer plexiform layer (OPL), which

separates the outer nuclear layer (ONL) from the inner nuclear layer (NL). Here, synapses

between photoreceptor cells and bipolar cells form via the horizontal cells. The bipolar

cells lie in the inner nuclear layer together with four other major ce11 classes (horizontal,

interplexiform, arnacrine and displaced ganglion). Distal to the inner nuclear layer is the

inner plexiform layer (IPL) which contains the direct or indirect synapses between the

interneurons and the ganglion cells in the most proximal layer, the ganglion ce11 layer. On

the level of both the inner and outer plexifon layer visual information is being integrated

and modulated by horizontal and amacrine cells. respectively. Al1 ganglion cells project

their axons towards the optic disk where they converge to form the optic nerve, which

serves as the sole pathway for transmission of visual information to the brain (Adler and

Farber, 1986; Robinson, 199 1 ; Rowe, 199 1 )

1.2 Development of the mammalian retina

RetinaI structures arise from the germinal neuroepithelium, and their neuronal

development follows the same principles that have been defined for the rest of the CNS.

The precise time at which the eye primordia become induced is still in question, but studies

in amphibians indicate that this event may occur as early as gastrulation (Spemann, 1938).

Additionally Spemann's work shows that a well defined area of the anterior neural plate

seems to be largely committed to generating the eye at the neural plate stage. This

cornmitment, however, is not complete until the optic vesicle stage, since transplantation of

this neural plate region with more posterior mesoderm leads to the formation of more

posterior structures (Aldennann, 1935).

The first visible signs of eye development are observed at the neural plate stage,

which corresponds to embryonic day eight (E8) in rnice. Two small indentations, called

the optic pits, or sulci, form bilaterally on the dorsal surface of the neural plate (Figure 1-

2A). As the neural plate thickens and curls up on itself to fuse into the neural tube, the

Figure 1-2

Schematic photographs illustrating the development of the mouse eye. Sections are coronal

in E8-9.5 and sagitd in E10-19.5. Abbreviations: ciliary body (cb), comea (c), ganglion

ce11 layer (gcl), iris (i), lens (l), lens cup (lc), lens placode Op), lens vesiçle (IV), neural

retina layer (ml), neuroblast layer (nbl), optic nerve (on), optic pit (op), optic stalk (os),

optic vesicle (ov), presumptive pigment epithelium (pe), primitive forebrain (pfb),

prospective hindbrain (phb). Adapted fiom Kaufman ( 19%).

optic vesicles form as lateral outgrowths of the indentations of the diencephalon, the optic

pits. At day E9 (in mice) the optic vesicle will have extended enough to establish physical

contact with the overlying ectoderm (Figure 1-2B). Mesenchymai cells, which initially fil1

the space between the optic vesicle and the overlying surface ectoderm, partially degenerate

as the two ce11 layers establish physicai contact. Those mesenchymal ceus that remain will

give rise to the muscles of the pupil, the pigment cells of the iris, the ciliary body and the

choroid (Robinson, 1991). The development of the eye placode and the lens disk, as local

thickenings of the optic vesicle and the surface ectoderm, respectively, depends on the

establishment of physical contact between both tissues. The optic vesicle will subsequentiy

invaginate to form the the optic cup (Figure 1-2C). This invagination occurs slightly off-

center, resulting in the formation of a groove dong the ventral wall of the optic stalk. This

groove, the optic fissure, deepens as the ventral optic cup thickens to complete the cup

structure and, in the aduli eye, will serve as the exit point of ganglion ce11 axons and retinal

blood vessels (Robinson, 199 1).

The inner and the outer cup layer assume different fates &er the invagination of the

optic vesicle. Proliferation suddenly decreases in the outer cup layer leading to the

formation of a single-ce11 layer, the future retinal pigment epithelium (Figure 1-2D).

Continuing proliferation in the inner layer of the optic cup, however, leads to the formation

of an apparently homogeneous mass of cells called the neuroblast layer (Figure 1-2E). All

neuronal cells found in the adult retina, as well as the Müller glial cells, onginate from the

neuroblast layer. Only the second type of glial cells, the astrocytes, migrate into the eye,

via the optic nerve (Reh, 1992).

Mitotically active undifferentiated cells of the developing retina go through M-phase

at the outer limiting membrane (OLM), and through S-phase in the inner part of the

neuroblast layer. From there, they return to the OLM for their next division. After their

final mitosis, neurons migrate inwards to assume their proper position within the retina.

Ce11 differentiation is thought to occur after the last rnitosis but prior to migration (Hinds

and Hinds, 1978; Tumer and Cepko, 1988). The birth of neuronal cells has been found to

occur in two waves in al1 mammalian species. With rninor variations between species,

ganglion cells, horizontal cells, cone cells and amacrine cells constitute the first wave,

while bipolar cells, rod photoreceptors and Müller glial cells make up the second (Sidman,

1961; Young, 1985). In dl species studied to date lineage analysis suggests that these cells

arise from multipotent precursor cells (Cepko, 1992; Wetts and Fraser, 1988).

Coincident with strong proliferation is neuronal ce11 death as an integral part of

normal retinal development (Silver and Hughes, 1973). More than half of al1 generated

neurons have died by the time adulthood is reached, most likely because they failed to

establish synapses (Robinson, 199 1).

The fnst morphologicai signs of ce11 differentiation in the neuroblast layer are found

at E l 1 in mice. This early differentiation occurs in ganglion ce11 precunors, and thus the

ganglion ce11 layer is the first layer to separate from the neuroblast layer. The remaining

layers form simultaneously and lead to the formation of the multilayered adult retina

(Figure 1-2F). Retinal differentiation in the mouse is not complete until the second

postnatal week, concurrent with the opening of the eyes (Sidman, 1961; Hinds and Hinds,

1974, 1978, 1979; Young, 1985).

Complex developmental processes, such as the morphological changes in the

developing retina outlined above, require complex genetic control. Work of the last ten

years has led to the identification of multiple classes of developmental regulators. One such

class consists of transcriptional regulators that, based on functional and structural

characteris tics, have been named homeobox genes.

2. HOMEOBOX GENES

2.1 Discover y and Characterization of the homeobox

The discovery and study of the homeotic genes has allowed more molecular insight

into developmental processes than might have been guessed 15 years ago. Most of the

pioneering work was done in Drosophila rnelanogaster due to the relative ease of genetic

studies. The narne Homeosis was originally coined by Bateson (1894) for ~ansforrnations

of body segments 'into the likeness of something else'. In 1923 Bridges identified the first

homeotic mutation in flies. Due to the transformation of the rudimentary pair of wings, the

halteres, into an actual second pair of wings, he named the mutation bifhorux (Bridges and

Morgan, 1923). The second homeotic mutant, Antennapedia, was identified by Gehring

in 1967, as a spontaneous mutation that converted the antenna into an additional pair of legs

(Gehring, 1967). The fact that mutations in single genes led to transformation of body

segments into the likeness of others led Lewis to postulate that these homeotic genes might

represent a tandem array of conserved genes (Lewis, 1978).

The ability to clone the genes responsible for these mutations came with the advent

of chromosome walking, and was used to clone both the bithorax (Bender et al., 1983),

and the Antennapedia locus (Garber et al., 1983). Complementary DNA's that were

located to the genomic region surrounding the Antennapedia locus exhibited cross-

hybndization when used on genomic blots, the first evidence suggesting the existence of

conserved sequences predicted by Lewis (1978). Sequence analysis of the isolated cDNAs

revealed that al1 of them share a common motif of 180 nucleotides, which was termed the

homeobox due to its occurrence in homeotic genes. By analogy, the encoded protein

domain was named the homeodomain. The homeotic genes in Drosophila were found to be

organized into two complexes, the Antennapedia and bithora complexes (Gehring, 1994).

The fîst clues as to the function of the homeodomain came from the identification

of homology to DNA binding motifs of the al and a 2 yeast mating type (MAT) proteins

(Shepherd et al., 1984) and the helix-turn-helix motifs (Scott et al., 1989) of prokaryotic

transcriptional regulators (Pabo and Sauer, 1984). The presence of these putative DNA

binding domains indicated a possible involvement in transcriptional regulation. in recent

years homeodomain proteins have been proven to be transcriptional regulators with

sequence specific DNA binding properties. Homeodomains bind DNA as monomers with

high affinity (Desplan et al. 1985). The ability of the homeodomain to regulate

transcription has been demonstrated in ce11 culture for frz, Ubx and Abd-B (Jaynes and

O'Farrell, 1988; Thali et al., 1988) and in vivo for the regulation of hunchback by bicoid

(Driever and Nuesslein-Vollhard. 1989). In vitro target sites were found to have a

conserved 5'-TAAT-3' core sequence, even for relatively divergent homeodomains. Initial

homeodomain swap experiments showed that the developmental specificity of

homeodomain protein function resides in the homeodomain (Kuziora and McGinnis,

1989). In some cases, however, the homeodomain requires additional regions of the

protein to achieve its developmental specificity (Mann and Hogness, 1990). Mutagenesis

studies demonstrated that much of the DNA binding specificity resides in amino acid 50 of

the homeodomain (Treisman, 1989).

Structural analysis of homeodornains has shown that the homology with regulatory

motifs is not restricted to the sequence level. Nuclear magnetic resonance (NMR)

spectroscopy for Antennapedia (Antp) has shown that the homeodomain forms three a-

helices, with helices two and three exhibiting a helix-tum-helix motif (Qian et al., 1989).

NMR studies of Antp bound to its target sequence showed that helix three, the so-called

recognition helix, contacts DNA in the major groove. The N-terminal arm of the

homeodomain contacts bases in the minor groove and the loop between helix 1 and 2

establishes contact with the phosphate backbone. While residues that are absolutely

conserved among al1 homeodomains contact the 5'-TAAT-3' core sequence, residue 50, the

ninth residue of the recognition helix, contacts the two nucleotides immediately 5' of the

core, providing additional binding specificity (Otting et al., 1990). These findings have

been confumed by the X-ray crystallography data with the engraiied homeodomain and the

MAT a2 homeodomain (Kissinger et al., 1990; Wollberger et al., 199 1).

2.2 Phylogeny of the horneobox

One of the biggest surprises in homeobox research was the finding that homeobox

genes are not confined to huitnies. An initial evolutionary survey showed that these genes

also exist in vertebrates, including chicken, rnice and man (McGinnis et al., 1984). To

date, homeobox genes have also been identified in orgmisms as divergent as sponges,

yeast and plants (Murtha et al., 199 1 ; Seimiya et al., 1994).

Subsequent work demonstrated that homeobox genes were not only present in

many organisms, but that the structural organization of the homeotic clusters had also been

conserved. Clustering comparable to Drosophila was found in nematode, mice and man

(Gehring, 1994). Whereas invertebrates contain only one or two clusters, mamtnals such

as mice and man have four homeotic clusters in their genome. These clusters seem to have

aisen from an Anfp type prototype gene through tandem duplications (Graham et al., 1989;

Boncinelli et al., 1988 and 199 1). In mice and man each of the four clusters, designated

Hox a through d, consists of 9 or 10 homeobox genes which c m be grouped into 13

paralogous groups. Astoundingly, the physical order of the paraiogous groups in the

cluster has been conserved between fruitfly and mammals (Duboule and Dolle, 1989;

Graham et al., 1989). Al1 the genes in the mouse homeobox cluster are transcribed in the

same orientation (Boncinelli et al., 1991) and their physical order in the cluster appears to

have functional significance, in that expression of the genes at the 3' end of the cluster is

developrnentally earlier, more anterior and more responsive to retinoic acid (Robertis,

1994). Two forms of selective forces have been suggested to be necessary for the

conservation of the cluster organization. One is cis-regulatory elements that are shared

between adjacent genes in the cluster (Peifer et al., 1987). The second may be that higher

order chromatin structures within the cluster are essential targets of trans-activators

(Jürgens, 1985; Gyurkovicz et al, 1990; Campbell et al., 1995). The extreme degree of

conservation at the nucleotide and genornic level suggests that very different body plans

might be achieved, both in insects and vertebrates, by evolutionarily conserved gene

networks that possibly have similar regulatory interactions.

Expression of the Hox genes in mice was found to demarcate specific regions of the

embryo, rather than tissues or ce11 types which are established later in development

(Gehring, 1994). Genes of the Hox clusters are not expressed anterior to the hindbrain,

and therefore they cannot be involved in the development of the more anterior CNS

structures of forebrain, rnidbrain and anterior part of the hindbrain.

To date, a large number of homeobox genes have been identified that, based on

their expression pattern, may contribute to the development of the antenor structures of the

CNS. Almost one third of the 30 distinct homeodomain classes known to date have been

implicated in some aspect of head development (Kappen et al., 1993; Robertis, 1994). Of

these the Pax/Prd, POU and LIM homeodomain proteins are charactenzed by additional

protein domains, while the Dlx, Otx, Emx, Gsc, Msx and the Nkx proteins are classified

solely on the basis of their homeodomain sequences (Robertis, 1994). Arnong these, the

Pax/prd class has received particular attention due to its involvement in genetic disease in

flies, rnice and man.

2.3 Padprd class of homeobox genes

The Paxlprd class of homeobox genes is characterized by the paired domain, which

was initially identified as a conserved region between the Drosophila paired and goosebeny

proteins. The paired dornain spans 128 amino acids (Bopp et al., 1986) and has been

shown to possess sequence-specific DNA binding activity (Chalepakis et al., 199 1 ;

Treisman et al., 1991). While the paired domain is characteristic of al1 nine marnmalian

Pax proteins, only four of them also contain a homeodomain. AU four of them encode a

serine in the crucial position nine of the recognition helix (Kappen. 1993). The vertebrate

Pax genes are thought to have arisen from the paired gene through tandem duplications

(Gruss and Walter, 1992). The Pax genes are dispersed throughout the genome,

suggesting that the chromosomal environment is unimportant in dictating their expression

pattern (Strachan and Read, 1994).

