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Fig. 5. Differentiation of sensory patches with the production of hair cells is
inhibited by DAPT treatment initiated at an early stage. Organ cultures
established at stage HH16-17 were maintained for 2-5 days in either control
medium or in medium containing 20 µM DAPT. (A-F) Bmp4
expression analysed after 2 or 5 days in vitro. In control specimens (A,D),
typically two or three patches of strong Bmp4 expression are observed
(arrowheads); in DAPT-treated specimens (B,E), the size and mean number of
Bmp4-positive patches are reduced and the intensity of Bmp4
expression within them is greatly diminished. (C,F) Quantitative analysis of
the mean number of patches of Bmp4 expression in control versus
DAPT-treated otocyst after 2 (C) or 5 (F) days in vitro. (G,H)
Serrate1 and hair-cell antigen (HCA) immunostaining in control (G) and
DAPT-treated (H) otocysts after 5 days in vitro. The micrographs are
z-projections of confocal optical sections encompassing the entire
thickness of the dissected otocysts. Serrate1 is expressed in several patches
containing differentiated hair cells (arrows in G) in control otocysts. In
DAPT-treated otocysts, Serrate1 expression is abolished or greatly reduced and
hair cells are few, although densely clustered (arrow and inset in H).
(I,J) Quantitation of the number of patches of Serrate1
expression (I) and of the number of hair cells (J) after 5 days of culture in
control versus DAPT-supplemented medium. Error bars represent s.e.m. All the
differences between control and DAPT-treated groups were statistically
significant (t-test; *P<10-6).
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