POBLACIÓN SUJETO

There are several phenotypes in the o / retina that are not addressed by these putative roles of ChxIO. One is the absence of an optic nerve. Ganglion cells are present in the mutant, and they do project their axons, but these fail to leave the globe and fasiculate into a cohesive optic nerve. The optic stalk degenerates into a residual line of cells that are morphologically distinct from the surrounding mesenchyme (see Figure 1, Chapter 4). ChxIO is not expressed in postmitotic ganglion cells (Burmeister et al. 1996), leaving a number of possibilities to explain this phenotype. One is that the premature closure of the choroid fissure presents a physical barrier through which the axons cannot advance, and their guidance cues cause axons to form the observed whorls. Another is that, owing to the reduction in cell numbers during early development, there is a disruption in the microenvironment, which prevents ganglion cells from differentiating correctly. A third possibility is that the absence of ChxIO in mitotic cells that are fated to become ganglion cells

results in a ganglion cell phenotype upon differentiation. The failure of ganglion cells to aggregate into a nerve fibre layer, or to form an optic nerve head, suggests that the notion of a simple mechanistic physical barrier to axonal growth is not sufficient to explain this phenotype. If it were, then one might expect the formation of a fasiculated optic nerve head, which was then unable to leave the globe. As it is in the o / retina, axons migrate away from ganglion cells through the retina.

As ChxIO is expressed only in the neural retina during development, it is perhaps surprising that the o / phenotype displays such severe microphthalmia. One might speculate that as the reduction in proliferation in the neural retina is notable when the eye is at a very early stage of development, that this has a profound effect on total eye size rather than just on the retina itself. This phenotype indicates that signalling occurs between the retina and other areas of the eye to convey information about growth, and that these signals are inhibited or lost in the o / retina owing to reduced proliferation, or inherent disruption of a relevant signalling pathway.

Lens development in the o / eye is relatively normal (although the lens is proportionally smaller than in the wild type), but the lens invariably becomes cataractous in adulthood (Burmeister et al. 1996). The reduction in the size of the lens again may be attributable to disturbances in signalling between the retina and the lens, which have been shown to be essential for lens development (Grainger, 1992). Burmeister et ai. (1996) suggest that the formation of cataracts in the o / eye may be a secondary phenomenon, bearing

in mind the fact that ChxIO does not appear to be expressed in the lens at any stage.

Similarly, Burmeister et al. suggest that the morphologically abnormal photoreceptors seen in the mutant may be secondary effect, as ChxIO is not reported to be expressed in differentiated photoreceptors. The photoreceptors have reduced or absent inner and outer segments, shown by the juxtaposition of the outer nuclear layer (i.e. the cell bodies of the photoreceptors) to the RPE. In the normal retina, these layers are clearly separated by the inner and outer segments. This is investigated in depth in Chapters 4 and 5.

It is interesting that given strong ChxIO expression in V2 interneurons in the spine during development and adulthopd^Sander at al. 2000) that no spinal phenotype has ever been associated with the o / mouse. This has not been investigated yet, but it may be that other factors are capable of compensating for the loss of ChxIO in these cells, or that loss of these cells themselves result in a subtle phenotype that is not severe enough to be notable.

1.15.1 ChxIO interacts with R0R-/3

Interactions with a member of the nuclear receptor family of transcription factors have been suggested (Chow et al. 1998). Co-localisation of ChxIO and RORp

and a dramatic reduction in RORp expression observed in the o / mouse suggest a role for this protein in retinal proliferation, possibly by via ChxIO.

1.15.2 Amelioration o f the oi^ phenotype by genetic modifiers

Bone-Larson et al. (2000) examined the offspring of back-crossed inbred o / mice of the strain 129/Sv-SX and sub-species Mus musculus castaneus. The ocular phenotype was ameliorated, although the eyes were not of normal size. Furthermore, the bipolar cell marker RetB1 was identified by immunohistochemistry, although the marker PKC was not. Optic nerves were present bilaterally, and electroretinograms indicated partial visual function. These data suggest that the presence of genetic modifiers, as found in a different genetic background, interact strongly with ChxIO during development, and have a profound effect on its function.

1.15.3 Identification o f retinal stem ceils

Recently, stem cells have been identified in various vertebrate organs and tissue types, including the liver and mesenchyme (Shafritz 2000). Tropepe et al.

(2000) and Ahmad et ai. (2000) both independently demonstrated the existence of neural progenitor cells from the adult mouse eye that display stem cell properties in vitro. These cells were capable of proliferation in culture, and showed subsequent differentiation into some of the cells of the retina including bipolar cells, rods and Müller glia. This finding opposes the dogma that the mature retina lacks regenerative capability, and contains no mitotic neurons. The putative stem cells expressed ChxIO, reflecting their multipotentiality as neuro- retinal precursors.