Studies of the in vitro target sequences of the Padprd genes have reveaied a

surprising difference between Pax and Hox homeodomain function. Many DNA binding

proteins bind DNA as dimers or higher order complexes. However, most Hox

homeodomains were found to bind DNA as monomers. The prd homeodornain on the

other hand has been shown to bind cooperatively to palindromic binding sites.

Cooperativity was also found upon exchange of the ninth residue of the paired

homeodomain from serine (S9), to glutamine (Q9) or lysine (Kg), residues commonly

found in other homeodomain classes. However, this amino acid exchange results in a

change in the magnitude of cooperativity and the binding specificities (Wilson et al., 1993).

Although the cooperativi ty found for the paired homeodomain was shown to occur on

DNA and not in solution, its basis is not known. Possible mechanisms for the

cooperativity include direct protein-protein interactions between the homeodomains, or

allosteric changes in DNA shape after binding of the first homeodomain (Wilson et al.,

1993). New data suggests that the changes in DNA shape occurring when paired

homeodomains bind are superimposable on those found for non-paired homeodomains,

implicating that direct protein-protein interactions are a more likely primary mechanisrn of

cooperativity (Desplan, un publis hed resul ts).

Pax genes are expressed primarily during embryonic development and al1 except

Pax1 and Pax9 exhibit a distinct spatial and temporal expression pattern in the developing

nervous system. Al1 the homeobox-containing Pax genes have been suggested to be

involved in the specification of positional information, possibly the A-P and D-V axis in the

developing spinal cord and brain (Strachan and Read, 1994; Chalepakis et al., 1993).

Five of the nine mammalian Pax genes, narnely Paxl. P d , Pax3. P d and P d ,

were found to be mutated in previously described disease loci in several species, and four

of these phenotypes involve the brain (Gruss and Walther, 1992; Read, 1995). The fact

that none of the marnrnalian Hox genes could be assigned to any disease locus has been

suggested to be due to the redundancy of the four conserved clusters (Strachan and Read,

1994). Most of the identified natural Pax mutations are loss-of-function mutations,

resulting in developmental defects, while the only gain-of-function mutation identified to

date confirms the oncogenic properties that have been shown in cell-culture studies (Galili

et al., 1993, Strachan and Read 1994). Mutation analysis and ectopic expression

experiments have recently established that one of the Pax genes is essential and suffîcient

for eye developmen t .

2.4 Pax 6/eyeless; Master control genes of eye development

Par6 encodes a paired domain and a S9 homeodomain. Expression is detected in

the optic pit as early as E8.5, and later extends into the eye vesicle, optic stalk, retina and

also the lens, comea, and ectoderm from which they develop. P d is therefore expressed

in al1 the structures of the developing eye (Gruss and Walther, 1992). Mutations in the

Pax6 gene and its orthologues were found to be responsible for eyeless in Drosophila

(Quiring et al., 1994), Small eye in mice (Hogan et al., 1988) and Aniridia in humans

(Chalepakis et al., 1993). Small eye animals, in the heterozygous state, have reduced body

size, smali eyes, vacuolated lenses, cataracts, abnomally folded retinae and a reduced RPE

layer. Homozygotes completely lack eye and nasal tissues and die postnatally due to the

failure to breathe. Aniridia is an autosomal dominant disorder with variable expressivity,

which is characterized by partial or complete absence of the iris and abnormal lens, retina

and optic nerve.

The fact that eye development is controlled by orthologous genes in both vertebrates

and insects suggests that eye morphogenesis may be under similar genetic control in al1

higher eukaryotes. Pnx6/eyeless is not only essential but also sufficient for eye

development in Drosophila. Ectopic expression of eyeless or Pax6 in Drosophila imagina1

discs results in the formation of ectopic eyes, with normal morphology and a full

complement of differentiated cells (Halder et al., 1995). This finding suggests that eyeless

is the master control gene for eye development in flies. Although Pax6 has been shown to

be crucial for proper mouse eye development, some rudimentary eye structures do develop.

Thus, Pax6 may not be a master regulator of eye development in mamrnals, even though it

can functionally substitute for eyeless in Drosophila. In contrast to the fruit fly, various

rnembers of the Pax/paired family as well as other transcription factors (Liu et al., 1994)

have been shown to be expressed in the mammalian eye. Consequently vertebrate eye

morphogenesis may require the orchestrated activities of other members of the Paxlprd

family, such as the homeobox gene CkiO, in addition to regulatory genes of other types.

3. C h x l O

3.1 ChxlO biology in the mammalian retina

Ch10 identifies a new subclass of prd-like homeodomain proteins. It was

originally isolated from an adult human retinal cDNA library in a differential hybridization

scnen for evolutionarily conse~ed genes that are expressed abundantly and specifically in

the retina (Bascom et al., 1992). The human and mouse cDNA's both contain an open

reading frarne encoding a protein of 361 amino acids which contains a homeodomain,

putative transactivation domains (proline rich, serinelthreonine rich, acidic), and a putative

nuclear targeting sequence (Liu et al. 1994). Comparison of the Chxi0 sequence with

those in Genbank revealed that the gene most similar to this novel clone is a Caenorhbditis

elegans homeobox gene, cailed cehl0 (Hawkins and McGhee, 1990; Svendsen and

McGhee, 1995), leading to the designation of CHXIO (Ç.elegans-like Homeoboz m. The similarity between the two polypeptides extends into a 57 arnino acid region

immediately C-terminal to the homeodomain (Figure 1-3A) which has also k e n identified

Name Homeodomain Sequence Identity

Figure 1-3

(A) Protein dot-matrix alignment of chxlO and its closest homologue cehl0. Alignment

was done on Geneworks 2.1 with a window size of 10 amino acids and cut-off at 70%

amino acid identity. The chxlO and cehl0 proteins are 361 and 344 amino acids in length,

respectively . (B) Alignment of the c h 1 0 homeodomain with similar homeodomains of the Paxlpaired

class of homeobox genes. Mismatches are shown and respective amino acid identities to

chx 10 are indicated. Adapted from Liu et al. (1994).

in the goldfish cDNA Vsxl as a 54 amino acid region (Levine et al., 1994). Due to the

high degree of evolutionary conservation from nematode to man, Svendsen and McGhee

(1995) suggested that this 54-57 amino acid residue sequence represents a functional

protein domain and named it the CVC-domain, for the first three clones containing this

domain (çhxlO, xsx 1, ~ehlO). Based on the overall homology in the homeodomain

sequence, Ch10 belongs to the PaxlPrd farnily of horneobox genes (Figure 1-3B). The

predicted chx 10 protein also contains an octapeptide sequence as do most of the Pax genes,

but it does not contain a paired domain. Among the PaxfPrd family mernbers, chxIO is

very similar to the Drosophila aristaless protein, with 75% amino acid identity in the

homeodomain and additional conservation in outside regions, which suggests that these

genes might have developed from a cornrnon ancestor (Liu et al., 1994).

The pattern of Ch10 expression in mouse development suggested that it may play

significant roles in the formation of the retina. Northem analysis in adult mouse tissues

revealed that ChxlO expression in the retina is abundant but not specific, since weak

hybridization was also observed in pons, medulla and spinal cord (Liu et al., 1994).

Immunofluorescence, using a polyclonal antibody raised to the N-terminus of the human

protein, showed that expression in the retina is further restricted. The chx10 protein is

observed only in the inner nuclear layer, in the nuclei of bipolar cells and amacrine cells,

and possibly in al1 ce11 types of this layer. Occasional weakly staining cells were observed

in the ganglion ce11 layer and are believed to be displaced amacrine cells. Within the inner

nuclear layer expression seems to occur in a gradient with higher levels at the outer border

(Liu et al., 1994).

The developmental expression of the murine Ch10 gene was studied by 3% in

situ hybridizations (Liu et al., 1994). Transcripts were first detected at E9.5 ai the tip of

the evaginating optic vesicle (these cells constitute the presumptive neuroretina), while no

staùiing was observed in the overlying head ectoderm which will give rise to the lem, or in

the presumptive optic stalk (Figure 1-4B). As the optic vesicle invaginates to fonn the

Figure 1-4

In situ hybridization with the ChxlO probe in the mouse retina. A, C, E, G are

micrographs taken under bright field illumination, B, D, F, H are rnicrographs taken under

dark field illumination. (A, B) A transverse section of the forebrain of a 9.5 dpc mouse

embryo at the optic vesicle stage. (C, D) A section of a 1 1.5 dpc embryonic mouse eye at

the optic stage. (E, F) A section of a 16.5 dpc embryonic mouse eye. (G, H) An adult

mouse retina section. Abbreviations: forebrain (0, ganglion ce11 layer (gcl), inner nuclear

layer (hl), lens (l), neuroblast layer (nbl), neuroretinal layer (ml), outer nuclear layer (onl),

optic stak (os), optic vesicle (ov), pigment epithelium (pe). Taken from Liu et al. (1994).

optic cup, Ch10 expression remains confined to the inner layer, the actively proliferating

neuroblast layer (Figure 1-4D/F). Upon differentiation and layer formation, expression is

extinguished in the ganglion and the photoreceptor ce11 layers, but remains in the inner

nuclear layer (Figure 1 -4H).

Outside the eye, the C h i 0 transcnpt is found in the hindbrain and spinal cord. At

E 1 1 Chxi0 is expressed throughout the posterior neural tube, from the rhombencephalic

isthmus into the spinal cord. Expression in the media1 pons and medulla oblongata

correlates with regions where cranial motor nuclei and reticular nuclei develop. in addition,

expression was observed at the border of the caudal thalamus and the pretectal area of the

rostral midbrain as well as lateral to the median groove of the upper lip. However the

chx10 protein has not yet been detected in these structures in adult mouse tissues using

immunofluorescence (D. van der Kooy and R. McInnes, unpublished observation).

The developmental regulation of Ch10 expression was studied by quantitative

Northern analysis on rabbit retinae. Transcripts were first detected at a time point

correlating with optic cup formation. Levels rapidly increase 3-4 fold as horizontal cells,

bipolar cells, amacrine cells and cone photoreceptors form. While rod outer segment

differentiate, expression drops to the constant level observed in the adult (Liu et al., 1994).

The highly restricted temporal and spatial pattern of expression of Chxi0 suggests

possible functions for the protein in the developing and mature mammalian retina. Liu et

al. (1994) proposed that ChxlO might specify ce11 identity within the inner nuclear layer

and give positional cues for lamination. They also speculated that absence of ChxlO

expression in the outer nuclear layer and the ganglion ce11 layer rnight be a result of, or a

requirement for, the formation of those layers. Functional concepts like these are usually

addressed by means of creating a mouse that lacks a hinctional gene product. In this case

however, the gene did not have to be targeted, since a natural mutation in ChxlO was

identified.

3.2 ChxlO mutations and ocular retardation

Ocrlur retardation (or) is a recessive mouse mutation that was initially described by

Truslove (1962) as a spontaneous mutation amongst a CBAICSïBL colony of mice.

Homozygous mice are viable. but born blind with small eyes that have thin and poorly

differentiated retinae (Figure 1 - S m ) . Additionally, the mutant developing retinae show

decreased morphogenetic ce11 death, cataracts, optic nerve aplasia and microphthalmia. The

disease is truly recessive in that no phenotype has ever been described for the heterozygous

animals (Robb et al., 1978).

Molecular analysis showed that a mutation in Chxl O is responsible for the ocular

retardation phenotype. Initially mapped to mouse chromosome 2, Chri0 was found to lie

physically close to the ocular retardation locus (B. Taylor, M. Burmeister and R. McInnes,

unpublished). In an attempt to refine the mapping with a CA-repeat of genomic ChxlO,

none of the 170 outbred mouse crosses displayed recombination between the CA-repeat

and ocular retordution, suggesting that Ch10 might be causative for the ocular retardation

phenotype (M. Burmeister and R. McInnes, unpublished). Andysis of the Chri0 locus in

genomic DNA from ocular retardation Nce, did not reveal any gross rearrangernents.

Sequence analysis of the Chxi0 locus in one of the disease alleles, 04 showed a C to A

conversion in codon 176. This change converts a tyrosine to a premature stop codon,

resulting in a truncation of the protein within the homeodomain, indicating that mutations in

Chxl O mutation are causative for the ocular retardution phenotype. Immunofluorescence

showed that no c h 1 0 protein is ever detected in the orJ retina at any stage of development,

even at times such as El 1 when the mutant eye is morphologically normal although

possibly slightly reduced in size.

The morphological abnormalities in oJ rnice suggest a critical requirement for the

ch10 protein in eye development. The o d mutation initialiy manifests at E10.5, at which

time there is a virtual absence of morphogenic ce11 death with only 2% of the normal

number of necrotic cells being detected in the mutant neuroblast layer. Mutant retinae

Figure 1-5

Phenotypic characteristics of the ocular retardation mice (right) and their wild type litter-

mates (left). (A) Section of a El3 embryonic mouse eye with stained nuclei. Taken fiom

Tmslove (1962). (B) Aduit mice. Taken from Burmeister et al. (unpublished).

cease to develop, stay single layered and only develop a rudimentary optic fïber tayer

(Figure M A ) . The use of ce11 type specific antibodies, indicated that many major ce11

types were present, except bipolar cells. Thus, rod photoreceptors, ganglion cells and

amacrine cells are present and situated in their correct location, but neither of two bipolar

ce11 markers detected staining, suggesting that these cells are either absent or poorly

differentiated. This suggests that in addition to being necessary for general proliferation

and celi death in the developing retina, ChxlO appears to be required for the ce11 fate

determination of at least bipolar cell progenitors (M. Hankin and R. McInnes, unpublished

observations).

The identification of the mutation in ocular retardation adds to the group of mouse

mutants with eye disease phenotype for which mutations have been assigned (Pax6, Gii3,

Rrra, rds and Mi). The mutant phenotype in ocular retardation, however, is restricted to

the eye and is only detected in homozygous animals. Both the severity and the specificity

of the disease phenotype suggest that Ch10 is crucial for the proper development of the

mamrnalian retina. Evidence from our laboratory and others now suggests that chx 10

specifies a novel subclass of paired-like homeodomain proteins.

