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First published online May 30, 2007
doi: 10.1242/10.1242/dev.001842

Development 134, 2369-2378 (2007)
Published by The Company of Biologists 2007
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Notch signalling is needed to maintain, but not to initiate, the formation of prosensory patches in the chick inner ear

Nicolas Daudet1,2,*,{dagger}, Linda Ariza-McNaughton1,* and Julian Lewis1

1 Vertebrate Development Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
2 Centre for Auditory Research, UCL Ear Institute, University College London, 332 Gray's Inn Road, London WC1X 8EE, UK.


Figure 1
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  		Fig. 1. Expression of Notch target genes of the cHes5 family is repressed by DAPT treatment. (A-F) Stage HH12 embryos cultured for 24 hours in control (A,C,E) or DAPT-containing (B,D,F) medium and processed for whole-mount in situ hybridisation for cHes5.1, cHes5.2 or cHes5.3; dorsal views, with anterior on the left. In control specimens, all cHes5 genes are expressed in the neural tube, and in the anterior (arrows) and posterior (arrowheads) domains of the otic cup, although the expression of cHes5.2 (C) appears fainter and more restricted than that of cHes5.1 (A) and cHes5.3 (E). After 24 hours in DAPT medium, the cHes5 genes are no longer detected in the neural tube or in the otic cup (asterisks in B,D,F). For each Hes gene analysed, the DAPT and control embryos shown were from the same experimental batch.

 

Figure 2
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  		Fig. 2. Delta1 regulates neuroblast production by lateral inhibition. (A,B) Whole-mount view of dissected ears from stage HH12 embryos cultured for 24 hours in control (A) or DAPT-supplemented (B) medium then processed for Delta1 in situ hybridisation. In control embryos, Delta1 is expressed in scattered cells located in the anterior and medial region of the otic cup, corresponding to the delaminating neuroblasts. In DAPT-treated specimens, expression of Delta1 is more intense in the anterior region of the otic cup, and cells expressing Delta1 contact one another, but the size of the neurogenic patch is reduced (square brackets). (C-E) Transverse views of the otic cup of stage HH12 embryos cultured for 24 hours in control (C) or DAPT-supplemented (D,E) medium, and immunostained for three proteins: Delta1 (blue), Islet1 (green) and TuJ1 (red). In DAPT-treated embryos, an abnormally large number of Islet1-positive neuroblasts delaminate from the otic cup and accumulate in the underlying mesenchyme (arrows in D, compare with C). In control specimens (C), Delta1 protein is almost undetectable, whereas its expression is increased in the inner ear (D,E), as it is in the neural tube (asterisk in D), of DAPT-treated embryos. (E) In DAPT-treated embryos, Islet1- and TuJ1-positive neuroblasts are frequently found in the lumen of the otic cup (arrow). (F-I) Dorsal views (anterior is up) of stage HH11 embryos cultured for 4 hours in control (F,G) or DAPT-supplemented (H,I) medium then processed for whole-mount Delta1 in situ hybridisation. A short DAPT treatment induces a strong upregulation of Delta1 in the neural tube (asterisk) and in the otic cup (arrow in F,H). Closer examination of the anterior part of the otic cup shows that the delaminating neuroblasts are scattered in control embryos, but are more numerous and are frequently organised as cell clusters in DAPT-treated embryos (arrows in G,I). A, anterior; D, Dorsal.

 

Figure 3
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  		Fig. 3. Serrate1 is not regulated by lateral inhibition in the inner ear. (A,B) Whole-mount view of dissected ears from stage HH12 embryos cultured for 24 hours. In the control (A), Serrate1 is most strongly expressed in two patches of cells located in the anterior (a) and posterior (p) regions of the otic cup. After 24 hours of DAPT treatment (B), Serrate1 expression is absent or greatly reduced in the anterior region, but persists in the posterior region. (C-D') Transverse sections of the neural tube and inner ear of stage HH12 embryos cultured for 24 hours in control (C) or DAPT-supplemented (D) medium; double in situ hybridisation for Serrate1 (red) and Delta1 (green) and immunostaining for Serrate1 protein (blue, and monochrome in C',D'). Delta1 expression is upregulated in both the inner ear and neural tube of DAPT-treated embryos (compare C and D). By contrast, in DAPT-treated embryos, both the intensity and the extent of Serrate1 expression are reduced in the inner ear (compare bracketed regions in C,C' with arrowed regions in D,D'), although they are increased in the neural tube (asterisks in C',D'). (E,F) Stage HH12 embryos cultured for 4 hours in control (E) or DAPT-supplemented (F) medium. After this brief DAPT treatment, Serrate1 expression is upregulated in the neural tube (asterisks), but not in the posterior rim of the otic cup (arrowheads), where its levels of expression appear unchanged as compared to control specimens. (G) Dorsal view of a stage HH10 embryo processed for Serrate1 whole-mount in situ hybridisation; anterior is to the left. Serrate1 expression is not detected in the presumptive otic placode field (ot, arrows), which is located anterior to the first somite (asterisk). Notice the expression of Serrate1 in scattered neurons within the neural tube (arrowheads) and in a region of the cephalic ectoderm (ce). (H,I) Whole-mount view of dissected ears from stage HH10 embryos cultured for 24 hours. In control otocysts (H), the anterior (a) and posterior (p) patches of Serrate1 expression are present. In DAPT-treated specimens (I), Serrate1 expression is absent or reduced in the anterior region, where abnormal extrusion of neuroblasts in the otocyst lumen can be seen (arrowhead; see also Fig. 2E). However, the posterior patch of Serrate1 expression persists. A, anterior; a, anterior region; ce, cephalic ectoderm; D, dorsal; ot, presumptive otic placode field; p, posterior region.

 

Figure 4
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  		Fig. 4. Blocking Notch signalling reduces the expression of Sox2 and Bmp4. (A-D) Whole-mount view of stage HH12 embryos cultured for 24 hours in control (A,C) or DAPT-supplemented (B,D) medium. All panels are dorsal views (anterior left). (A) Sox2 is expressed throughout the early otic epithelium of control embryos, sometimes with an increased expression in the anterior and posterior regions of the otic cup. (B) In DAPT-treated embryos, Sox2 expression is greatly decreased in the otic cup, as it is in the neural tube. (C) Bmp4 is expressed in control embryos in the posterior rim of the otic cup and in a small patch in the anterior part (arrows). (D) After DAPT treatment, Bmp4 expression in the otic epithelium is greatly diminished.

 

Figure 5
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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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© The Company of Biologists Ltd 2007