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Notch signalling and the initiation of neural development in the Drosophila eye

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

View larger version (143K): [in a new window]   Fig. 1. Activation of Notch signalling is sufficient to induce the accumulation of Atonal at high levels. (A,B) In N54/9 mutant clones (which lack green GFP staining), Atonal (red) expression is maintained at low levels and is not upregulated as it is in neighbouring wild-type tissue. The broken line indicates the border of the clone. (C,C') Clones of ectopic expression of Ser (green) do not induce ectopic neural differentiation. Photoreceptor differentiation is visualised by Elav expression (red); white arrows indicate the approximate position of the morphogenetic furrow. (D-I) Clones of ectopic expression of Dl (green). The expression of Atonal and Elav are shown in red and blue, respectively, in D,G. (D-F) Clones of Dl-expressing cells within 12-15 cell diameters of the furrow induce the expression of Atonal at high levels autonomously as well as in the cells surrounding the clone (e.g. white arrow in D). In clones that are partially within the competent zone (arrowhead in D,E) Atonal is only activated in the cells nearest to the morphogenetic furrow. Occasionally, we observe Elav-positive cells anterior to normal Elav expression (arrowhead in F). (G-I) Large clones of Dl-expressing cells that cross the morphogenetic furrow causes its anterior displacement. The expression of Atonal behind the morphogenetic furrow is disorganised, and the number of Atonal-expressing cells isolated seems increased. We also observe Elav-expressing cells in advance of its endogenous expression (arrowhead in I), and this expression is disorganised. White arrows indicate the approximate position of the morphogenetic furrow. Here, and in all figures, anterior is towards the left.

View larger version (111K): [in a new window]   Fig. 2. Co-expression of dpp and Dl can induce neural differentiation in all regions ahead of the morphogenetic furrow. The white arrows indicate the approximate position of the morphogenetic furrow. (A) Induction of neural differentiation (visualised by Elav expression in red) in clones ectopically expressing Dl alone (green) is limited to a band of cells near of the morphogenetic furrow (arrowhead). (B-F) Clones ectopically expressing Dl and dpp (green). (B,C) Clones co-expressing dpp and Dl can trigger neural differentiation in all regions anterior to the morphogenetic furrow, even in clones far from the morphogenetic furrow (arrowhead); compare with A. (D-F) The first effect caused by these double overexpressing clones is the activation of Atonal (red). Again, this induction occurs in all the cells surrounding the clone even when they are distant from the competent region. In most cases, Atonal expression is observed several cells away from the border of the clone; within the clone, only isolated cells express Atonal. We explain this result as the consequence of the Atonal expression inducing an ectopic morphogenetic furrow (see text).

View larger version (143K): [in a new window]   Fig. 3. The proneural function of N is reduced when dpp signalling pathway is blocked. (A) Clones of brk-expressing cells along the eye discs margins prevent the initialisation of the MF. (B-G) Clones co-expressing brk and Dl. (H-J) Clone of Dl-expressing cells. In all panels, green marks the clones, red indicates Atonal and blue indicates Elav. (B) Clones of brk- and Dl-expressing cells along the posterior eye margin show the same phenotype as clones of brk-expressing cells – preventing furrow initialisation, which leads to complete loss of neural differentiation (white arrow); this is a characteristic phenotype of the loss of function of the Dpp-signalling pathway. Clones ahead of the morphogenetic furrow only induce activation of Atonal in a thin band of cells immediately anterior to the morphogenetic furrow. Compare the clone indicated in B (arrowhead) with clones of Delta-expressing cells in H and Fig. 1; the distance from the furrow at which Atonal expression can occur is substantially reduced in the presence of brinker. (E-G) Several clones of Dl- and brk-expressing cells. Note the relatively poor ability of these cells to induce Atonal expression compared with clones expressing Dl alone (compare clones labelled with white arrows in E and F with clones of similar size and localisation labelled with white arrows in H,I). Surprisingly, despite the activation of Atonal expression, none of the double mutant clones analysed (n=30) express the neural marker Elav anteriorly to the endogenous Elav expression. Although the clones expressing Delta and brinker activate Atonal expression autonomously, there is a preferential activation of Atonal expression in neighbouring wild-type cells (when they lie near of the morphogenetic furrow – e.g. arrowhead in E); a higher magnification of this clone is shown in C,D. Red arrows indicate the approximate position of the morphogenetic furrow.