Throughout the following text 1 will be referring to homology between cDNAs or

proteins in the sense of sequence similarîty, without infemng close common ancestry. The

term orthologues will be used to describe genes from distinct species that represent the

most recent bifurcation of one common ancestral gene.

3.3 ChxlO homologues in mice and goldfish

Genomic ChxlO homologues were isolated in a screen of a genornic DNA library

for the Ch10 gene. Using a full-length mouse ChxlO cDNA as the probe, genornic

clones covering the entire ChxlO locus were obtained. In addition, a second class of

clones was observed which hybridized specifically, but less strongly, to the Ch10 cDNA.

These clones fell into three different classes, arbitrarily named 15 1, 17 1 and 48 1, and one

151

3' UTR

370 455 579 760 1143 /+ (A),,

3089

B. homeodomains

C ~ X 1 O / V S X ~ KRRHRTIFTS YQLeBLElW? NEAliYPDWA -EL PWRXQVWFQ

...... .............................. vsx 1 V... Il......... H.........

. * .... .. . . ........ ceh 10 .........Q XD...... QDs 1.. v..o.. Q......... T.

171 ----- .v..A H. ........ G . . . . . ......... A.. . . ..,.,,-..-- -i-----ir-

151 ---..*_-a-- ---------- --------*- -_-------- ------ . . . a . . . . . . . . X .

helix 1 helix 2 helix 3

CVC domains

C ~ X ~ ( ) / v s x ~ KCWGRSnmR EYGLVGAMVR HSIPLPESIL KSAK-DGIHD SCAPWLLG- - --MHKKSLE

............................. V S X ~ 1 N....N.M.G ........ EP AG....... ............ ... ....... cehl0 .T..K.T.ï.. L....T.T EAADPQQ . A . . . . . . . . M.

Figure 1-6

(A) Schematic representation of the CHXlO cDNA with the ORF indicated as an open box.

Shaded areas highlight the homeodomain and CVC domain, while vertical bars in the ORF

indicate the exon junctions. The Chrl O genomic homologous clones 15 1 and 17 1, and

their respective areas of similarîty to CMO, are indicated. The solid lines indicate the parts

of the clones 15 1 and 17 1 that correspond to the CM0 ORF, and the dotted lines indicate

putative introns. (B) Aiignment of al1 known CVC domain homeobox proteins prior to this

project. The chxlO protein is show on top and only mismatches are indicated for the other

sequences. Da& shaded areas in the homeodomain indicate the predicted a-helices.

representative of each class was malyzed. Sequence analysis within the shonest

hybridizing fragments showed the existence of potential open reading frames. In al1 three

clones these partial ORFs displayed significant homology to the ChxlO homeodomain and

displayed some conserved splice junctions with Ch10 (Figures 1-6A/B; J. Novak and R.

McInnes, unpublished).

Ch10 homologues have also been isolated from the goldfish retina. Levine and

Schechter (1993) canied out a PCR screen for homeobox genes in the goldfish retina and

identified Vsxl and Vsx2, which both encode a homeodomain and a CVC domain. The

vsx2 protein has 100% amino acid identity to ch10 in homeodornain and CVC domain and

is therefore likely to be the ChxlO orthologue of goldfish. Vsxl, on the other hand, shows

significant changes in both domains, and thus represents a Vsx2/ChrlO homologue (Figure

1 -6B; Levine et ai., 1994). Remarkably, Vsxl is expressed in the inner nuclear layer of the

goldfish retina, a location like that of ChrlO. This result indicates that these genes are

likely to be true homologues and that the homology extends beyond the nucleotide and

amino acid level to functional characteristics.

4. RATIONALE FOR THIS PROJECT

The isolation of partial mouse genornic ChxlO homologues by Jakub Novak and

the cloning of one vertebrate retinal homologue by Levine et al (1994), suggested that

ChxlO homologues are likely to exist in mammals. Homologous genes arise through

duplications of ancestral genes, which leads to functionally redundant units that

subsequentiy diverge in sequence, expression pattern, and function. Due to this process,

highly homologous clones ofien overlap considerably in their expression patterns (Strachan

and Read, 1994). For this reason, ChxlO homologues are likely to be expressed in the

retina, as is already indicated for Vsxl. Mammalian Ch10 homologues, or alternatively a

mammalian Vsxl orthologue, may therefore be represented in a retinal cDNA library,

which is why the screen for these homologues was undertaken.

CHAPTER 2: ORIGINAL RESEARCH

INTRODUCTION

This chapter describes my work on the identification of human C H X I O

homologues. To identiQ such genes, a human retinal cDNA library was screened with a

human CHXlO probe at high and low stringency. Human CHXlO clones were eliminated

from the screen using PCR analysis for CHXIO 3' UTR sequences. PCR-negative clones

were plaque purified to homogeneity and phage inserts were subcloned into a suitable

sequencing vector. Of the clones that have been analyzed to date, three were found to

encode homeodomain proteins, two of them novel (CHXIO-I and OTX3) and one

representing the previously identified human LIM homeobox gene LH2 (Xu et al., 1993).

To date 30 clones remain to be subcloned and anaiyzed. Sequence analysis of the CHXIO-

1 cDNA established its close hornology to the CHXlO cDNA in homeodomain and CVC

domain. The expression of CHXIO-1 in adult human tissues was analyzed by Northem

blotting and preliminary in situ hybridization, which showed that CHXIO-I expression

levels are far below those of CHXlO in adult retina. In addition CHXIO-I exhibits a

distinct layer specificity from CHXlO in that i t is expressed at equal levels in the inner

nuclear and the ganglion ceIl layer (Liu et al., 1994).

Cloning of a homologous cDNA from a mouse retinal library led to the

identification of the cDNA encoded by the 17 1 genomic locus, Ch.171. Sequence analysis

for Ch171 established that it is quite distinct from CHNO-1 in nucleotide and predicted

protein sequence. In contrast to CHXIO-1 in humans, C h i 7 1 is expressed at high levels

in the adult mouse retina. Genomic Southem blots were perfomed in order to establish the

phylogenetic relationship between CHXIO-1 and Chxl7l. This analysis showed that no

mouse sequence is more similar to CHXIO-I than Chxl7I is. However it seems that

CHXI O- 1 is not the closest homologue to ChxI7l in human genomic DN A, suggesting

that a human CHX17I cDNA might exist.

EXPERIMENTAL PROCEDURES

Human retinal cDNA library

tissue source: adult neuroretina

primer: oligo-dT

arnplified: no

vector: hgt 10

cloning site: EcoM

host: BNN 102 or C600 hfIA

independent recombinants: 2.106

selection: tetracycline

source: Jeremy Nathans

reference: Bascom et al., 1992

Human fetal brain (cortex) cDNA library (Stratagene #936206)

tissue source: 17- 18 week late abortion, normal femde

primer: oligo dT and random

size selected: SSkb, average 1 .O kb

amplified: once

primary plaques: 2- 106

estimated background: 4% non-recombinants

vector: WIi

cloning site: EcoRi

helper phage: R408 or VCSM 13

host: XL1-Blue

selection: tetracyciine

source: Stratagene, La Jolla, CA.

Mouse brain cDNA library

tissue source: adult mouse cortex

primer: oligo dT and random

size selected: average s ix 1 kb

amplified: yes

vector: AZAPII

cloning site: EcoM

host: XLl Blue

selection: tetracycline

source: Hayden M., UBC

Mouse newborn eye cDNA library

tissue source: Mouse eye PO-3

primer: oligo dT

size selected: >1 kb, average size 1.5-2.0 kb

amplified: once

background: >O. 1 %

number of independent clones: 1 1.6- 106

vector: pcDNA, derivative of pCDM8

cloning site: BstXI (destroyed)

host: MC10611P3

selection: ampicilllli

source: Yang Xianjie, Harvard Meàical School

Mouse retinal cDNA library (h BALBZ)

tissue source: 1 month-old Balbk mice

primer: oligo dT

amplifieci: once

vector: Charon BS(-)

cloning site: HindIII and EcoRI

host: Kg02 or LE392

selection: none

source: Anand Swaroop

reference: Qais Farjo et al., 1993.

Phage library screening

Phage were diluted in SM buffer (Sarnbrook et al., 1989). Host bacteria were

cultured overnight at 370C in 0.2% maltose LB with lOmM MgCI2, then harvested and

resuspended to an OD600 of 0.6- 1.0 in lOmM MgS04 (Sambrook et al., 1989).

Adsorptions were carried out at 370C for 20 minutes. Bacteria and adsorbed phage were

mixed with top agarose (0.7% agarose in LB, 1OmM MgCl2) which had been melted and

cooled to 50%, plated onto 1.5% agar LB plates, and incubated at 370C for 6 to 12 hours.

Hybond-N+ membranes (Amersham) were used for plaque blotting as outlined in Ausubel

et al. (1989): replica filters were incubated twice for 5 minutes in denaturing solution (0.5

M NaOH, 1.5 M NaCI), twice for 5 min in neutralizing solution (1.5 M NaCI, 0.5 M Tris

pH 7 3 , 5 min in 2x SSC (Sarnbrook et al., 1989), fixed for 5 min in 0.4 M NaOH, and

rinsed for 30 sec in 5x SSC. Filters were prehybridized for a minimum of 3 hours in 30%

or 50% formamide, lx Denhardt's solution, 5x SSPE, 0.1 % SDS and 100 mg/ml

denatured salmon-sperm DIVA at 420C (Sarnbrook et al., 1989). Hybridizations were

carried out for a minimum of 8 hours at 420C in buffer of the same composition as the

prehybridization buffer with the addition of 10% w/v dextran sulphate with approxirnately

1-2x106 cpm/rnl of hybridization buffer. Probes were labeled to a specific activity of

- 1x109 cpm/pg by the random hexanucleotide prirning method (Feinberg and Vogelstein,

1983) using [ ~ - ~ ~ P I ~ c T P (NEN). Labeled probes were spun on Sephadex G-50

(Pharmacia) columns that had been prewashed with an equal volume of 0 . 0 1 ~ TE pH 8.0,

0.1% SDS, denatured with 0.2 volumes of 1 M NaOH for 5- 10 min, and neutralized by

adding Tris pH 7.5 to a final concentration of 0.5 M before being added to hybridization

buffer. Membranes were washed twice for 20 min in 2x SSC, 0.2% SDS at room

temperature for both stringencies and twice for 20-30 min in either 2x SSC, 0.2% SDS at

500C for low stringency or 0 . 2 ~ SSC, 0.2% SDS at 65OC for high stringency. Washed

membranes were exposed to XAR5 X-ray films (Kodak) with intensifying screens at

-7OOC.

Phage DNA analysis

Phage DNA was prepared according to Grossberger ( 1987). For analytical

purposes 1 pg of phage DNA was digested with 10-20 U of restriction enzymes using

conditions specified by the enzyme manufacturers. Digests were loaded on 0.7-18

agarose gels in lx TAE (Sambrook et al., 1989) and electrophoresed in 1 x TAE.

Photographs were taken with a Siratagene Eagle Eye photodocumentation system.

Plasmid library screening

The transformed plasmid library was diluted in LB and plated onto numbered 132-

mm Hybond-N+ membranes on top of LB agar plates containing ampicillin (100 pg/ml) at

a density of 80,000 colonies per plate, and incubated upside down for 12 hours at 370C.

Replica filters were prepared according to Ausubel et al. (1989) and incubated for a few

hours until the colonies had just appeared. DNA on the replica lifts was denatured,

neutralized and fixed as recornmended by Ausubel et al. (1989). Filters were then soaked

in 2X SSC for 5 min and washed in 5x SSC, 0.5% SDS and 1mM EDTA for 30 min at

650C to remove ce11 debris. Prehybridizations, hybridizations and washes were done as

outlined above for phage library screening.

Subcloning

Cloning vector (pBluescript KS(-), Stratagene)was linearized and dephosphorylated

as outlined in Sambrook et al. (1989). Digests of phage DNA vector were electrophoresed

on 0.7% agarose gel in l x TAE. Selected fragments were cut out and DNA purified from

the gels using either GENECLEAN (BI0 101, CA) or QIAEX (Qiagen, CA) purification

kits as specified by the supplier. The DNA was resuspended in 5pl of lx TE pH 7.5. The

yield of DNA was estimated by the band intensity of a 1 pl aliquot in a lx TAE gel using a

k - H W Z size ladder as quantitation standard. Roughly 50 ng of vector and enough phage

insett for a molar ratio of 1 to 2-3 were rnixed with 0.5 pl of T4 Ligase (BRL) and 1 pl of

5x T4 ligase buffer (BRL) in a total volume of 5 pl, and the reactions incubated 3- 12 hours

at room temperature. 1-2 pl of the reaction mixture were used to transform 25 pl of DHSa

competent cells (BRL) as specified by the manufacturer. The transformed cells were plated

ont0 LB plates with 0.08 mg/ml X-gal, 0.2 rnM IPTG, and 100 pg/ml ampicillin, and

incubated ovemight at 370C.

CHXlO 3' UTR PCR

The 250 bp 3' UTR fragment of CHXlO was amplified by PCR (Saiki et al., 1988)

as single fragment from 5 CI of the pnmary library picks in at total volume of 25 pl using

the primer set 5'-AACAATTGGGAGCACATAGCC-3' and 5 ' -

AAG'ITGCAACGCACïTGCTC-3'. The components used in the amplification were: 50

mM KCI, 10 mM Tris pH 8.3, 1.5 rnM MgCl2, 0.011 gelatin, 200 pM of each dNTP,

and 200 ng of each primer. In a Perkin Elmer themocycler, an initial denaturation was

carried out for 5 min at 980C, followed by 35 cycles. Taq polymerase (10 units) and light

minera1 oil were added after the initial denaturation step. The amplification profile for the

PCR was 950C for 30 seconds, 5S°C for 30 seconds and 720C for 30 seconds.

