Expression of BRN3b and PAX6 in the developing retina

Plasmid containing a 200bp fragment of the mouse Brn3b gene was kindly donated by David Latchman (Institute of Child Health, UGL, London). Over the length of this fragment, the sequence conservation between mouse and human is high enough to make a useful human riboprobe (Liu et al. 2000). In the mouse, Brn3b is expressed in the retina exclusively in a proportion of postmitotic ganglion cells, and is the first known marker of a ganglion cell fate (Xiang, 1998). Evidence from expression studies and analysis of mice containing targeted deletion of Brn3b indicate that it is not required for ganglion cell fate specification, but is required for correct differentiation and maintenance of this cell type (Gan e ta l. 1999).

PAX6 is involved in the earliest stages of eye development, but in the adult retina in both humans and mice expression is maintained in a proportion (-30% ) of ganglion cells (Graw 1996, Nishini et al. 1999), and in amacrine cells of the inner nuclear layer. Expression patterns for BRN3B and PAX6 were generated by in situ hybridisation as described above and in Chapter 2, Methods.

At Bwpc, expression of BRN3B expression (Figure 3.4b) can be seen throughout the neural retina, but is more concentrated in the inner layer. This expression pattern may reflect the developing ganglion cell layer; the punctate and lower levels of BRN3B expression in the outer retina may reflect the migration of postmitotic ganglion cells into the presumptive ganglion cell layer.

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Figure 3.4. Transcription factor gene expression in the 8 wpc human retina by in situ hybridisation. At this stage the retina is dived into two layers (a), and while BR N 3b expression can be readily detected in both (b), it is certainly expressed at lower levels in the outer layer. This is in contrast to to the expression pattern of C H X10 at the same stage (Fig. 3.2), but again reflects the presence of postmitotic ganglion cells within this layer. PAX6 expression (c) is uniform throughout both layers, possibly reflecting both the presence of diving cells and ganglion cells.

Abbreviation; rpe, retinal pigmented epithelium. Scale bar = 100pm.

PAX6

The expression gradient observed is complementary to that observed for CHX70 (Figure 3.2).

In contrast to the gradients of expression seen at 8wpc for both CHX10

and BRN3B, PAX6 expression (Figure 3.4c) is seen uniformly throughout the retina. As Pax6 in mice is expressed in retinal neuroblasts and ganglion and amacrine cells, it is difficult to dissect this expression pattern at this stage of development with reference to specific cell types. Judging from intensity of staining, cells are either expressing PAX6 at lower levels, or not every cell is PAX6-positive. However, single-cell resolution is not visible. Given that PAX6 is only expressed in a relatively small proportion of ganglion cells, the lower levels of expression seen in the inner layer may reflect PAX6 expression in ganglion cells.

In the 10wpc retina, the morphological distinction between the inner and outer retina is far clearer (Figure 3.5a), and as discussed above, CHX10

expression is maintained only within the outer (neuroblastic) layer (Figure 3.2d). Expression of BRN3B (Figure 3.5b), by contrast, is almost entirely restricted to the inner layer. This suggests that by 10wpc the majority of ganglion cells are found within a single layer of the retina. There are a very few BF?A/3B-positive cells within the outer layer, which may reflect late born ganglion cells within this layer as they migrate towards the putative ganglion cell layer.

PAX6 expression at this time (Figure 3.5c) is similarly restricted to the inner layer, with a few individual cells expressing in the outer layer. The intensity of PAX6 expression is higher than that of BRN3B within this inner

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BRN3b

Figure 3.5. Transcription factor gene expression in the 10 week human retina by in s itu

hybridisation. By this stage (c), the inner layer has expanded considerably, and the outer layer remains densely packed. expression is limited to the inner layer, the putative ganglion cell layer. PAX6 expression is also largely restricted to the inner layer, and its higher expression levels may reflect both ganglion and amacrine cells. Abbreviation; rpe, retinal pigmented epithelium. Scale bar = 100pm.

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PAX6

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layer. Although PAX6 is expressed in proportionally fewer ganglion cells than

BRN3B, this increased intensity of staining may reflect development of amacrine cells within the inner retina at 10wpc.

In the adult retina, BRN3B expression (Figure 3.6b) is weakly detected in the ganglion cell layer, suggesting that this gene has a role in the maintenance of this cell type. Expression appears to be in the majority of cells within this layer, consistent with the expression pattern seen in the mouse, and the loss of 70% of ganglion cells within the Brn3b targeted deletion mouse (Gan et al.

1996, 1999).

PAX6 expression in the adult human retina (Figure 3.6c) is seen in the ganglion cell layer in the majority of cells. This is in contrast to the 30% of Pax6

expressing cells within the ganglion cell layer observed in the mouse. Although the differential gene expression patterns for the many different classes of ganglion cells has not been established, this may reflect differences in the relative proportions of distinct ganglion cell classes between mouse and human.

Expression of PAX6 is also readily detected in the inner nuclear layer, in a discreet band on the inner aspect. This position is consistent with the location of amacrine cells (Hollenberg and Spira 1972). In comparison to the expression of CHX10 in the inner nuclear layer there is no overlap between CHX10 and

PAX6 expression domains, indicating that the cells expressing CHX10 are certainly not amacrine cells.