View larger version (159K): [in a new window]   Fig. 4. The simultaneous loss of Dpp and N signalling does not prevent the initiation of Atonal expression. All clones are marked by the absence of ß-galactosidase (green), Atonal is stained in red. (A-C,G-I) medea mutant clones. (D-F,J-L) med Dl double mutant clones. (A,G) In med clones Atonal expression is reduced (white arrow) or occasionally absent (arrowhead). (B,H) Some med clones posterior to the morphogenetic furrow ectopically express Atonal (white arrowhead). (C,I) med clones along the posterior eye margin prevent the initialisation of morphogenetic furrow. (D,J) Double mutant clones of med8 Dlrev10 express Atonal at low levels. (E,K) Occasionally posterior med8 Dlrev10 double clones ectopically express Atonal in a cluster of cells (arrowhead in K); note the difference between this and clones of med alone, where the ectopic expression is in isolated cells (compare K with H). (F,L) In some med8 Dlrev10 double clones there are regions where Atonal expression is totally lost (white arrow in L), a phenotype also observed in med clones (white arrowheads in A and G).

View larger version (150K): [in a new window]   Fig. 5. Notch signalling regulates the expression of Emc and Hairy. (A-D) Clone of N54/9 mutant cells. (E) Clone of Dlrev10 mutant cells. (F-L) Clones of Dl-expressing cells. (A-D) In clones of N54/9 mutant cells (which lack green GFP, outlined in white), Hairy (red) is upregulated, and the sharp border between Hairy expressing and non-expressing cells is broken (B, and arrowhead in D). Conversely, in these mutant cells Atonal (blue staining in A,B) is maintained at low levels and not upregulated (white arrowhead in C). (E) Large Minute+ Dlrev10 clone (which lacks red ß-galactosidase staining) shows increased Hairy expression behind the morphogenetic furrow. Note that in the mutant cells adjacent to the clone (arrowhead) Hairy is not expressed. This non-autonomous rescue is characteristic of Dl mutant clones. (F-H) Clones of Dl-expressing cells (green), cause the downregulation of Hairy (red) expression, autonomously, as well as in the wild-type cells surrounding the clone (arrowhead). The downregulation of Hairy is correlated with increased levels of Atonal (blue staining in F,G and arrowhead in H). (I-L) The expression of Emc (blue ß-galactosidase staining in the emcP5c strain in I,J) and Hairy (red in I,J) are reduced in Dl-expressing cells (green). Note that whereas the expression of Hairy disappears in all the cells surrounding the clones and in most of the cells within the clones, the levels of Emc are downregulated within the clones but not in the adjacent wild-type cells. The white arrows indicate the approximate position of the morphogenetic furrow.

View larger version (41K): [in a new window]   Fig. 6. During the progression of the morphogenetic furrow, Hh signalling activates at least two different signals. One is Dpp which, at long range, primes cells to adopt a ‘pre-proneural’ state. These cells are ready to initiate neural differentiation, because they express Atonal and Daughterless, but are held in check because they also express the inhibitors Hairy and Emc, which keep the Atonal expression and activity at low levels. Delta/Notch activation provides a second Hh-dependent signal, which only works at short range (within the morphogenetic furrow), as Delta is a membrane-bound ligand. The activation of Notch causes the downregulation of Hairy and Emc, and consequently the upregulation of Atonal expression and activity. A different short range signal, unidentified but transduced by Raf, has also been proposed to upregulate Atonal (Greenwood and Struhl, 1999) (here shown in grey, see text).

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