Contrastación de la Hipótesis Específica 2

Morphological changes have been used to define stages in retinal maturation but another criterion would be the expression of genes associated with phases of maturation towards the final, differentiated postmitotic state. Little is known about genes expressed early on in development which affect cell differentiation. Certain of the paired class of homeodomain-containing transcriptional regulators (reviewed by Gruss and Walther, 1992; Beebe, 1994) have been proven to be functionally significant in the development of the eye,

largely by virtue of cloning disease loci. An example of this is pax-6, mutations in which

Introduction

heterozygotes have small eyes whilst affected homozygotes show anophthalmia, craniofacial and central nervous system defects. A similar situation occurs in humans, where different mutations in single dosage cause congenital cataracts and aniridia, but double dosage gives

rise to anophthalmia, craniofacial and central nervous system defects (Glaser et al, 1994).

Another example is the chxlO gene which is expressed in all neuroblasts of the optic cup,

where a mutation in exon 3 causes the ocular retardation phenotype in the mouse (Mclnnes et

al, 1994).

Since chxlO is also expressed in the inner nuclear layer of the mature retina (Liu et

al, 1994) there is an exciting possibility that other, as yet undiscovered homeobox genes may

play a significant role in maintaining the retina. However, most of the known hom eobox genes are expressed very early in eye development, in mitotically active cells (Gruss and Walther, 1992; Beebe, 1994) whereas retinal dystrophies by definition have, at worst, a recognisable retinal structure before cell loss begins. Thus, genes important in these diseases would be expected to be expressing themselves at a very late stage in retinal maturation.

Two other homeobox genes have been identified and shown to be expressed during

murine eye development. Hox-8, is expressed in the developing optic cup vesicle in layers of

cells which histologically mark the domain of the neural retina and surface ectoderm of the

corneal epithelium whilst hox-7 is expressed at a later stage in the ciliary body domain

(Monaghan, 1990).

1.4.2 Photoreceptor gene expression

Studies have been carried out on mice (Bowes et al, 1988, 1989) rats (Gonzalez-

Femandez et al, 1993) and dogs (Farber et al, 1992) which give some indication as to the

order of gene expression in the neonatal retina.

The mRNA levels of the phototransduction proteins opsin, arrestin, transducin (T) a ,

p and Y subunits and rod cGMP phosphodiesterase subunit (pdeb) in the mouse were

examined by slot blotting and densitometry. Messenger RNA expression at birth was detected at low levels for pdeb, Tp and arrestin which increased over the first 9 postnatal days after which it begins to plateau towards adult levels by 24 days. Opsin, Ty and Ta appear slightly

Introduction

later on, becoming detectable at 5, 6 and 8 postnatal days respectively (P5, P6 and PS).

Transcript levels also rise rapidly, attaining almost adult (day 24) values by day P12 (Bowes et

al, 1988, 1989). Immunocytochemical methods in the same study conferred greater

sensitivity, demonstrating detectable levels of arrestin, Ty, opsin proteins at P4 and T a proteins at birth, and days P4 and P7 respectively. Amounts equivalent to adult protein expression were attained 3 days subsequent to detection except for arrestin which remained

constant into adulthood (Bowes et al, 1988). Presence of phosducin protein by western

analysis is detected at P3 days, increasing to adult levels over the first 2 weeks. It is also

complexing with Tpy at a very early stage (Lee et al, 1990). This tends to indicate a relative

maturation to an almost 'adult' gene transcription state at between 12 and 14 days.

Northern and slot blot studies on the Irish setter dog demonstrate a similar, though

prolonged timescale of gene expression in the neonate (Farber et al, 1992). Expression of

mRNA at birth was not ascertained as the earliest samples were taken at 7 postnatal days. Opsin, arrestin, pdeb, and T a transcripts all increase fairly rapidly to reach more or less adult levels by 60 postnatal days. This is in good agreement with the estimates from photoreceptor morphology and cytology. The rod cGMP phosphodiesterase a and y subunits seem to rise much more slowly, not attaining adult levels for 100 days or more. However, the relative increases in transcript are all 3- to 7-fold between 7 and 60 days. Levels of the Tp and phosducin transcripts appear to reach their maximal level much sooner, at about three weeks but since the former is expressed in other neuronal cell types, this may not reflect the message in the photoreceptors.

Studies of other photoreceptor genes in the rat indicate that interphotoreceptor retinol binding protein (IRBP) and its cellular counterpart (CRBP) are both expressed at birth whilst opsin expression becomes detectable by postnatal day 6. This may support a role for IRBP and CRBP in the establishment of final photoreceptor maturation, possibly via the transport of growth factors. One particular factor which may be important for the maintenance of the mature photoreceptor is basic fibroblast growth factor, whose expression in the retina

Introduction

These studies together, not only infer roles for certain proteins based upon the presence of their transcripts, but also provide a framework against which photoreceptor maturation may be measured at the nucleic acid level. Against such data, specific transcripts may then be measured in attempts to reveal mRNA defects in candidate genes prior to cell loss.

The goal of retinal maturation is to develop a highly ordered arrangement of specific cells designed to transduce light energy into a biochemical signal which can be transmitted to the brain to be integrated into a visual signal. Although all the cell types and interphotoreceptor cell matrix play essential roles in maintaining development, structure and viability, the crux of the signalling process is the biochemical pathway of visual transduction which takes place in the photoreceptors.