DESCRIPCIÓN GENERAL

The first step towards the elucidation o f om6-related T-box gene function in the developing eye was to determine their exact patterns of expression in the early retina. This allows identification o f cell types and retinal regions in which they are active. I aimed to provide a detailed and comparative analysis o f T-box gene expression in the developing mouse and human eye; reports to date have considered early stages of mouse development only and have not examined expression in humans (Chapman et a l, 1996).

In Drosophila, the optomotor blind {omb) mutant exhibits an inability to respond appropriately to rotary stimuli in a specially designed ‘optomotor’ maze. Anatomical analysis revealed disrupted neuronal connectivity within the fly optic lobes (Pflugfelder and Heisenberg, 1995). This phenotype is due to non-lethal mutations in a single gene, found to encode a member o f the T-box family o f transcription factors. Genetic cascades that underlie Drosophila development are frequently conserved in vertebrate species, so much that Drosophila genetics is increasing being used in the study of human disease (Bernards and Hariharan, 2001). This is true o f the developing eye, despite the radically different structure o f fly and vertebrate visual systems (Wawersik and Maas, 2000). The importance o f omb in the developing fly visual system meant that homologues to omb might be important during development o f the vertebrate eye. In the mouse, members of the o/M^-related mammalian subclass o f T- box genes (the Tbx2 subclass), Tbx2, Tbx3 and Tbx5, were expressed in the developing retina (Chapman et a l, 1996).

In this study I explored in detail the expression of o/w/?-related mammalian T-box genes within the developing eye. Expression was examined in mouse and human tissue throughout the period of eye morphogenesis and retinal stratification. I examined human tissue to prove the relevance of T-box genes to human eye development. Since very young specimens of human tissue were not available, T-box gene expression during early stages of eye development was only examined in mice.

T-box expression in mice was also analysed to provide a basis for the functional T- box gene studies undertaken in this species in chapters 5 and 6. It was necessary to

compare mouse and human expression to validate the mouse as a meaningful model for human T-box gene function. The use of mice as an experimental model is favoured for the study of mammalian development. Small rodents have long been popular for experimentation because their anatomy and physiology is similar to larger mammals and their small size makes for easy handling and husbandry. Though rats have traditionally been the rodents of choice, the relative ease o f mouse transgenesis has led to the increasing use of mice. This is particularly true in developmental biology, where transgenic technology has been most effectively applied.

R E S U L T S

The expression of om6-related T-box genes Tbx2!TBX2^ Tbx2ITBX3 and Tbx5/TBX5

was examined throughout mammalian eye development. The presence and distribution o f the specific T-box mRNAs in mouse and human tissue were determined using reverse transcriptase-polymerase chain reaction (RT-PCR) and RNA

in situ hybridisation. Expression of the owZ)-related T-box genes was compared with expression of Tbx20, a new gene identified in my laboratory. T-box gene expression was also compared with protein distribution of human POU4F2 (BRN3B), a marker for developing retinal ganglion cells (RGCs). An analysis of POU4F2 during human retinal development was carried out.

3.1.

Tbx2, Tbx3

TbxS

Expression o f Tbx2, Tbx3, and Tbx5 was examined during eye morphogenesis in the developing mouse embryo. Analysis at the earliest stages o f eye development was not possible in human tissue, as the youngest available specimens were at 6 weeks post­ conception (wpc). At this stage the human optic vesicle had already invaginated to form the optic cup and the lens vesicle had formed: eye morphogenesis was essentially complete.

Mouse embryos of the CBA strain were analysed at embryonic day (E) 9.5 to E l2.5 by performing whole mount in situ hybridisation to visualise the mRNA distribution

o f T-box genes. Some in situ hybridisations were performed on cryosections of mouse tissue. Embryos examined at these timepoints were found to be between the 9- and 53-somite stage o f development. During this period the newly formed optic vesicles expand from the prosencephalon (embryonic forebrain) to contact the surface ectoderm. Reciprocal signalling between the optic vesicle and the surface ectoderm is necessary for the formation of an optic cup from the former and a lens from the latter (Chow and Lang, 2001). The maturing optic cup surrounds the lens and comprises an inner multicellular neural retina and an outer monolayered retinal pigmented epithelium (RPE). See 1.1. for a description of eye morphogenesis.

The variability of the somite stage between embryos examined at a specific timepoint, even within a single litter, makes length o f gestation a poor measure o f developmental stage. At early post-gastrulation stages of development I have used the number of paired somites adjacent to the neural tube as a consistent indicator of developmental stage.