CHX10-1 genomic PCR

The 164 bp fragment of the CHXIO-1 open reading frarne was amplified by PCR

(Saiki et al., 1988) as single fragment from 50 ng of human genomic DNA in a total

volume of 50 pl using the primer set 5'-CCATGGGGATGATAAGGAAGC-3' and 5'-

GAGGTCAATAGCCACATCTTCC-3'. The components used in the initial amplification

were: 50 m M KCl, 10 mM Tris pH 8.3, 1 .S mM MgC12,0.01% gelatin, 200 FM of each

dNTP, and 200 ng of each primer. In a Perkin Elmer themocycler. an initial denaniration

was carried out for 5 minutes at 94OC, followed by 35 cycles. Taq polymerase (IOU) and

light mineral oil were added after the initial denaturation step. The amplification profile for

the PCR was 94OC for 40 seconds, 600C for 30 seconds and 720C for 30 seconds.

PCR for the genetic mapping was done using a MouseMamster Hybrid Panel (#2

from Coriell Ce11 Repositories, Panel#2, Carnden, NJ, USA). The components used in the

initial amplification were: 67 mM Tris pH 8.8, 6.7 m M MgCI2, 10 m M P- mercaptoethanol, 16.6 rnM (NH&S04,6.8 pM EDTA, 170 pg/rnl BSA, l .S mM dNTPs

each, 10% DMSO, and 200 ng of each primer. Initial denaturation, amounts of Taq

polymerase, and the amplification profile were as described above.

Plasmid DNA analysis

Plasmid DNA was prepared by the boiling prep method from Sambrook et al.

(1989) with slight modifications (J. Rornrnens, pers. comm.). In brief, bacteria were

collected from 3 ml of ovemight culture. The pellet of bacteria was resuspended in 250 pl

of STET and 30 pl of lysozyme (10 mg/rnl) were added. Bacteria were placed in a rolling

boil for 55-60 seconds. After centrihigaiion the supernatant was precipitated in two

volumes of ethanol, incubated at room temperature for 5 minutes and centrifuged in a

micro-centrifuge for 5 minutes. The pellet was resuspended in 300 pl of 0.3 M Sodium

acetate pH 7-7.5 and 750 pl of ethanol were added (room temperature for 5-15 minutes).

After centrifugation the pellet was washed in 70% ethanol, re-centrifuged and air-dried at

room temperature for 5- 10 minutes.

Sequence analysis

DNA was sequenced using the T7 polymerase DNA sequencing kit from

Phannacia. Sequencing reactions were carried out with [ ~ ~ S I ~ A T P (NEN) according to the

Pharmacia protocol, except that reactions were performed in a Nunc micro-titer plate.

Reactinns were run on 6% polyacrylamide gels (acrylamide/N'Nt-bis-methylene-

acrylamide ratio 36.5/1,0.053% APS, 0.052% TEMED) at 1500-2000 V, gels were fixed

with a 10% methanol, 10% glacial acetic acid solution, dried at 800C and exposed to an X-

ray film (Kodak). Sequences were compareci to released sequences at Genbank using the

BLAST server (blast@ncbi.nlrn.nih.gov; Altschul et al., 1990). Sequence alignrnents

were performed on Geneworks 2.1 from Intelligenetics using standard parameters.

Map location of known human or mouse phenotypes

The chromosomal location of human genetic diseases or mouse phenotypes were

determined using the on-line Mendelian inheritance in man (OMIM) server at the Genome

Data Base (http://gdbwww.gdb.org/gdbdoc/topq.html) or the Jackson Laboratory Mouse

Locus catalog (http://www.infonnatics.jax.org/mgd.html), with a Netscape 0.96 software

package.

Eye donations

Human eyes for retinal RNA preparations were obtained from the Ontario Eye Bank

(Toronto).

Total RNA purification

Total RNA was prepared from tissue samples using the T W O L method (GIBCO

BRL, Cat.#15596, 1992).

Poly-(A)+ RNA purification

Poly-(A)+ RNA was prepared from total RNA using a Oligotex-dT purification kit

(Qiagen, Catalog #70022). Purification is based on hybridization of the poly-(A) tail to

oligo-dT primers that are coupled to a solid phase matrix. Since hybridization depends on

high salt concentration, the poly-(A)+ RNA is subsequently eluted in low ionic strenght

buffer.

Northern blot analysis

Total RNA and poly-(A)+ RNA gels were prepared and blotied according to

Sambrook et al. (1989). Hybridizations were carried out in the high stringency

hybridization buffer under the conditions described above, and washed using the same high

stringency washing conditions.

Tissue preparation for in situ hybridization

The neuroretina layer was removed from human eyes (Ontario Eye Bank) and

rinsed once in PBS, followed by a two hour fixation in 4% paraformaldehyde in PBS at

room temperature. Sarnples were dehydrated by passage through an ethanol series (70%,

85%, 95%, two times 100%) and embedded in paraKin. Retinai sections (-7pm thickness)

were cut and placed on pretreated, RNAse fkee slides. The slides were subsequently baked

ovemight in an oven at 55-600C (Y.Zhou, pers. c o r n ) .

Digoxygenin in situ hybridization

Digoxigenin labeled sense and antisense nboprobes were generated by in vitro

translation of a non-conserved part of the CHXIO-1 cDNA as probe (nucleotide 758-

1696). In situ hybridization were carried out following a protocol from Boehringer

Mannheim.

Genomic Southern Blot snalysis

Human and mouse genornic DNA were digested, electrophoresed and blotted as

outlined in Sambrook et al. (1989). Hybridizations were done in the buffer described

above at 50% and 40% formamide concentrations, while membranes were washed at 650C

in 0 . 2 ~ SSC, 0.2% SDS and 550C in 2x SSC, 0.2% SDS, respectively.

RESULTS

1. Attribution of data

Primary screen of 1.5- 106 p h (Lin Liu)

PCR analysis and plaque purification for class 1 (David diCiommo and Melanie Schumach)

Physical mapping of the OTX3 gene to chromosome 19q13.3 (Aiessandra Duncan)

Physical mapping of CHXIO-1 to chromosome 20 (Catherine Duff)

Physical mapping of CHXIO- I to chromosome 20p 1 1.2 1 q 1 1.2 1 (Henry Heng)

Northem analysis for Ch171 (Bryan Snow)

2. Human retinal screen for CHXZO homologues

Three million plaque forrning units (phi) of an unamplified oligo-dT primed adult

human retinal cDNA library were screened by hybridization to a 680 nucleotide Apal

fragment of the human CM10 cDNA, which spans the homeobox and the CVC-box.

Duplicate lifts were screened at high and low stringency (see Figure 2- 1) and clones were

grouped according to the presence or absence of a signal at either stringency (Figure 2- 1).

Three classes of clones were identified: Class 1 clones, which gave signals in low

stringency only, Class 2 clones, with signai at low and high stringency, but with markedly

reduced signals at high stringency, and Class 3 clones, with almost equally strong signals

under both conditions. Based on the high degree of sequence similarity between CHXlO

and both mouse clone 17 1 and goldfish V s x l , a true CHXI0 homologue should be

expected to appear in Classes two and three.

In order to exclude CHXI0 clones from the screen, ail primary picks were screened

for CHXIO-specific sequences by PCR, using primers to the CHXlO 3' UTR,

imrnediately upstream of the poly-(A) tail. Most clones from an oligo-dT primed library

should contain this part of the cDNA. Al1 primary picks of Classes 2 and 3, totaling 450,

and Class 1, totaiing 85, were screened using this PCR approach. The expected band size

3 a 106 pfu of an unamplified oligo-dT primed adult human retinal cDNA library in hgt 10

0 . 2 ~ SSC/ 0.2% SDS @ 6SoC

probe: CHXlO 680bp ApaI fragment 85

PCR: CHXlO 3' UTR primers l plaque purification

subclonelsequence

11 CHXIO-1 23 CHXlO 2 LH2 5 unknown 30 yet to be subcloned

Figure 2-1

Flow-chart of the human adult retinal screen for CHXlO homologues, with a summary of

di clones analyzed to date.

of 144 bp was reproducibly found for 357 of the 450 Class 2/3 clones and 23 of the 85

Class 1 clones, suggesting them to be CHXlO cDNAs. Ninety-three of the high stringency

clones were shown not to contain this most 3' part of the CHXlO cDNA, and 66 were

plaque purified to homogeneity (Figure 2- 1). Similarly, 49 of the 62 PCR negative Class

1 clones were plaque purified to homogeneity (Figure 2-1).

Plaque pure phage of Classes 1,2 and 3 were dot-blotted on a lawn of host bacteria

and incubated uniil phage plaques becarne visible. Multiple lifts were hybridized to the 680

nucleotide Apal fragment of the human C M 1 0 cDNA at high and low stringency. This

analysis confinned the initial classification (Figure 2-2). Subsequently phage DNA was

prepared, inserts were excised and gel purified. and subcloned into pBluescript for

sequence analysis. My studies of 25 of these clones have led to the identification of three

homeobox containing cDNAs, OTXJ, LH2 and CHXIO-1 , which are described in the

follow ing text.

3. OTX3, a novel orthodenticle homologue

The clone described in this section was actually isolated from a previous low

stringency screen for CHXIO homologues that was initiated by Midhat Osman and

Elisabeth Garami. In general, this screen was not technically satisfactory, but it did yield

one homeobox-containing clone. Due to the conditions used to isolate this clone, it is

categorized here as a Class 1 clone. Sequence analysis revealed that this clone encoded a

homeodomain-containing ORF that was tmncated at the 5' end. Due to this truncation only

a partial homeobox sequence is available, which is highly homologous to the mouse Otx

homeoboxes, with 81% nucleotide identity to Otxl and 77% nucleotide identity to 01x2

(Figure 2-3A). Cornparisons were only made to the mouse genes since the sequence of the

human genes has not been made public. The changes in the nucleotide sequence result in

the substitution of four of the 36 amino acids of the homeodomain that are encoded in this

cDNA clone (Figure 2-3B). This finding suggests clearly that this cDNA represents a

Figure 2-2

A Dot-blot of 59 plaque pure phage of Classes 1, 2 and 3 were hybridized to a 680

nucleotide Apal fragment of the human CHXlO cDNA probed with 680ApaI at low

stnngency (see Figure 2-1) in panel (A) and high stringency in panel (B). Typical

representatives of the isolated clones, are clone #l for CHXIO, clone #97 for C H X l O- I

and clone #22 for LH2.

A. Consensus CGA . GGGAG . G . AC. AC. TT . AC. . G .. CA CAGCT . GACG T .... G . . . . . . . . T ... c . 60

O a l otx2

. . . C. .... C .C..C..C.. C..GC.CT.. ..... G.... .GCTC.AGGC Gt7i'G.TCG.A

... A. .... A .G..G..T.. T..TA.GG.. ..... C.... .TCTG.AAGC TCTG.TTG.C

. Consensus . .G. c . C. . T ACCCAGAC . T CT. .... .G . GA. GAGGTGG C.CT. m . AT CAA. TGCC. 120

OTX3 GT.V.C.AG. ........ G. ..ATGCCC.T . . G....... .T..G..G.. . . . TC. ... T ot~f AA.A.T.GC. ........ A. ..TCATGC.C . . G....... .A..C..G.. . . . CC....A 01x2 AA.A.C.GG. . . . . . . . . A. ..TCATGA.G .. A....... .A..G..A.. ... CT....A

Consensus GA. TCCAG . G T . CAGGT . TG GTT . MGM. CG. . G . GC . A A. TGC . G . CA . CAGC . . CAG I 80

om3 .. G... ..G. .T.....T.. ... C.....C ..GA.G..T. .A...A.G.. G....GA... Ou1 . . G.. . . . A. .C.....T.. ... C.....C ..CC,C..C. .G...C.C.. G....AG... Oa2 .. A.....G. .G.....A.. ... T.....T ..AA.A..T. .G...c.c.. A....AG...

otx 1 otx2

otx3 otx 1 otx2

ou3 otx 1 otx2

otx3 otx 1 otx2

oix3 otx 1 otx2

YAREEVALKI NLPESRVQVW FKNRRAKCRQ QRQQQKQQQQ PPGGQAKARP AKRRA----- 7 1 FMREEVALXI NLPESRVQVW FKNRRARCRQ QQQ------- -SGSGTKSRP AKKKSSPVRE 117 FMREWALKr NLPESRVQVW FKNRRAKCRQ QQQQQ----- QNGGQNKVRP AKXKSSPARE 90

WSPASESPLX EAQRAG---- LVASG--PSL TSAPYAMTYA PASAF---CS SPSAYG---- 159 WSPASISPGS APASVSVPEP LAAPSNTSCM QRSAAGAAT AAASYPMSYG QGGSYGQGYP 237 WSPASISPLS DP-------- LSTSS--SCM QRS-YPMTYL QA----------- SGYSQGYA 160

-SPSSYFSGL D--PYLSPMV PQLGGPALSP LSGPSVGFSL AQSP---TSL ---SG----- 205 TPSSSYFGGV DCSSYLAPMH SHHHPHQLSP MAPSSMAGHH HHHPWHPL SQSSGHHHHH 296 -GSTSYFGGM DCGSYLTPMH HQLPGPG-AT LSPMGTNAVT SHLNQSPASL ---ST----- 210

QSYG- ---AYSP-VD SLEFKDPTG- ----m3KFTY NPMDPLDYKD Q-SAWKPQIL. 248 HHHHHQGYGG SGLAFNS -AD CLDYKEPGAA AASSAWKfrNF NSPDCLDYKD Q- ASWRPQVt . 354 ---a- QGYGA SSLGFNSTTD CLDYKDQTA- ----SWKLNF NA-DCLDYKD QTSSWKFQVL. 3 9

Figure 2-3

Multiple alignments of 0TX3 with its two closest homologues Otxl and Oa2. (A)

Nucleotide alignment of the respective homeobox sequences. (B) Amino acid alignrnent of

the respective 5' truncated peptides, with identical areas shaded.

novel homeodomain protein, which we named OTX3. In situ hybridization (ISH) mapped

this gene to human 19q 13.3 (A. Duncan, Queens University, pers. cornm.). The different

map locations of OTXl (12~13, Kastury et al., 1994) and OTX2 (14q21-22, Kastury et

al., 1994) confirm the conclusion that 0TX3 represents a novel gene. No diseases

involving eye or neural tissues have been linked to this location in humans. Similarly, no

abnormal mouse phenotypes map to the homologous syntenic region of the mouse genome,

chromosome 7, between 1 and 22 centi Morgans (CM).

