Three distinct roles for notch in Drosophila R7 photoreceptor specification

Abstract

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.

Figure 1. Details of the developing and adult ommatidia.

(A) Scanning EM of the adult eye. Each facet corresponds to a single ommatidium. (B) A schematic adult ommatidium. The four cone cells (c, light blue) overlie the eight photoreceptors. Green labels the outer photoreceptors (R1-R6); R7 is colored purple and R8 is dark blue. R7 lies higher in the retina than R8. (C) TEM section through an ommatidium corresponding to the middle cross-section in (B). R7 is evident at this level. (D) TEM section through a developing ommatidium. At this stage the cone cells are yet to join the cluster. Presumptive photoreceptors are numbered. Note that R8 is the central cell at this stage. (E) Schematic summary of the RTK signaling that promotes the photoreceptor fate. Ras activation via Sev or DER signaling leads to the expression of Phyll, which targets Ttk for degradation. (F) Schematic summary of the incorporation and differentiation of the first seven cells added to the precluster. (i) The precluster is surrounded by many cells. (ii) Cells join the cluster at precise positions; the first three join on the R2/5/8 face. (iii) The two that contact R2/5 begin to differentiate as R1/R6. (iv) The cell between them waits and then subsequently begins differentiation as R7. Two cone cell precursors join the cluster in flanking positions. (v) Two more cone cells join to complete the four cone cell grouping that surrounds the photoreceptors. (vi) The 12 cell cluster with 8 photoreceptors and four cone cells is formed.

Figure 2. N regulates sev transcription.

(A, B) Activated N induces up-regulation of sev.lacZ. (A) shows a wild type eye disc stained for sev.lacZ (green) and α-Svp (blue). The R1/6 precursors (asterisks) are labeled with α-Svp. (A') shows the same disc labeled for sev.lacZ alone. The asterisks mark the R1/6 precursors which do not express detectable levels of sev.lacZ. (B) Image of a sev.N^* eye disc stained as in (A). Here the cells in the R1/6 positions (asterisks) can be seen expressing high levels of β-Galactosidase (green). They do not express Svp because they differentiate as R7s. (C) Down-regulation of N signaling reduces sev.lacZ expression. Wild type clones (black) induced in a sev.Su(H)EnR background (green). The twin spots with two copies of sev.Su(H)EnR are brighter green. (C') Within the wild type clones there is an increase in sev.lacZ staining (red), and in the twin spots there is an enhanced reduction of expression. (C″) is a merge of (C) and (C'). The inset shows a detail from the box indicated in (C') additionally stained for svp.lacZ (blue) which highlights the R1/6/3/4 cells. To the right (in the wildtype tissue) R1/6 flank the R7 cell expressing sev.lacZ (red). To the left (where there are two copies of sev.Su(H)EnR) the cell in the R7 position (asterisk) expresses little sev.lacZ but has high levels of svp.lacZ (blue). (D–F) Reduction of N activity using N^[ts] reduces Sev protein levels. (D) Shows a wild type disc stained for Sev (red). (E) In a N^[ts] disc held at 30°C for 24 h Sev staining is very low. (F) Clones of N^[ts] (labeled by the absence of GFP) held at 30°C for 24 h show an autonomous reduction in Sev expression.

Figure 3. Rescue of sev transcription using heterologous promoters.