T-box gene expression in the optic vesicles

Expression of Tbx5 was first detected in the developing mouse eye using in situ

hybridisation at the 9-somite stage of development. At this stage the post-gastrulation mouse embryo has already turned within the yolk sac so that the embryonic head faces its tail. The neural tube has closed rostrally and the optic pits (the first morphological sign of eye development) are deepening to become optic vesicles. Tbx5 expression was faint and diffuse in the optic vesicles at this time (Fig. 3.1A).

By the 15-somite stage, Tbx5 expression was clearly detected in the dorsal optic vesicles (Fig. 3.1B). This expression was predominantly in the region of the dorsal optic vesicle in direct opposition to the surface epithelium, but extended a short distance into more proximal dorsal neuroepithelium (Fig. 3.1C). It is the neuroepithelium in direct apposition to the surface epithelium that invaginates and develops as neural retina. I hereon describe this region as presumptive (or prospective) neural retina, and the more proximal optic vesicle as presumptive (or prospective) RPE. Tbx2 was similarly expressed to Tbx5 in the dorsal optic vesicles at 15 somites (Fig. 3.1D, E). TbxS mRNA was not detected in the optic vesicles by 22 somites (Fig. 3.1 F).

Figure 3.1

Tbx5 was expressed in the dorso-distal optic vesicles (A-C):

A Faint and diffuse Tbx5 expression was first detected in the expanding optic pits o f the 9-somite mouse embryo (arrow). Expression was also seen in the posterior (ventricular) region o f the developing heart (h). Dorsal (d) orientation o f the developing eye is indicated for all figures. Scale bar = 0.5 mm.

B Tbx5 was expressed in the dorsal optic vesicle o f the 15-somite mouse embryo. Expression was seen also in the posterior developing heart (h) and the emerging forelimbs (f). Dotted line indicates section shown in C. Scale bar = 0.5 mm.

C Vibratome section o f embryo in B through the coronal plane and the dorso-ventral axis o f the optic vesicle. TbxS expression was seen in the presumptive neural retina (demarcated by point o f contact with surface ectoderm, se, with dotted lines) and in the dorsal-most region o f presumptive pigmented epithelium (proximal optic vesicle). Scale bar = 50 pm.

Tbx2^ but not TbxS^ was expressed in the dorso-distal optic vesicles (D-F):

D Tbx2 was expressed in the 15-somite mouse optic vesicles (arrow, dotted line=section in E), facial primordia (p), otic placodes (o), heart (h), forelimb bud (f) and allantois (a). Scale bar = 0.5 mm. E Vibratome section o f embryo in D through the coronal plane and the dorso-ventral axis o f the

optic vesicle. Thx2 expression was seen throughout the presumptive neural retina (distal optic vesicle adjacent to surface ectoderm, se), in the dorsal-most presumptive retinal pigmented epithelium (RPE; outside dotted line) and in the maxillary mesenchyme (m). Scale bar = 50 pm. F TbxS mRNA was detected in the developing maxillary region (m) the otic placodes (o) and the

allantois (a), but not the optic vesicles, o f the 22-somite mouse embryo. Scale bar = 0.5 mm.

Tbx2 and Tbx5 persist in the late stage optic vesicles (G-K):

G At 24 somites TbxS remains expressed in the dorsal optic vesicles (arrow), the posterior heart (h) and in the forelimbs (f). Dotted line indicates plane o f section in H. Scale bar = 0.5 mm.

H Vibratome section o f embryo in G through the optic vesicle. TbxS expression was seen in the dorsal region o f presumptive neural retina (adjacent to surface epithelium, se; demarcated by dotted lines) and over the dorsal boundary with presumptive RPE. Scale bar = 50 pm.

J Tbx2 was expressed in the 27-somite mouse retina (arrow, dotted line=section in K), the facial primordia (p), otic placode (o), heart (h) and limb buds (1). Scale bar = 0.5 mm.

K Vibratome section o f embryo in J through the optic vesicle. Tbx2 was expressed across the presumptive neural retina (demarcated by dotted lines and adjacent to the lens placode. I) with higher levels dorsally, in the dorsal-most presumptive RPE and maxillary mesenchyme (m). Presumptive neural retina is. Scale bar = 5 0 pm.

mTbx2 and mTbx5 are expressed