4. Human LIM homeobox gene t H 2

Sequence analysis of clones #22 and #29 showed that they both represent part of

the previously identified cDNA for the human LIM homeodomain gene LH2 (Xu, et al.

1993). The homeobox of this gene has 601 nucleotide identity to the CHXIO homeobox

and no significant similarity to any other region of the C M 0 cDNA.

5. CHXIO-2, a novel CHXIO-like homeobox gene

5.1 Cloning

A novel CHNO-like cDNA was initially identified from the overlapping clones #4

and #28. This cDNA contains both a homeobox and a CVC box, and was named CHXIO-

1, due to its overall similarity to CHXIO. Reprobing of the plaque pure phage dot blots at

high stringency showed that another 14 clones hybridized strongly to both #4 and #28 and

thus most likely represented the sarne cDNA (data not shown). Subcloning and sequencing

of seven additional clones enabled the identification of a 1947 bp long composite cDNA

(Figure 2-4AB). The CHXIO-1 transcript contains an intemal EcoRI site which resulted

in the loss of the most 3' 250 bp in many of the clones (Figure 2-4A), most likely when

excising the insert from the EcoRI cloning site. This fiagrnent most likely constitutes part

of the cDNA since it has been isolated from three unique clones. The CHXIO-1 cDNA

encodes a full length OW. In the 5' UTR the CHXIO-1 cDNA contains an in-fiame ochre

CAAGCGCGAC GTCTCTGGAT CCCGTTTGCT TGCTiGGAA CCATGACCGG CCGGGACTCG CmCCGACG GGCGCACTAG CAGCAGGGCG 90 - M T G R D 5 L S D G R T S S R A CTGGTGCCTG GCGGTTCCCC TAGGGGCTCG CGCCCCGGGG ClTCGCCATC ACGGACCTGC TGGGCTTGGA GGCCGAGCTG CCCGGCGCCC 180 L V P G G S P R G S R P G A S P S R T C W A W R P S C P A P GCTGGCCCAC GACAGGGATC TGGCTGCGAG GGTCCGGCAG TCGCGCCGTG CCCGGGCCCG GGCTTGACGG CTCCAGCTGG CGCGTGGGGG 270 A G P G Q G S G C E G P A V A P C P G P G L T A P A G A W G CCCTACCGCT GGGACTCGGC CTCCTCTGTG GCTTCGGCAC GCAGCCGCCG GCGCCGCTCG AGCACCCTGC CTGCTCCTAG CGGACGTGCC 360 P Y R W D S A S S V A S A R S R R R R S S T L P A P 5 G R A GTTCCTGCCG CCCAGGGGCC CCGAGCCCGC TGCCCCGCTG GCTCCCAGCC GTCCGCCGCC TCCGCTCGGC CGCCAGAAGC GCAACGCAGC 450 V P A A Q G P R A R C P A G S Q P S A A C A R P P E A - , Q - R - S GTCTCCACGT CCGATGAGGA CAGCCAGTCT GAAGACAGGA ATGACCTAAA GGCATCCCCC ACCTTGGGCA AGAGGAAGM%@GCG~'C 540 V S T S D E D S Q S E D R N D L K A S P T L G K R K ~ ~ R ~ ~ R ~ ~ ~ , AGGACACjiTt $ACTGCTCAa CCAGCTGGM GAGTTCGAGA A6GCATTCAC;~CC;CACGCCÇAC TACCCyCAtE.TbTATCC5~~~~CCT~ 630

.-i.'y2.~ . j= :A .H'' Q-L.:E*, :E- ~,E.:Y - ' : ~ : . ~ : , ~ : : , ~ ~ L ; ' ~ ~ ' . - ~$.p;~$jY~;&'~ . +. &.:gr. 6 - w CKAGff CCC CWGACCGG ATACAGGTCT :Gm..AA ; c c G ~ G $ G c ~ * Q $ ~ ~ ~ A c c ~ ~ G M GCGCTGCGGC 720 ; k : 1 v w - : Q ~ & R W GGCAGCAGCG TGATGGCCCA GTACGGGCTG TACGGCGCCA- TGGTGCGCCA CTGCATCCCG CTGCCCAGAC TCCGTGCTCA ACTGCCCGAG 810 G S S V M A E Y G L Y G A M V R H C I P L P R L R A Q L P E GCCGGCCTGC TGGGCTCCTG CGCGCCCTGG CTCCTGGGGA TGCATAAAAA ATCCATGGGG ATGATAAGGA AGCCAGGMG TGAAGATAAG 900 G G L L G S C A P W L L G W H K K s u c M I R K P G S E D K TTGGCAGGAC TCTGGGGCTC TGACCACTTC AAAGAAGGTT CTAGCCAGAG TGAGTCACGA TCACAGAGAG GCTCAGATAA AGTGAGCCCT 990 L A G L W G S D H F K E G S S Q S E S G S Q R G S D K V S P GAGAATGGCT TGGAACATGT GGCTATTCAC CTCTCCAGCT CTGCCCGGCA GGAGACCAAG AAAGTGCACC CTCGGGCTGG TGCtCAAGGA 1080 E N G L E D V A I D L S S S A R Q E T K K V H P G A G A Q G GGCTCCAACT CCACGGCACT CGAGGGGCCC CAGCCAGGGA ACCTGGGAGC CACATGAGAC CCACAGGTCC CACTGTCAAA GGCTGAAAAT 1170 G S N S T A L E G P Q P G K V C A T . GTGCATATTC CTCACTGGCA T m C C A A A A GACAAAAATT GTCCAAGACA TATACTCTCA G m G A T r T CITCTGACAA TGTCAAGATA 1260 AAATGCAATG CCACTTGCTT TAAGAGGACA GATGAGTGAC OAGGGAAAA TTCTAf fTCA TTCTAGAATG GAAACGGITC CTTAATGGCA 1350 CTTTAACCAG TTAACTTGGT GCAAAACTTT TWCTCTT TTGTAGCTTG AGCAGGGAGG ATCCGAAGïT CAAAAATGAT TTCACTGCTG 1440 GGCATCATTT GAGATGTGTG GTTCAATGTA CTGTATAGAT GAATAGGTTA GGTCTGATGT CCTCATTCTC AATTAGGAAA TATAAAATTG 1530 TTTAATATCC AGAAGAATCT CATATCATCA TAGTGAAGAC TCCATACAGA CACATGAATG AATTATATK T C m A C C T T GAACTTGGCC 1620 T T G G A T A ï l l C A A f m C r r GTTAGGGAGT AATTTTGTGT TTTCTTGACT CTCACrrGAT AGGCTATCCA TATTGGAATT CAAAGAAACC 1710 CATACTCTAG T C G m C T G C A T T T C C m A AAAATAGCAT GGGATCATAT TTAAACAATT GTTGGATAGC TGGAAACAGT C r r m A C G T 1 8 0 CAGACCAGGC T i T K G T C l T TCAAAATTTG TGATTGAAAG C A C m A G T G A C r r G G A l l l TGTAAATCTT T C C m C C T G C G f l l C C T T T 1090 GCCATTTATT TATTCTTTAA A A U C A T ITTGTGGT GT GTTAAAAAAA AAAAAAA 1947

Figure 2-4

(A) Schematic representation of the CHXIO-1 cDNA and al l clones that were isolated from

the human retinal screen. (B) Nucleotide and predicted amino acid sequence of the human

CHXlO-1 cDNA. Shaded areas represent the homeobox (dark) and the CVC box (light).

The upstream in-frame stop codon is double underlined. The putative poly-adenylation

signal in the 3' UTR is underlined.

stop codon (TAA) nine nucleotides upstream of the start codon at nucleotide 1 (ATG). The

sequences surrounding the ATG (GAACCATGA) correspond well to the Kozak sequence

(CC(80%A, 2O%G)CCATGG(40%)) for translational starts (Kozak, 1984). The open

reading frame between nucleotide 1 and the opal stop codon (TGA) at nucleotide 1092

encodes a predicted protein of 364 amino acids, with predicted molecular weight of 39

kDa. At the nucleotide level the CHXIO-1 cDNA is 54% identical to CHXlO over the

entire ORF, with the highest conservation in the homeobox (78% nucleotide identity) and

the CVC box (77% nucleotide identity). The CHXIO-1 3' UTR contains a poly-

adenylation site at nucleotide 1869, 22 nucleotides upstream of a poly-(A) tail,

corresponding well with the average distance of 1 1-30 nucleotides (Lewin, 1990).

CHXIO-1 encodes a homeodomain of the prd-like type (Bürglin, 1994; residues

163 and 222), with a glutamine at position 50; the residue at this position has been shown

to be crucial in determining the DNA binding specificity (Treisman et al., 199 1). The

homeodomain sequence is most sirnilar to the chxlO homeodomain with 92% amino acid

identity and less similar to the cehl0 homeodomain with 78% arnino acid identity (Figure

2-5A). The chx 10-1 protein also contains a CVC-domain (residue 223-276), immediately

C-terminal to the homeodomain, with 70% amino acid identity to the chxlO CVC domain

and 6 1 % amino acid identity to the cehl0 CVC domain. Outside of the homeodomain and

CVC domain chx 10 and chx 10- l are highly dissirnilar with 17% amino acid identity in the

N-termini and 19% amino acid identity in the C-termini. In these outside regions, the

chxl0-1 protein is slightly more sirnilar to the ceh10 proiein in the N-terminus (22% amino

acid identity) but slightly less similar in the C-terminus (12% amino acid identity).

5.2 Putative protein domains

Comparison of the chxl0-1 peptide to sequences in Genbank led to the

identification of several other putative protein domains. The predicted chxlO-1 protein

contains sequences that resemble putative activation domains (Figure 2-5B). N-tenninal to

chxl0-1 - - nuciear targeting

N--~CVC= -C octapeptide

O 100 200 300 aa

Figure 2-5

O

100 -

chxlO 200 -

300- aa

(A) Dot matrix alignrnent of the human chx 10 and chx 10- 1 peptides. Alignment was done

on Geneworks 2.1 with a window size of 10 amino acids and cut off at 70% amino acid

I 1

\ HD CVC

I I I 7 I I

identity. The length of the human chxlO and chxl0-1 proteins are 361 and 364 amino

-

-

- 92% 70% -

C

acids, respectively. (B) Schematic representation of the predicted chxlO and chxlO-1

polypeptides. A putative nuclear targeting sequence is located immediately N-terminal to

the homeodomain. Both of the predicted proteins contain potential trans-activation domains

of the proline-rich serinelthreonine-rich or acidic type.

the horneodomain is a region with 24% proline content, reminiscent of the proline-rich

activation domains found in CTFlR/3 (Mitchell and Tijan, 1989; Mermod et al., 1989).

Similar to CTFI, this region is split up into two blocks (residue 18-77 and 100-131).

Interestingly, two irnperfect repeats (CEAPAGPG and AECPGPG), comparable to those

described in the AP-2 proline-rich region, can be found in block 1 (Williams and Tijan,

199 1). C-terminal to the proline-rich region is a 12 residue sequence (residues 140 to 15 1)

that contains 50% acidic amino acids (two glutamic acids and four aspartic acids),

comparable to the longer acidic activation domain found in the AP-I/Jun/TFIID proteins

(Mitchell and Tijan, 1989). In the C-terminus of chxl0-I there is a 31 amino acid region

(residue 300-330) with 32% serinehhreonine content resembling the trans-activation

domain described for the POU homeodomain protein GHF-1 (Theill et ai., 1989). In

addition, this region is also fairly acidic (248 between 284-326, with 5 glutamic acids and

4 aspartic acids). The chx 10- 1 protein aiso encodes a putative nuclear targeting sequence,

as defined by Dingwall and Laskey (1991). This sequence, between residues 160-167

(KRKKRRHR), extends five amino acids into the homeodomain.

5.3 Mapping to the chromosomes

In order to determine whether CHXIO-1 might be implicated in human disease, the

gene was mapped by both PCR and in situ hybridization. PCR primers were devised for

use on a chromosome-hybrid panel. Since this technique requires amplification from

genomic DNA. it is best to devise primers within one exon. 1 assumed that the genomic

structure of CHXIO-1 might be quite similar to CHXIO, and therefore primers were

constructed to a region of CHXIO-1 (nucleotides 820-840 and 959-980) corresponding to

exon 4 of the CHXlO gene (Liu et al., 1994). 1 demonstrated that these primers ampli@ a

fragment of the predicted size (160 bp) from human genomic DNA. These pnmers were

found to ampli@ a single band of 160 bp from chromosome 20 of a Coriell PCR panel of

human chromosome hybrids (C. Duff, Hospital for Sick Children, pers. comrn.). This

result was confirmed and refined by Henry Heng (Hospital for Sick Children) using clone

#4 (Figure 2-4A) for fluorescent-in situ-hybridization (FISH). A strong signal was

observed on human chromosome 20, in a band corresponding to the region of 20p 1 1.2 1 -

q 1 1.2 1 (Figure 2-6A/B).

5.4 Northern analysis on total RNA

In order to determine the tissue specificity of CHXI0-I expression in adult human

tissues, a multi-tissue Northern blot, containing 5 pg of total RNA from retina, brain, liver,

kidney, spleen, fibroblasts and lymphoblasts, was hybridized with a CHXIO-1 probe

(nucleotide 758- 1698) at high stringency. No signal was detected in any of the tissues after

a 2 day exposure. Even with a 10 day exposure, only a faint band corresponding to

roughly 2 kb, could be recognized in the retinal RNA lane, which is absent from the other

lanes (Figure 2-7A). The low abundance of the CHXIO-I transcript is in sharp contrast to

the high level of expression of CHXlO (Liu et al., 1994), but was confirmed upon

reprobing of the Northem membrane with a CHXlO probe, which reproduced the strong

signal seen by Liu et al. (1 994) after only ovemight exposure (data not shown).