Upper panels show the levels of Sev expression (green) in third instar eye discs, and the lower panels show the corresponding adult eyes. Blue arrows indicate ommatidia with R7s, red arrows indicate ommatidia without R7s. (A) shows a wild type eye disc stained for Sev. The inset shows a high magnification image of an ommatidial cluster counterstained with α-Arm (red) to highlight the adherens junctions. The arrow points to Sev protein in the R7 precursor. In the corresponding adult retina all ommatidia have R7 cells. (B) In sev⁰ mutant flies carrying two copies of a tub.sev transgene, little Sev staining is evident in the disc, and there are no R7s in the adult retina. (C) When four tub.sev transgenes are introduced in a sev⁰ fly there is more Sev expression detected in the discs, and many ommatidia show rescued R7 cells. (D) sev^0; GMR.sev eye discs show high levels of Sev expression in the eye disc. The inset shows a high magnification image of an ommatidial cluster counterstained with α-Arm (red) to highlight the adherens junctions and the arrow indicates the level of Sev protein in the R7 precursor. This level of Sev protein rescues sev⁰ as evidenced by the presence of the R7s in the corresponding adult retina.

Figure 4. The effects of N manipulations in the sev°; GMR.sev background.

(A–C) Dl clones (labeled by lack of pigment—evident in photoreceptors by the absence of the black granular mass adjacent to the rhabdomeres) in the GMR.sev-rescued background. (A) Mosaic analysis shows that normal ommatidia still form if either R1 or R6 is mutant for Dl (arrows point to R1 or R6 cells lacking Dl), but not when both are mutant. (B,C) The fate of R7 precursors when both R1 and R6 are mutant. (B) The lower ommatidium labeled with black numbers shows the cell in the R7 position (asterisk) appearing as an R1/6 type when the cells in the R1/6 positions are both Dl. Compare with the wild type ommatidium (top right) labeled in red. (C) At the level of the R8s, the lower ommatidium (black labels) still shows the large rhabdomere cell (asterisk) consistent with it being an R1/6 type, and R8 can be seen projecting between the inferred R1/2 cells. Compare with the wild type ommatidium (red labeling) in which the cell in the R7 position is no longer evident at this depth and R8 projects between R1 and R2. (D,E) Clones of sev.Su(H)EnR labeled by the absence of pigment. (D) When the cell in the R7 position carries sev.Su(H)EnR, it can transform into an R1/6-like cell (asterisk) with (E), a rhabdomere that projects into the R8 levels. Labeling and details are as given in (B,C) above. (F) In a sev.Su(H)EnR eye disc cells in the R7 position often differentiate as a normal R7 (lower left asterisk marks a Runt-expressing R7), but at a lower frequency the cell in the R7 position expresses Bar the R1/6 marker (upper right asterisk). Cells are also stained with α-Elav to mark the neural fate. (G,H) The effects of N^* on sev°; GMR.sev R1/6 cells. (G) shows a mosaic analysis of sev.N^* (marked by the absence of pigment). Arrows point to sev.N^* R2, R3, R4 and 5 cells in normally constructed ommatidia. (H) Image of a third instar sev°; GMR.sev; sev.N^* eye disc. The cells in the R1/6 positions express Runt (asterisks) the R7 marker. The tissue is counter-stained with α-Svp to mark R1/6/3/4 cells and α-Elav to label neurons. (I) Schematic summary of the effects of N manipulation in the sev°; GMR.sev background. If N signaling is reduced in the R7 precursor, it differentiates as an R1/6 type. If N signaling is activated in an R1/6 precursor, the cell is specified as an R7.

Figure 5. Evidence that N^* generates a barrier to photoreceptor differentiation.

(A–D) The effects of sev.N^* in the absence of sev. (A) shows an apical section through a sev.N^*eye; ommatidia have a variable number of photoreceptors, often four large rhabdomere cells and two or three small rhabdomere cells - see inset. (B) When sev is concomitantly removed (sev⁰; sev.N^*), there is a loss of the small rhabdomere cells. (C) sev°; sev.N^* eye disc shows cells in the R1/6 positions (asterisks) expressing the cone cell marker Cut (green). (D) When sev.Ras^* is supplied to the cells shown in (C), the R7 fate is restored to the cells in the R1/6 positions (asterisks) as evidenced by Runt expression (green). (E–H) Down-regulation of N signaling converts cone cells to R1/6 type cells. (E) shows a section through a sev.Su(H)EnR eye; many large rhabdomere cells are present in the ommatidia. (F) A sev.Su(H)EnR disc labeled for Cut (green) and SVP (red). Circles highlight early R3/4 pairs showing no evidence of incorporation of mystery cells. (G) Image from the posterior of a sev.Su(H)EnR disc labeled as in (F). Cells in cone cell positions expressing Cut can also express Svp (asterisks). (H) Image of a sev.Su(H)EnR 36 h pupal disc showing supernumerary Svp-expressing photoreceptors (red). (H') The same disc as (H) with the level of Cut expression (green) flattened onto the Svp layer. There are a reduced number of Cut expressing cone cells.

