Figure 4

r3 and r5 are reduced in size in Krox20^Cre/floxembryos. (a, b) The size of r3 was estimated at E9.5 on flat-mounted hindbrains by labeling adjacent rhombomeres. r4 was delimited by in situ hybridization with a Hoxb1 probe and r2 by detection of the alkaline phosphatase activity from an r2-specific transgene [47, 48]. The negative territory located in between corresponds to r3 and is reduced in Krox20^Cre/flox(b) compared to control Krox20^Cre/+ (a) embryos. A few Hoxb1-positive cells are also observed within r3 in embryos (arrows). (c, d) Flat mounts of Krox20^Cre/+ (c) and Krox20^Cre/flox(d) hindbrains immunolabeled with an antibody directed against the 155 kDa component of neurofilaments (2H3). r3 and r5 can be distinguished from even-numbered rhombomeres by their less advanced differentiation of reticular neurons, revealed by lower neurofilament immunoreactivity. The r5/r6 boundary is clearly visible since it is followed by axons (arrow in (c, d)). Both r3 and r5 are reduced in Krox20^Cre/floxembryos, the effect being more dramatic in r3 (arrowheads). (e) Breathing frequency at birth in heterozygous Krox20^lacZ/+ (left) and in Krox20^Cre/flox(middle) mice is the same as in wild-type mice (WT, white columns); it is lower than normal in homozygous Krox20^lacZ/lacZmice (right). *** : p<0.001