5.5 Northern analysis on poly-(A)+ RNA

To confirm the expression of CHXIO-1 in the retina, and to measure the size of the

transcript, blots of poly-(A)+ RNA blots were performed. Poly-(A)+ RNA was prepared

from 120 pg of total RNA from human lymphoblasts and retina, leading to an estimated

yield of 2-3 pg of poly-(A)+ RNA. Hybridization with the CHXIO-f probe at high

stringency revealed a distinct band of 2.05-2.25 kb specific to the retina lane only (Figure

2-7B). This corresponds well with the predicted CHXIO-1 cDNA length of 1950 bp,

when considering an average poly-(A) tail of roughly 200 (A)'s, as found for other

transcripts (Lewin, 1990).

C. Mouse chr. 2

t

syntenic regions

Figure 2-6

Mapping of CHX10-2 to human chromosomes. (NB) FISH analysis of CHXIO-1 on

human metaphase chromosomes. (A) DAPI staining (B) Fluorescencelhybridization is

distinctiy observed on chromosome 20 corresponding to the centromeric region (arrows).

(C) Schematic representation of human chromosome 20 and its syntenic region in mice, on

chromosome 2. Map locations of human CHXIO-1. mouse Ch172 and the mouse

developmental eye defects Coloboma (Cm) und blind-sterile (bs) are indicated.

Figure 2-7

Northem analysis of CHXIO-1 in adult human tissues. (A) Total RNA blot after 10 day

exposure shows a faint signal in only the retina lane. (B) Poly-(A)+ RNA blot after

ovemight exposure exhibits a distinct band in only the retina lane.

5.6 In s i tu hybridizations

In order to detexmine whether CHXlO-1 is expressed in a layer specific manner

within the adult retina, in situ hybridizations were performed. Using paraformaldehyde

fixed sections of adult human retinae, staining was observed specifically with the antisense

but not the sense probe. The staining was observed in the inner nuclear layer and the

ganglion ce11 layer (Figure 2-8).

6. isolation of mutine CHXlO-1: Chx171

6.1 Cloning

Due to the difficulty of examining gene expression in man, and the relative ease of

studying development in the mouse, 1 decided to isolate the mouse orthologue of CHXIO-

1. However neither two million plaque forming units of an adult mouse brain library (M.

Hayden, pers.comrn.) nor one million colony fomiing units of a newborn eye library (Y.

Xinajie, pers.cornrn.) produced any positives when screened with clone #28 (see Figure 2-

4A) at high stringency. However 220 hybridizing clones were observed when screening

2.6 million plaque forming units of an adult mouse retina library (A. Swaroop,

pers.cornm.). Subcloning and sequencing of 1 1 arbitra~ily picked clones showed that they

al1 represented the same cDNA (Figure 2-9A). Sequence analysis of the two longest clones

MR3 1D and MR42, lead to the identification of a 2.6 kb long cDNA (Figure 2-LOB). This

cDNA may not be full-length since clone MR27 exhibits a 3' UTR that is 0.8 kb longer.

However, this 3' UTR sequence was not included in the analysis since it has only been

found in one clone. Cornparisons with Our database revealed that the isoiated cDNA

exhibited 100% nucleotide identity to the open reading frame fragment of 123 base-pairs

within the genomic clone 171, leading to the narne Chxl71. The 5' UTR of the cDNA

contains an in-frame arnber stop codon (TAG) 18 nucleotides upstream of the Start

(GAAGCATGA), which corresponds well to the Kozak sequence (CC(80%A,

20%G)CCATGG(40%)) for translational starts (Kozak, 1984). The open reading frame

Figure 2-8

In situ hybridizations of sections of adult human retina with CHXIO-1 probes. (A) Some

non-specific hybridization to the sense probe is observed in the imer nuclear layer (INL).

(B) Specific hybridization is observed in the i ~ e r nuclear (N) and the ganglion ceil layer

(GCL) after hybridization with the antisense probe. The bar in (A) represents 100 Pm.

B. GAAilCAAGC GAGGACTCAA CTCCGGCTCC T A G C O A A G GACTGAGAAG CATGACTGGA CGGGATGGGC llTCGGATGC GCGCTCCAGG - M T G R D G L S D A R S R

AGTAGAGCCC TGGCGCCAGG CTGTCCTCCC ACCGGCTCTC GCCnCCAAG C m G C C A T C AATGACCTGC TGGGATfGGA GGCACACCTG S R A L A P C C P P T G S R L R S F A 1 N O L 1 G L E A D L CCAACTCCGG CGGAGCCTGG GCTAAGATCC AACAGCGGAG ATCCAGCCGA GGCAATAGGG TCGGGACCGG GACCGGCGCC AGGACTCTGC P T P A E P G L R S N S G D P A € A I G S G P G P G P G L C GGCTCCTGCC CTGCTCGCGG CGCTCTCCCG CTGGGCCTGG CGCTCCTCTG CGGCTTCGCG GCGCAGCCCC CTTCCGTGCC GCCGCCGCTC G S C P A R G A L P L G L G L L C G F G A Q P P S V P P P L GTGCGCGCTG CCTGCTCCTG CCCGACTGCG GCTTCTGCCA TCTGCAGGAC CCGAGCCCTG GTCTCCCCCA GGGCCCGGTC CACCCGCGCC V R A A C S C P T A A S A I C R T R A L V S P R A R S T R A TGCGCTCGGC AGCCAGCAGC GCAGCGGAGC GTCTCCACGT CGGATGGGCA CAGTCCATCT CMGAAAAGA ATGACCCGAA GATGTCCCTT

AGAAAACCAG AAAGTGAAGA CAAGTTGGCA GGACTCTGGG AGTTTGACCA TCTCAAAAAG GGTGCTAATA AGGATGAGGT GGACCCTGAG R K P E S E D K L A G L W E F D H L K K G A N K D E V D P E AGGGGGCCAG ACGAAACAAC CCAGAACCCT GAGAATAGCT TGCAGGATGT GGCCATTGAC CTGTCCAGCT CTfCCAGGCA GGAGACTAAG R G P O E T T Q N P E N S L E D V A I 0 L S S S S R Q E T K AAAATGCCCC CAGGGTCCAG TACTCAGCTG CCCCAGCCCC AACAGGTGGG AGCCTCATGA GACTGACAGA TTGCGTCCCC CAAAGCTGGA

K H P P G 5 S T Q L P Q P Q Q V G A S . CAAACACCTT CAGTTYGATT l lCTCTCTGA AATCTCTGGA TAAAAAGCAA CACTACAACA CTGAATAAAC AATGACGTAG GGAAAGTTCC C m A G f f C T AGAGTGGGGA ACATKATAC CACCTCTCAA GCCAGTCAGT TTGGTCTAAA ATCTTGTTï l TCCTTAGTGC AATCTGAACA TGGAAGGTTT GAAGTTCAAA CAAAATTTTA GGGATGGGGT ATGACGTCGC TTAGTTAATA GTACACAAGA AGGCCTGGAT TCAATACCCG GCACCACTCA AACCAGATAC AGTAGCACGC ACCTGCATCC CAGCACTCAG GGGCCGGAGA CAAGAGATCA GATGTTCAAG AGCATCCCAG AGAGAGAGAG AGACAGAGAG AGACAGAGAG AGATTATTAC TCCCTAGCAT TATTfAAGAT GTGGTTCACT CATTAAAGAG GGCTGGTCTG ACCTCCTCAC TCTAAGTTAG AATATTTGGT TTGGTTl?GG TGAAATGGG TTCTfGCTGT GCAGCCCAGG CICGCTTCAT TTTCATGATC TCCTGCCTCA GTCTCTCTAG TGCTGGGTTA CAAGTClGAA TATAATTGTT ATACCATCTC TAGGCTTCAG AGGAGTGAAT GAATCAATGA ATGAATGACT GCTATAiTAC CATTCCTTGA GCTGGGTCfA GGACATCTCT GTCTTTGTGA TAGGTGATM TTTTGTCrrC TCAGTCACn G A T G G G C n GAATATTCGA GGTATAAACA ACAAAGAAAG CCACAAGCCC CTCCTCTCTG AAGTCACCTG GAGCCATCTT TAAGCAAl lG TTAAATATCT GGAGACTCTC TGAAGGTCAG ACCCAACTTC TTGGTCTTTT TGAATTTATG TTTGAAAGCA C m A G T A A C TTGCATTCAT GAATGilGTC TTCCCTGTAT T T C r m G C C ATTTATTTGT TAITTAAAAA TAAATGTGTC GTGTGTCTGG CTCAAAGCTA CTfCCTGGCC CCTCAGTAGG CTCTCCCCrr ATGCCCCGIl TTAGTCTGGA AACATCCCGA GCAAGCTGTC TTCAGCCCG CAGAACTTAG CACAGGGTCT GTGTCATGTA CTGTGTGGCA GTAGCCCTGG ClTi7GGAGT TCAGAGGCTG AAGAAGGGTT G T C T G t m G GGCAGCTTCC TCTGCAGATT ATCTCAGGAA CCCATCAAGT TAGGCTGTCC CCACCITCAC CCTACTCTCT 'I'TAGATAAGA AAACGGGCAG T C T C n C C l T TAGTCTAGGT TTGTCCTATG TTAGTTCCAT A G T W A C A C TCTATATCAA TGAACfATTA ACCCTGTGAG ATGATGGTGA AATGTTGCTA GGTGAGAAGC CCrrAGAGGG GGAAAGAGAT AAGAGGAAAT A A A G A T G m AAAGTACACT GAAAATAAAA TAAATAATAT TCGAAAAAAA AAAAA

Figure 2-9

(A) Schematic representation of the Chxl71 cDNA and al1 the isolated clones. (B)

Nucleotide and predicted amino acid sequence of the Mouse CM71 cDNA. Shaded areas

represent the homeobox (dark) and the CVC box (Iight). The in-frame stop codon

upstream of the translation start site is doubly underlined and the predicted octapeptide

singly underlined. Putative poly-adenylation signals in the 3' UTR are underlined.

between nucleotide 1 (ATG) and the opal stop codon at nucleotide 1086 (Tee) exhibits

74% nucleotide identity with the nucleotide sequence of the CHXlO-I ORF. The ORF

encodes a predicted protein of 362 arnino acids, with predicted molecular weight of 39

kDa. The 3' UTR contains a poly-adenylation site at residues 2548 and 2574 ( M A A A ) ,

46 and 20 nucleotides upstream of a poly-(A) tail, respectively. The second poly-

adenylation site at nucleotide 2574 corresponds better with the average distance of 1 1-30

nucleotides (Lewin, 1990).

The chxl7 1 polypeptide contains a homeodomain of the prd-like type (Bürglin,

1994), situated between residues 170 and 229. The homeodomain of chxl71 is 97%

identical to that of chx10-1 and less similar to the ceh10 homeodomain with 78% amino

acid identity. Like dl known CVC domain-containing homeodomain proteins, the CVC

domain of chxl7 1 is situated immediately C-terminal to the homeodomain, and is 80%

identical to the CVC domain of chx 10- 1 and 6 1% identical to the CVC domain of ceh 10.

In addition, chxl7 1 encodes an octapeptide of the HSIDGILG type (residue 3 1-38:

FAINDLLG) as it has k e n found in some members of the Pax/prd class of homeodomain

proteins including chxlO (Noll, 1993) and is similar in seven out of eight residues to the

octapeptides of chxl0, cehl0 and vsxl (Liu et al., 1994; Svendson and McGhee, 1995,

Levine et al., 1994). The chxlïl protein and the chxl0-1 protein are 42% identical in their

N-termini and 62% identical in their C-termini. Over the eotire length of the predicted

protein chx 17 l is 60% identical to chx 10- 1. The complex of homeodomain and CVC-

domain is found 7 amino acids more C-terminal in chxl71 than in the chxl0-1 peptide

(Figure 2- 10A).

6.2 Putative protein domains of chu171

The predicted chx17 1 protein contains sequences that resemble putative activation

domains (Figure 2-10B). N-terminal to the homeodomain is a 81 amino acid region

(residues 22-102) with 21% proline-content, reminiscent of the proline-rich activation

acidic

proline-rich - - s - - serinehhreonine-rich -

nuclear targeting

octapeptide

Figure 2-10

(A) Dot matrix plot for the human chxl0- 1 and mouse chxl7 1 predicted proteins.

Alignment was done on Geneworks 2.1 with a window size of 10 amino acids and cut off

at 70% amino acid identity. The length of the human chxl0-1 and mouse c h 1 7 1 putative

proteins are 364 and 362 amino acids, respectively. (B) Schematic representation of the

predicted chx 10- 1 and chx 17 1 polypeptides. A putative nuclear targeting sequence is

located immediately N-temiinal to the homeodomain. The predicted proteins contain

potential trans-activation domains of the probe-rich, seriw/threonine-rich or acidic type.

domains found in CTFIR/3 (Mitchell and Tijan, 1989; Memod et al., 1989; Williams and

Tijan, 199 1). C-terminal to the proline-rich domain, is an 1 1 residue region (residues 148-

158) that contains 45% acidic amino acids (2 glutamic acids and 3 aspartic acids),

comparable to the longer acidic activation domain found in the AP-I/Jun/TFIID (Mitchell

and Tijan, 1989). An acidic dornain is also found C-terminal to the homeodomain, in a 22

amino acid region (residues 308-329) with 41% acidic amino acids (five glutamic acids and

four aspartic acids). Further towards the C-terminus is a 17 iunino acid region with 4145

serine/threonine content, resembling the Iarger trans-activation domain found in the POU

homeodomain protein GHF-I (Theill et al., 1989). A putative nuclear targeting sequence

(Dingwall and Laskey, 199 1) identicai to that of chx 10- 1, is situated between residues 167-

174 (KRKKRRHR), this sequence extends five amino acids into the homeodomain.

