Notch activity opposes Ras-induced differentiation during the Second Mitotic Wave of the developing Drosophila eye

Abstract

Background: EGF receptor acts through Ras and the MAPK cascade to trigger differentiation and maintain survival of most of cell types in the Drosophila retina. Cell types are specified sequentially by separate episodes of EGFR activity. All the cell types differentiate in G1 phase of the cell cycle. Before differentiating, many cells pass through the cell cycle in the "Second Mitotic Wave" in response to Notch activity, but no cell fates are specified during the Second Mitotic Wave. It is not known how fate specification is limited to G1-arrested cells.

Results: Competence to differentiate in response to activated RasV12 was diminished during the Second Mitotic Wave accounting for the failure to recruit cell fates from cycling cells. Competence was not restored by blocking cell cycle progression, but was restored by reduced Notch activity.

Conclusion: Competence to differentiate does not depend on cell cycle progression per se, but on the same receptor activity that also induces cell cycle entry. Dual effects of Notch on the cell cycle and on differentiation help ensure that only G1 phase cells undergo fate specification.

Figure 1

A. ELAV-labelling of differentiating photoreceptor neurons in the eye imaginal disc. Anterior to the left. Bar indicates columns 1–4, where cells progress through the Second Mitotic Wave cell cycle. ELAV protein is first detected in column 2. GMR-GAL4 drives UAS-reporter gene expression in all cells in column 1 and more posteriorly. Progressive addition of neural cells to each ommatidial cluster occurs more posteriorly (rightwards). B. GMR-GAL4>UAS-RasV12. Ectopic neural differentiation is first detected in column 5 (arrows), after the SMW. C. At 29°C, GMR-GAL4>UAS-RasV12 is expected to drive transgene expression at a higher level. Ectopic differentiation is nevertheless restricted until after the SMW, however (arrow). D. RasV12 expression in response to 'strong' GMR-GAL4 likewise affects differentiation only posterior to column 5 (eg arrow). E. 5 h after a 10 min heat shock, hs-RasV12 has not yet affected the pattern of ELAV expression. F. 9 h after a 10 min heat shock, hs-RasV12 has induced ectopic ELAV expression both anterior to the furrow, and posterior to column 7(arrows). G. 14 h after a 10 min heatshock, ectopic ELAV expression (arrows) is observed anterior and posterior to a band of ommatidia that remain little affected. H. More intense Ras activity, due to longer heat shock, leads to general neurogenesis. 14 h after a 1 h heat shock. I. More intense Ras activity, due to transcription from the Sev promoter in SavRasV12, also induces neurogenesis in cells occupying columns 3–5.

Figure 2

A-C. Normal pattern of neurogenesis and cell cycle progression in GMR-GAL4. Cells enter S-phase of the SMW after column 0, and most cells (~90%) perform mitosis and degrade their Cyclin B between columns 3–5. Arrowhead indicates column 0 in the morphogenetic furrow. D-F. In GMR-GAL4>RasV12, ectopic neurogenesis occurs after the Second Mitotic Wave is completed (arrow). 100% of SMW cells perform mitosis and degrade Cyclin B in this genotype[11]. G-I. GMR-p21 prevents entry into the SMW, so that no cells contain Cyclin B posterior to the morphogenetic furrow [10]. Ectopic neurogenesis is still delayed until after column 5, however (arrow).

Figure 3

A. In wild type, photoreceptor neurons (ELAV, magenta) differentiate clustered around central R8 cells (SENSELESS, green). Arrowhead indicates column 0 within the morphogenetic furrow, where single R8 cells are first individually resolved. Anterior to the left. B. In GMR-p21, photoreceptor differentiation is first observed in column 2, as in wild type (compare panel A). C. In wild type, cone cells (CUT, magenta) are first detected around ommatidia in column 10. D. Cone cells are detected no earlier in GMR-p21, despite not having to divide before differentiating.

Figure 4

A In wild type, photoreceptor neurons (ELAV, magenta) differentiate clustered around central R8 cells (SENSELESS, green). Arrowhead indicates column 0 within the morphogenetic furrow, anterior to the left. B. Reduced N function causes recruitment of additional neurons (ELAV, magenta). Not all label with the R8 marker Senseless(green). Many are associated with large clusters of R8 cells (arrows). Additional neurons are also recruited by ommatidia that contain single R8 cells, however (arrowhead). Such recruitment reflects N function after R8 cell patterns has been finalized in column 0. C. Ectopic non-R8 neurons are EGFR dependent. When egfr function is reduced in addition to N, the only extra neurons are R8's. D-F. Ectopic expression on N-DN does little to interfere with normal neurogenesis, or with ectopic neurogenesis in response to activated RasV12.

Figure 5

A. In wild type, photoreceptor neurons (ELAV, magenta) differentiate clustered around central R8 cells (SENSELESS, green). Yellow bar indicates columns 1–4, corresponding to the Second Mitotic Wave. B. GMR-RasV12 promotes ectopic neurogenesis posterior to column 5 (eg arrow). Between columns 2–4 most ommatidia (72%) had the normal number of ELAV-positive cells, and only 0.50 extra neurons per ommatidium were seen on average. C. Reduced N signaling results in ectopic R8 specification within the morphogenetic furrow. An average of 0.36 extra neurons were seen in column 4 where R8 cells were unaffected(eg arrow; see also Figure 4B). 68% of the ommatidia in column 4 had the normal number of ELAV-positive cells. There are many more ectopic neurons anteriorly, but as columns 2–3 contained many R8 cells it is unclear how many other neurons differentiated as a direct consequence of loss of N function and how many were recruited by R8's. D. GMR-RasV12 promotes more and earlier ectopic neurogenesis when N function is reduced (compare panel B). Arrow indicates extra ELAV-positive cells around ommatidia with single R8 cells. There were 4.1 extra ELAV positive neurons per ommatidium, on average, and in some preparations all the cells appeared to be ELAV positive.