Figure 6. Ttk expression and its correlation with the failure to differentiate as a photoreceptor.

(A) Wild type expression of Ttk. Ttk (green) is expressed at high levels in the cone cells in the apical regions. (A') But is only weakly expressed in the nuclei of the basal layer. Note, the strong staining at the back of the disc is from apical tissue curving down in the disc. (A″) shows the same disc also stained for Svp and Runt to label the R1/6/3/4 and R7/8 cells respectively, to allow clear identification of the Ttk-expressing cone cells. (B) A N^[ts] disc held at 30°C for 24 h and stained for Ttk (green), which is significantly reduced. (B' and B″) show the same disc, respectively, stained for Svp (red) and Sens (blue) to show the persistent expression of other proteins. (C) A sev°; sev.N^* eye disc stained for Ttk (green) and Svp (red). (C') shows a blow-up in which the cells in the R1/6/7 positions (asterisks) express high levels of Ttk. (D) is a wild type disc for comparison with (C). Here R1/6/7 do not express high levels of Ttk, but instead express high levels of either Svp (R1/6) or Runt (R7).

Figure 7. Summary diagrams.

(A) R7, R1/6 and the cone cells form an equivalence group. Manipulations of the RTK/N pathways can freely transform fates between the R7, R1/6 and the cone cell types. Any cell can form any of the three fates depending on the signals that it receives. (B) N activates a repressor of photoreceptor development that DER cannot overcome but Sev can. In the upper level, the wild type situation is depicted. Here we infer that a DER activating signal from the R2 (red arrow) specifies the R1 precursor as a photoreceptor (R1/6 type). In the middle level, when N is ectopically active in the R1 precursor in the absence of sev, the DER activating signal is unable to specify the R1 precursor as a photoreceptor. In the lower level, when N is ectopically active in the R1 precursor in the presence of the sev gene, the cell is specified as a photoreceptor (R7 type). (C) A model of fate R1/6/7/cone cell specification. (i) The precluster cells express low levels of Dl (black arrows). (ii) Cells are recruited into defined niches; the first three being those on the R5/8/2 face. Three cells occupy these positions and Dl from the precluster activates mild N signaling (weak shade of gray), which provides a weak block to photoreceptor specification. Spitz expressed by R2/5 (red arrows) activates DER in the cells in the R1/6 position and overcomes the N block. (C) R1/6 begin differentiation as photoreceptors and express high levels of Dl as cells join the niches of the two flanking cone cells. The cell in the R7 position and these presumptive cone cells receive Dl from R1/6 and N is activated to high levels (dark gray). This provides a potent barrier to photoreceptor specification and also activates sev transcription. Binding of Sev to Boss on R8 provides high-level RTK activity (blue arrow). (iv) The RTK signaling specifies the R7 precursor as photoreceptor and the concomitant presence of activated N directs the R7 rather than the R1/6 photoreceptor type. R7 proceeds to express high levels of Dl, as do the two cone cells and R3. As the subsequent cone cells join, they receive these Dl signals and activate both the barrier to photoreceptor differentiation and sev expression. (V) None of the cone cell precursors contact R8 so none have activated Sev, and Spitz diffusing from R2/5 is unable to trigger the photoreceptor fate because of the high N activity.