6.3 Northern analysis

Chxl71 (nucleotides 1269 to 2337) was hybridized to a Northem blot of mouse

tissues, with 5 pg of total RNA from retina, brain, heart, liver, kidney, lung, testis, spleen

at high stringency. Transcripts were detected in the retina lane after only overnight

exposure at levels comparable to those of ChrlO, at a position corresponding to a length of

-3.1 kb (Figure 2-1 1; B. Snow, pers. comm.).

6.4 Phylogenetic relationship to C H X I 0-1

Sequence alignments between CHXIO-1 and CM71 left doubt whether they really

do represent orthologues or actually are homologues. Compared to the sequence

conservation of CHXI0 across species (Figure 2-12) it seems striking that CHNO-1 and

Ch171 have such a relatively weak conservation of sequence outside the homeodomain

and the CVC domain. In addition, the presence of 2 amino acid differences in the

homeodomain (60 amino acids) and 10 amino acid differences in the CVC domain (54

amino acids) is unexpected, particularly since several of these changes are nonconservative

Figure 2-11

Northem analysis of Ch171 in adult mouse tissues. Total RNA Mot after overnight

exposure exhibits a distinct band in only the retina lane.

chxlO homeodomains

Human -RTI

Mouse . . . . . . . . .

Chicken .........

Goldfish . . . . . . . . .

chxlO CVC domains Human KCWCRS- EYGLYG- HSIPLPESII KS-ImS C A P W L L G m KSLE

...................................................... Mouse Chic ken ..................................... .E. ............ v.

...................................................... Goldfis h

Figure 2-12

Multiple protein alignments demonstrating the sequence conservation of the chxlO

homeodomain and CVC domain between goldfish, chicken, mice and man. Dots indicate

identical amino acid sequences, while mismatches in the amino acid sequence are indicated.

Dark shaded areas in the homeodomain indicate the predicted a-helices.

(Figure 2- 13). In order to determine the relationship between CHXIO-2 and Chxl71

duplicate mouse genomic blots were hybridized with full-length probes of either cDNA.

When using high stnngency conditions only Ch.171 hybridized to distinct bands in the

mouse genome, CHXIO-1 did not hybridize (Figure 2-14). Under slightly lowered

stringency (40% formamide) CHXIO-1 hybridized to bands which matched exactly the

band sizes detected by Chri71 (Figure 2- 14), suggesting that Chx271 is the locus most

closely related to CHXIO-1 in the mouse genome.

This finding does not necessarily imply that CHXIO-1 is the locus most closely

related to C h i 71 in humans. In order to determine this, duplicate human genomic blots

were hybridized with full-length probes of either cDNAs. At slightly lowered stringency

(40% formamide) CHXlO-1 detected only one single band (Figure 2- 1 S), whereas,

surprisingly Chxl71 detected repetitive sequences in human genomic DNA at these

conditions. Disregarding the elevated background oii the Chxl71 blot, it is clear that

several bands are detected, including those of CHXIO-1. Even when high stringency

washing conditions were used, bands that do not correspond to the CHXIO or the CHXIO-

2 locus remain visible (Figure 2-158). This finding indicates that human genomic DNA

contains at least one additional locus that is at least as sirnilar to Chxl71 as is CHXiO-1.

DISCUSSION

A multitude of screens for homeobox containing genes have been described in the

past several years. Whereas many attempts were based on the isolation of homologous

clones of Drosophila homeobox genes (e.g. Simeone et al., 1992), other screens for novel

sequences were performed using degenerate PCR approaches (e.g. Levine and Schechter,

1993). However most of the screens were performed on whole-embryo cDNA, which

potentially leads to two problems. First, highiy tissue specific homeobox genes might be

underrepresented and therefore rnissed. Second, homeobox genes that are involved oniy in

late differentiation events might not be represented in early embryonic cDNA libraries.

homeodomains

chx101vsx2 vsx 1 chxl0-l chxl71 cehl0

helix 1 helix 2 helix 3

CVC domains chx lOIvsx2 vsxl chxl0-1 chxl71 cehl0

KSAK

N...

QLPE

H. .D

block 1 block 2 block 3

Figure 2-13

Alignment of the homeodomains and CVC domains of ail known CVC domain-containhg

proteins. Mismatches from the chxlO sequence are indicated. Dark shaded areas in the

homeodomain indicate the predicted helices, while light shaded areas in the CVC domain

emphasize the consewed domains.

Figure 2-14

Duplicate mouse genomic Southem blots were hybridized with CHXîO-I (AIC) or Chi71

(B/D). A. and B. were carried out at high stringency (hybridization: 50% formamide at

420C; washes: 0 . 2 ~ SSC/O.P% SDS at 650C), while C and D were carried out at lowered

stringency (hybridization: 40% formamide at 420C; washes: 2x SSCIO.Z% SDS at 550C).

Abbreviations: BamHI (B) and EcoRI (E).

Figure 2-15

Duplicate human genomic Southern blots were hybridized with CHXIO-1 (AIC) or Ch171

(B/D). A. and B. were carried out at high stringency (hybridization: 40% formamide at

420C; washes: 0 . 2 ~ SSC/0.2% SDS at 650C), while C and D were carried out at lowered

stringency (hybridization: 40% formamide at 420C; washes: 2x SSC/O.2% SDS at 550C).

Abbreviations: BamHI (B) and EcoRI (E).

Exarnples of this type are the retinal homeobox genes, since the retina constitutes only a

minute part of the whole organism and some differentiation events do not occur until after

birth particularly in rodents (Sidman, 1961). Deitcher et al. ( 1994) tried to address the

problem of retina-specific homeobox genes by conducting a PCR-screen for homeobox

genes in the chick retina. However, their screen seemed to be technically problematic, in

that when using the PCR clone to screen a chick retinal cDNA library they were unable to

isolate the corresponding cDNA and only isolated a homologous cDNA. In addition, they

failed to identify chick CHXI O, which should have been amplified with the employed

primers and had not been published at the time (R.McInnes, pers. comm).

The data outlined in the introduction suggested that more retinal homeobox genes

are Iikely to exist. We were particularly interested in the isolation of close CHXlO

homologues, such as a human Vsxl orthologue, that would display almost complete amino

acid identity in the homeodomain. Using the library screening technique described, 1 have

been able to isolate two novel homeoboxîontaining cDNAs, namely OTX3 and CHXIO-1,

from an adult human retinal library. While the OïX3 is only distantly related, CHXIO-I

displays al1 the features of a close CHXlO homologue. However, due to the non-

embryonic stage of the libraiy, this screen also might not saturate the retina for homeobox

genes, a drawback accepted because of the demonstrated quality of the library, and because

CHXIO, although expressed in the adult, is also critical to mouse development (Burmeister

et al., unpublished).

The PCR approach that was used to distinguish clones of CHXlO from its

homologues, and thereby reduce the number of clones that had to be analyzed, does harbor

some potential problems. First, it will select not only CHXlO homologues, but also

CHXlO clones that lack the 3' UTR, or that have a rearrangement of the 3' UTR; such

clones have indeed k e n found in those selected. Second, if CHXI0 homologues share a

highly conserved regulatory element in the part of the 3' UTR that was amplified, the PCR

method rnight produce a sirnilar size band for actual homologues, which might thereby be

ignored in further analysis. However, based on sequence analysis we do not have any

indication of conservation in the 3' UTR of known C H X l O orthologues (man, chicken,

mouse). Even if such conserved sequences did exist, clones that have a 3' UTR

rearrangment should still be identifiable. In retrospect it might have been a good idea to

perform an additional PCR, with a primer set located in close proximity to the ORE Since

the screen was done with a probe covenng two thirds of the ORF, such a site would have

been less likely to be rearranged and therefore might have led to a more definitive

identification of CHXlO clones,

The identified human LIM homeobox gene LH2 is only 60% identical to the

CHXlO homeobox at the nucleotide level, showing clearly that the screening strategy was

sensitive enough to allow divergent sequences to be identified. The published expression

pattern of this gene includes high levels during lymphoid ce11 and CNS development, with

expression in the retina frorn optic cup io adult stages (Xu et al., 1993). Since this gene

has already been substantially characterized and does not represent a CHXIO homologue, it

was not chosen for further study.

The isolation of an OTX-like clone was unexpected, since substantial research

effort has been devoted to identifying the vertebrate homologues of orthodenticle, and

mammals were thought to oniy have two homologues (Simeone et al., 1992). Recently

another murine orthodenticle homologue, named Orthopedia. has been described by

Simeone et al. (1994). However, this clone differs substantially from the otx

homeodomains in many residues, including the ninth residue of the recognition helix, and

therefore can not be considered a true orthodenticle homologue. The partial cDNA that was

isolated in the screen described here contains a truncated homeobox with a high degree of

similarity to OtxlL2 at the nucleotide level, mostly differing at wobble-positions. The

nucleotides encoding the first 9 amino acids of the OTX3 cDNA differ strongly from the

other Otx homeoboxes, and are likely to be an artifact created by the technique used to

constnict the library. This method allowed snap-back structures to occur after first strand

cDNA synthesis, and therefore the 5' ends of some cDNAs might contain sequences that

do not correspond to the actual transcript (J. Nathans et al., pers. comm.). If these 9

amino acids are ignored, the remaining protein exhibits 88% amino acid identity to both o a

homeodomains. Although the rest of the protein is highly divergent, it does exhibit three

conserved blocks, when compared to otx 1 and 01x2 (Figure 2-3B); the significance of these

regions is unknown. The alignments clearly show that the OTX3 cDNA is more closely

related to mouse Otxl than to mouse 01x2. Al1 the alignments are based on the mouse

cDNAs of Otxl and 2, because, except for the 100% identical homeodomains, sequences

for the human OTX genes are unavailable.

CHXZO-1

The isolation of CHXIO-1, with both a homeobox and a CVC-box, confirmed Our

expectation that true CHXlO homologues might exist. The homeodomain sequence is

highly conserved with CHXIO, having 92% amino acid identity with only five differing

residues. Only one of these changes occurs within the helix-tum-helix motif, between

helix 2 and the mm, while the other four changes occur at the beginning and end of helix 1,

in regions that have been shown to be highly variable amongst homeobox gene farnily

members (Figure 2-13; Kappeii et al., 1993). Although no function has yet been assigned

to the CVC domain, its conservation in another homeodornain protein reiterates its probable

func tional significance . Interestingly, the predicted chxlO-1 peptide contains regions of high proline,

serinefthreonine and acidic amino acid content reminiscent of the putative activation

domains found in chx 10. Their arrangement within the peptide, however, is quite distinct

from chx 10 (Figure 2-5B). This different arrangement of protein domains suggests that

chxl0- l has acquired distinct functional properties from the chx 10 peptide.

Retinal expression of CHXIO-I in the adult is rnuch lower than that of CHXIO

(Figure 2-7). This finding is not necessarily surprising, since for most homeobox genes,

the highest expression is found during embryonic development, and this may be the case

for CHXIO-I as well. Al1 the murine Pax genes manifest down-regulation with

progressing developrnent, and two of the nine Pax genes are not expressed in the adult at

d l (Chalepakis et al., 1993).

Although the results of the in situ hybridization can only be regarded to be

preliminary, they allow for an interesting observation (Figure 2-8). CHXID-I was found

to be expressed in the inner nuclear and ganglion ce11 layers of adult human retina. Since

C m 0 was found to be specific to the inner nuclear layer in adult retina (Liu et al., 1994),

the expression pattern of CHWO- I and ChxlO are very similar. This is rather interesting,

in that many homologous homeobox genes such as OmlR have similar expression patterns

(Simeone et al., 1992), presumably reflecting a common origin. If confirmed in

subsequent expression studies, the result with CHXIO-I suggests that, like CHXIO,

CHXlO-I may also be involved in speciQing ce11 fate within the developing retina.

Human chromosome 20 does not contain any obvious disease loci in the region to

which CHXIO-I was mapped (Figure 2-6B). However, the homologous region of mouse

chromosome 2, contains two developmental eye diseases, Coloboma (Cm) and blind-sterile

(bs), as well as the locus of the ChrI O-homologous genomic clone 17 1 (B.Taylor, pers.

comm.).

Coloboma is a semidominant disease originally described in the offspring of an

irradiated ((C3HMeH x 1OlM) F1) mde. Although homozygotes have never been

described, the eyes of heterozygotes are small at birth and a ventral segment of their

choroid is absent. The eyeballs are rotated ventrally and affected animals have abnormal

posture, head-shaking, and circling, indicating additionai underlying neuronal defects

(Searle, 1966).

Blind-sterile mice arose spontaneously in an AKR/J strain. Homozygotes at day

El6 have bilateral nuclear cataracts that are present in the center of the lem, eyes that are

slightly smaller than normal, and glossy coats (Vamum, 1983). Adult males copulate

normally but have smaller than normal testis, with a reduced number of sperm, which, if

formed, completely lack acrosomes (Sotomayer and Handel, 1986).

C h x l 7 1

When a mouse retina library was screened at high stringency for the murine

CHXIO-I orthologue, only one cDNA was identified. This cDNA appears to represent the

transcnpt encoded by the 17 1 genornic locus. Although the CHNO-1 and Chri71 cDNAs

exhibit only 74% nucleotide identity over the whole ORF, both the homeodomain and the

CVC domain are well conserved, with 974 and 80% amino acid identity, respectively.

Over the entire length of the predicted proteins, however, they only exhibit 60% amino acid

identity (Figure 2- 1 OA).

