Impuesto a la Renta

Po is expressed in the otic placode/pit at ElO rat, which has about 10 somite pairs. This Po positive structure is already in the process o f invagination. The whole thickness o f the convex part, together with a neighboring flat area around the invagination, is Po positive (Figure 4-2). The otic placode invaginates and forms a vesicle from E l l in the rat. Po is expressed nearly all over the vesicle at E l l (Figure4-3A and 5-1 A). As there is no distinctive morphogenesis before E l 2, several markers that were reported to be specific to certain structures or cell types were compared to the pattern o f Po. Digoxygenin-labeled riboprobes o f BMP-4, c-ret. D elta-1, Serrate, Krox-20, Msx-1, FGF-3 were used. The body segment containing the otic vesicle and the first two branchial arches were dissected from E l 2 rat embryo before fixation. Segments were processed in different containers with different probes in the same experiment. An ElO mouse embryo with a (3-galactocidase gene (p-gal) inserted under PLP/DM-20 promoter was immunolabeled in a whole mount preparation and anti-p-gal antibody was used to reveal the activation o f PLP/DM-20. After in situ hybridization or whole mount staining, the tissues were sectioned at 20-

50 fj.M horizontally using the sucrose protected, gelatin embedded cryosection method (see chapter 2). When roughly dividing the vesicle into quarters using the dorsal-ventral and medial-lateral axes, the Po signal, apart from being present in the endolymphatic duct, is located mostly in the dorsal-medial and ventral-lateral part o f the vesicle. The expression o f BMP-4, c-ret and PLP/DM-20 in the rat otic vesicle have not been reported previously. The BMP-4 expression in rat is similar to previously reported in chick (Wu and Oh, 1996). The D elta-1 and Serrate expression are similar to that previously reported (Myat et al., 1996; Lindsell et all, 1996). Unfortunately the embryos I used for D elta-1 and Serrate were older is development and thus not strictly comparable to the embryos used for Po expression (not shown). C-ret is expressed in the budding endolymphatic duct and in the medial-ventral and lateral-dorsal aspect o f the vesicle, which is roughly reciprocal to the Po expression (not shown). The DAB reactions for the PLP/DM-20 signal were located in the dorsal-medial and ventral-lateral positions, similar to that o f Po, but weremore restricted (Figure 5-2B). No Krox-20 and Msx-1 expression was detected in the otic vesicle at this stage (not shown). As no adjacent section to the Po one was hybridized with markers for the sensory epithelium, it is not feasible to suggest any correlation o f any structure with Po expression at this stage. However, the tentative conclusion is that the overall pattern o f Po expression is very different, if not completely reciprocal, to the expression o f BMP-4, c-ret, delta-1 and Serrate, and is similar to the PLP signal. Although the specification o f the cochlear duct to the ventral half o f the vesicle and the semi-circular canals to the dorsal half o f the vesicle already take place before this stage (Li et al., 1978), there is no telling whether the Po pattern is specific to any o f the structures at this stage.

To further probe the expression o f Po in the developing inner ear, the head o f an E14 embryo was horizontally sectioned using the sucrose protected method (chapter 2). A series o f adjacent sections were collected on adjacent slides for different probe processing. Every other section was hybridized with Po probe. Fig 5-3B is a representative section that slices through the endolymphatic duct, the semi-circular canals and part o f the utricle. A series o f neighboring sections confirms the identities o f these structures (Figure 5-3). Po is expressed in the base o f the endolymphatic

duct, and in parts o f the semicircular canals. Fig 5-4 is a representative section more anterior to Fig 5-3. The cochlea, part o f the utricle and the semicircular canals are shown. In addition, the acoustic ganglion, which sits very near the cochlea, is Po positive. Like DRG o f the same age the motor roots o f acoustic ganglion which project toward hind brain and the sensory roots that innervate the inner ear are highly Po positive. Sporadic Po positive cells could be seen within the ganglion. One section o f the ganglion that sits nearest to the cochlea, however, is completely devoid o f Po positive cells except at the periphery (Figure 5-4). In the cochlea the pattern o f Po expression is restricted to only half o f the section and there is a very sharp boundary o f expression and non-expression (for example. Figure 5-4, 5-6). At this stage the acoustic nerves have started to innervate the cochlea (Von Bartheld et al., 1991). The sensory epithelium, which is near to the ganglion and is supposed to be innervated, is serrate and c-ret positive but Po negative (Figure 5-6, 5-7). W ithin the Po positive patch there are different levels o f expression, with the highest levels usually seen in cells facing the lumen, but in some areas the signal is found all through the thickness o f the cross section (for example, see Figure 5-6C).