This degree of divergence seemed higher than expected for orthologous

homeodomain proteins. In a study on the evolutionary conservation of Hox genes Kappen

et al. (1989) found an arithmetic mean of 10.7 nucleotide changes between human and

mouse orthologous homeoboxes. Even considering the standard deviation of +/- 4.5

nucleotides, this is much lower than the 24 nucleotide changes found between the CHXIO-

I and C h 1 71 homeoboxes. Since CHXIO-I and Ch271 do not belong to the Hox-class.

this finding might not necessarily represent the evolutionary pressure acting on these genes.

However, additional evidence indicates that these two proteins might be homologues rather

than orthologues. First, although chx 17 1 and chx 10- 1 contain similar activation domains,

their arrangements within the predicted proteins are quite distinct. This might indicate that

these proteins have acquired different funciional properties (Figure 2-IOB). Second,

aithough chxl7 1 is two amino acid residues shorter than chxl0- 1, its homeodomain and

CVC-domain complex are located hirther towards the C-terminus. Third, the variable

block 2 of the chx171 CVC domain displays only 25% identity to block 2 of chxl0-1, even

though blocks 1 and 3 are 95% conserved (Figure 2-13). In conclusion, Chxl7I and

C H X I O - 1 map to homologous chromosomal regions, but they differ in sequence,

transcript length, abundance and organization of predicted sequence motifs, to a degree

unexpected for orthologous homeobox genes of man and mouse.

In order io detennine whether C h 1 71 and CHXlO-1 represent homologues or

orthologues, their respective loci were examined in mouse and human genornic DNA.

Probing mouse genomic Southern blots with both the human CHXIO-1 and the mouse

Ch171 cDNAs showed that both appear to recognize the same genomic locus (Figure 2-

14). This result suggests that the screen of the mouse retinal cDNA library was successful,

in that the retinally expressed (at least in adult retina) sequence most sirnilar to CHXIO-1 in

the mouse genome, other than ChxlO, has indeed been identified. Probing human genomic

Southern blots with both the human CHXIO-1 and the rnouse Chxl7l cDNAs showed that

Chxl71 hybridizes to the human CHXIO-1 genomic locus and at least one other locus.

The hybridizing bands remain even after the hybndization stringency is increased (Figure

2-15). This result presents us with the possibility that the Ch171 and CHXIO-1 genes are

independent products of a duplication event of an ancestral gene, and that Chri 71 has a

human orthologue (CHXI 71).

The degree of sequence divergence between Ch171 and CHXIO-1 is consistent

with the occurrence of a duplication event. Dayhoff and Barker (1976) reponed a

cornparison of the extent of divergence between proteins that were derived from gene

duplications. They found that due to reduced selective pressure, substitution rates for one

of the genes are strongly elevated, presumably allowing the rapid acquisition of a different

function. This might explain why, considering the fairly recent divergence point between

man and mice of around 50 million years ago (Dickerson, 197 l), chxl0- 1 and chxl7 1 have

diverged 40% throughout the entire protein and 14% within the conserved homeodomain

and the CVC-domains.

Mer sequencing two thirds of the cDNAs from the human retinal cDNA screen that

1 conducted, no human CHXI71 cDNA has yet been cloned. Due to the demonstrated

retioa-specificity and abundance of Chxi71 in the rnouse it seems unlikely, if CHXI 71

exists, that it is not expressed in human retina. One possible explanation is that instead of a

change of the spatial expression pattern versus CHXI O- I , C M 71 might have acquired a

distinct developmental timing after the duplication event. A precedent for this type of event

is shown by Pax2 and P a 8 . Whereas both of these genes are expressed in the developing

neural tube and hindbrain in identical patterns, only Pax2 expression persists past E13.5

into the adult (Chalepakis, 1993).

With the isolation of the two clones (CHX10-1 and Chxl71) described in this

thesis, the group of CVC domain homeodomain genes has enlarged to one invertebrate and

four vertebrate members (Figure 2- 13). Although no function has yet been assigned to the

CVC domain, it could be analogous to class-specific domains found in paired- and POU-

homeobox genes which are involved in DNA binâing (Robertis, 1994), or the LM-domain

which is thought to be involved in protein-protein interactions (Sanchez-Garcia and

Rabbitts, 1994).

Alignments of the CVC domains of al1 known CVC domain-containing

homeodomain proteins show that the two lateral blocks (residues 1-27 and 40-59) are

highly conserved, while the middle region (residues 28-39) is highly variable (Figure 2-

13). Although block 2 does not display any conservation of particular amino acids, its size

is conserved, indicating that the spacing between blocks 1 and 3 seems to be crucial for

CVC domain îunction. Among al1 the vertebrate genes, block 2 has 11 residues length in

vertebrates, while that of the Celegans gene cehl0 is 10 residues in length (Figure 2-13).

Al1 the CVC-domain homeobox genes encode a homeodomain of the paired-like

type with a glutamine as residue nine of the recognition heüx (Bürglin, 1994). In a study

by Wilson et ai. (1993) these homeodomains were shown to be capable of cooperative

dimerization on their target sequences. In fact, paired homeodomains of the Q9 type

exhibited the highest degree of cooperativity, ranging up to 300-fold and were able to

heterodimerize with Paxlprd homeodomains of the S9-, and the K9-type. The observation

of dimerization for Pdprd-type homeodomain proteins, as well as the larger palindromic

DNA binding sequence may reflect the means by which the CVC domain-containing

proteins attain their functional specificity.

Al1 the CVC homeobox genes studied to date have been shown to be expressed in

neurons. More precisely, as far as data exist, al1 the vertebrate members are expressed in

the retina in a layer specific manner. Vsxl, CHXlO and CHXI 0-1 have been shown to be

expressed in the inner nuclear layer, while CHXIO-I is also expressed in the ganglion ce11

layer. This conservation of expression suggests that the vertebrate members of this

homeobox gene subclass are involved in the establishment or maintenance of specific ce11

types of the inner nuclear layer, or in the definition of the inner nuclear layer as a specific

domain within the retina. The Celegans member of this homeobox gene group, cehl0,

was found to be expressed in the AIY intemeuron, which receives synaptic input from the

thennosensitive AFD cell. The AFD ce11 has, due to its morphology and location, been

suggested to be potentiail y photosensitive (Svendson and McGhee, 1995). Although we

await further Chxl71 and CHXIO-I expression studies, members of the CVC homeobox

gene subclass may be involved in the determination and maintenance of sensory

interneurons.

CHAPTER 3: FUTURE DIRECTIONS

This thesis describes the cloning and partial characterization of two novel CVC

domain-containing homeobox genes. Further elucidation of the exact expression pattern of

both genes and comparison to the known patterns of expression of ChlO, Vsxl and

cehl0, should provide some insight into the function of this group of homeobox genes in

retinal development. In situ hybridization and immunohistochemistry cm be used to

determine the ce11 types in which these proteins are expressed.

C H X l O- I

The CHXlO-l gene has been shown to be highly divergent in sequence from

ChrlPI. In particular its abundance in the adult retina is very low compared to that of

Chxl71. More detailed expression studies of CHXIO-1 and Chxl71 will be necessary to

determine whether the genes have also diverged in hinction.

In order to determine the funciional equivalency of CHXIO-1 and Chx171, the

retinal layer and ce11 type in which both genes are expressed must be refined. The

localization of the CHXIO-1 transcripts reported as a preliminary result in this thesis will be

confirmed by additional digoxygenin in situ hybridization studies. Determination of the

expression pattem of mouse Ch171 might give an indication on whether CHXIO-I has

adopted a different function. Immunohistochemistry using antibodies that distinguish

chx 17 1 and c hx 10- 1, will provide additional data critical to assessing the relationship

between these two proteins.

In order to detexmine the role of CHXIO-I in transcriptional regulation its binding

sites (both to DNA and other proteins) must be identified and its activation properties

determined. Although the homeodomains of CVC proteins may have the same general

target DNA sequence as do the Pax/prd homeodomains, no activation of transcription was

observed with ch10 using the Pax reporter construct of Wilson et al. (1993) (G. Clarke,

unpublished results). Due to the presence of the CVC domain, binding properties of the

homeodomain might be altered, which is why target sequences must be selected from a

random oligonucleotide sequence library as outlined in Wilson et al. (1993). Using a

reporter construct with this target sequence trans-activating properties of CHXIO-1 could

be studied in Chloramphenicol acetyltransferase (CAT) assays as it has been done for other

homeodomain proteins (Wilson et al., 1993). Comparison of these data with those of

Chxl71 should indicate whether CHXIO-1 has retained the in vitro activation properties of

Chxl7l.

Narrowing down the tirne point of the duplication event might allow a correlation of

the presence of CHXIO-1 and morphological changes in the retina. To do this genomic

zoo-blots should be performed and include species which are phylogenetically closer to the

human than the mouse, such as new and old world monkeys.

Chxl71

In contrast to CHXlO-1, mouse Chxl71 is expressed at high levels, specifically in

the adult retina. To determine the importance of C M 7 1 for mouse retinal development, a

more detailed analysis of the Ch171 expression pattem will be necessary. If these

expression studies suggest a possible involvement of Chxl71 in the development of the

eye, proof of the requirement of Ch1 71 could be obtained by targeting the C h 1 7 1 locus.

Altematively the possibility that Chx171 is mutated in the known eye diseases blind-sterile

and Coloboma could be investigated.

In situ hybridization studies will be needed to define the spatial and temporal

expression pattem of C M 7 1 in mouse development. The focal points of this study should

be the time and place of the onset of expression, the expression pattem in neuronal tissues

and in the developing eye, and the cells in which expression is maintained in the adult

retina. This study will be complemented by irnmunohistochemistry for chxl71, which

should identiQ the specific cells that express chxl7l. Overlaps in the spatial expression

patterns of Chxi71 and ChxlO, rnight suggest both an interplay of the two proteins in

development, and their formation of heterodimen in cells in which they are both expressed.

To permit mutational analysis, or altematively to create a knock-out mouse, the

genomic locus of Ch171 must be isolated. This work has already k e n started and plaque

pure phage are currently king analyzed (C. Freund, unpublished results).

To determine whether mutations in Ch171 cause Coloboma or blind sterile, the

Ch171 location in the genetic map must be further refined. For this purpose the full-

length C h 1 71 cDNA will be used for linkage analysis. If this linkage analysis does not

produce any recombinants with either blind-sterile or Coloboma, it will be necessary to

investigate directly the possible involvement of Chiri 71 in these two diseases. Initially this

problem could be addressed by examining the retinal expression of C M 71 in mutant rnice

by Northern blotting. To characterize the inherent molecular mechanism, one should

initially examine the CM71 locus in genomic DNA from mutant animals, by Southem blot

analysis. If no gross rearrangements are found, small aiterations in the sequence (such as

substitutions) cm be sought by SSCP ancilor direct sequencing, in order to determine the

specific molecular defec t.

Altematively, if C h 1 71 does not seem to be implicated in known genetic diseases,

its requirement in retinal development will have to be detedned by gene targeting. This

project could be done using existing strategies such as those currently employed in our lab

for the targeting of the Rom1 locus (G. Clarke, unpublished results).

CVC domain function and significance

The significance of the CVC domain, a highly conserved sequence element found in

homeobox genes from nematode to man, is unknown but its conservation suggests that it is

likely to be important. The major question will be whether the CVC-domain, like the

homeodomain, binds DNA or altematively functions in the interaction with other proteins.

Research will have to focus on the determination of interacting partners. structural motifs

and its role in transcriptional regulation.

To identify the interacting partners of the CVC domain it will be necessary to

establish whether it interacts with DNA or protein. Interaction with DNA could be

demonstrated by gel-shift assays (using the target site selected for the homeodomain and

CVC domain complex by the Wilson method (1993)), while an interaction with proteins

could be shown by affinity-chromatography. The interacting proteins, possibly other CVC

homedomain proteins, could then be identified using the yeast two-hybrid system.

Since the CVC domain represents a novel domain it is important to determine its

tertiary structure. The relatively small size of the CVC domain should allow both NMR

spectroscopy and X-ray crystallography to be done. If the CVC domain does interact with

DNA, structural analysis should include the complex of the CVC domain bound to DNA.

Structural data might indicate residues critical for hinction and may also explain if the low

degree of conservation of block two arises from this block acting mainly as a spacer

sequence.

Alternatively the CVC domain might represent an activation domain. This

possibility could be determined by observing whether deletion of the CVC domain has a

deleterious or enhancing effect on tram-activation in a CAT-assay.

Human CHXlO homologues

The screening of the human retina cDNA library for CHXlO homologues must be

completed. Although additional novel clones may be identified, one has to consider the

possibility that no human CHXl71 or human VSXl is present in the human genome.

However, since circumstantial evidence suggests that these clones do exist, other strategies

will have to be used to clone these genes. While problems with the tissue specificity might

be circumvented most easily by screening a genomic Library, distinctive feanires such as the

Vsxl alternative exon might allow PCR strategies to be used successfully.

To date 35 clones of the high stringency class (Classes 2 and 3) remain to be

characterized. Additionally there are 48 clones of the low stringency class (Class I) , that

have not been analyzed. Since many rearranged clones have been identified, the initial

anaiysis should include additional PCR with primers specific for the 3' untranslated regions

of both CHXlO and CHXIO- I in order to exclude these known cDNAs. Analysis of these

clones might still lead to the identification of additionai CHXlO homologues.

This screen has not yet produced a human CHX171, either due to problems with

the screening procedure or tissue specificity. It is therefore probably best to screen for

such a clone in a human genornic library, which could be done in conjunction with the

isolation of the respective CHXlO-I locus.

Alignments of Vsxl with C H X i O - 1 and Ch171 clearly suggest that the

mammalian onhologue of Vsxl also remains to be identified. The most distinct feature of

Vsxl is a 12 nucleotide insertion in the CVC domain, which is why 1 have decided to

attempt a PCR based approach. Primers to the 5' end of the horneobox and overlapping

with the alternative exon of the Vsxl CVC box have been constructed, based on the unique

Vsxl sequence and the preferred human codon usage. Used on the uncharacterized clones

of the human retinal screen or alternatively, reverse-transcribed retinal RNA, these primers

should lead to the isolation of V W , if such a gene indeed exists.

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