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doi: 10.1242/10.1242/dev.00426

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A Notch-independent function of Suppressor of Hairless during the development of the bristle sensory organ precursor cell of Drosophila

Stefan Koelzer and Thomas Klein*

Institute für Genetik, Universitaet zu Koeln, Weyertal 121, 50931 Koeln, Germany



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  		Fig. 1. The development of the mechanosensory bristle organ of the adult peripheral nervous system of Drosophila. (A) An adult bristle (machrochaete) stained for neurA101-lacZ to reveal the nucleus of the socket cell (blue) and anti 22C10 antibody staining to mark the neurone (brown). The bristle sense organ consists of two more cells: the prominent bristle cell and a sheath or thecogen cell, which is not visible in the picture. (B) A wing imaginal disc of the late third larval instar stage, stained with anti Hnt antibody (red) to reveal the SOPs of the machrochaete. The disc contains a scaGal4 insertion that activates UAS GFP in the cells of the proneural clusters (green). The double staining reveals that the clusters are arranged in a stereotypic pattern that allows the identification of each cluster individually. ANP and PNP, anterior and posterior notopleural; APA and PPA, anterior and posterior postalar; DC, dorsocentral; SC, scutellar clusters. (C) Development of the bristle sense organ. The SOP is selected from a proneural cluster during the process of lateral inhibition, which is mediated by the Notch signalling pathway (not shown). The SOP, recognizable by the high level of expression of the proneural protein Ac, signals through the Notch ligand Delta to its neighbours (pink lines). Activation of the pathway results in the Su(H)-dependent switch to the epidermal fate in the neighbours of the SOP. The high levels of Ac and Sc proteins in the SOP are achieved through the activation of the SOP-E of the sc gene (Culi and Modolell, 1998Go). Once the SOP is selected, it switches off the expression of the proneural genes and initiates expression of neurA101-lacZ, sens and hnt. It then divides to generate the second order precursor cells pIIa and pIIb. pIIa divides to give rise to the socket and bristle cells. pIIb divides to generate a third-order precursor pIIIb and a glial cell. The glial cell migrates away and does not contribute to the formation of the sense organ. pIIIb further divides to give rise to the neurone and the sheath cell that protects the neurone. In this lineage, the Notch-signalling pathway is employed several times to help the cells to choose the correct fate. In the first step, pIIb sends a Notch-mediated inhibitory signal (pink line) that prevents pIIa from joining the pIIb fate and developing the pIIa fate. Later Notch is required to send an inhibitory signal from the bristle to the socket and from the neurone to the sheath cell to prevent the receiving cells from choosing the same fate as the sending cell. The differentiated neurone can be detected through the expression of the neurone specific 22C10 and Elav marker. (D) The consequence of loss of Notch function during bristle development. Owing to the lack of Notch signalling, all cells of a Notch mutant proneural cluster choose the SOP fate. As a result of the missing communication between the progenies of the SOP, an excess of neurones develops at the expense of the other fates of the sensillum. These supernumerary neurones can be visualized by anti 22C10 or anti Elav antibody staining.

 


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  		Fig. 2. Expression of the SOP markers neurA101-lacZ, Sens and Hnt in Notch mutant cell clones as well as in Psn, Su(H) and kuz mutant wing imaginal discs. Expression of the SOP marker is detected by antibody staining. Alleles used in this analysis are: Df(1)N81K, PsnC1, Su(H){Delta}47 and the combination kuz1405/kuz1403. They are amorphic or strong alleles of the corresponding genes. Anterior is towards the left, ventral towards the bottom. (A-D) neurA101-lazZ expression. The wing disc shown in A is also stained with an anti Wg antibody to visualize the expression of Wg (red). neurA101-lacZ expression in A is shown in green. The arrows indicate the SOPs that have formed. (E-I) Expression of Sens. (J-N) Expression of Hnt. (A,E,J) Expression of neurA101-lacZ, Sens and Hnt in wild-type wing imaginal discs of the late third larval instar stage. (B,F,K) Wing imaginal bearing Notch mutant clones that are labelled by the absence of the green GFP marker. Expression of the corresponding SOP marker is shown in red. (G,L) PsnC1 mutant wing imaginal discs. (C,H,M) Su(H){Delta}47 mutant wing imaginal discs. (D,I,N) kuz1405/kuz1403 mutant wing imaginal discs. In the wild type, neurA101-lacZ, Sens and Hnt are expressed in all SOPs of the wing imaginal disc. Sens is also expressed in cells along the wing margin (wm in E). Arrows in A indicate the position of each individual SOP. The figure reveals that cells of all proneural clusters in the notum that are mutant for Notch, Psn or kuz express the three SOP markers (arrows in F-I,K-N). The situation is different in Su(H) mutants: only a few cells of the DC, the ANP and ANP cluster weakly express neurA101-lacZ (arrows). Moreover, expression of Sens (H) and Hnt (M) is lost in almost all cells of the notal clusters. We occasionally found weak expression of Sens in some cells of the ANP and PNP cluster (arrow).

 


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  		Fig. 3. Expression of a neurone-specific marker in cells of proneural clusters in Notch, Su(H) and Psn wing imaginal discs of the late third larval instar stage. Anterior is towards the left, ventral towards the bottom. (A-C) Wing imaginal discs stained with anti 22C10 antibody. (D,E) Wing imaginal discs stained with anti Elav antibody. (A) Df(1)N81K mutant clones in the notum of a wing imaginal disc revealed by the lack of the GFP marker. The arrows indicate proneural clusters where the cells express 22C10. (B) Likewise, cells of the proneural clusters of PsnC1 mutant wing imaginal discs express 22C10 (arrows). By contrast, no expression of 22C10 is detectable in the Su(H){Delta}47 mutant wing disc (C). (D) Expression of Elav in PsnC1 mutants. The cells of some clusters, such as the APA+tr1, PPA and PSA clusters, which are fused to one big cluster (arrow), do express Elav. (E) By contrast, Su(H){Delta}47 mutant wing imaginal discs are devoid of any Elav expression in cells of the notum. The results suggest that the loss of SOP markers in the cells of Su(H) mutant proneural clusters is accompanied by a loss of neural differentiation markers.

 


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  		Fig. 4. Analysis of SOP development in Su(H){Delta}47; Psn12 double mutant wing imaginal discs. Anterior is towards the left, ventral towards the bottom. Expression of Sens and 22C10 is detected by antibody staining. (A) Sens expression is lost in all clusters with the exception of some cells of the PNP cluster that have residual expression (arrowhead). (B) Expression of the SOP-E in the same disc as shown in A. The cells of the notal proneural clusters of the double mutant discs still express the SOP-E (arrows), indicating that the absence of Sens expression is not caused by the loss of the cells. (C) Merge of the pictures A and B, showing Sens expression in red and expression of the SOP-E in green. (D) Similar to Su(H) mutants, Su(H){Delta}47; Psn12 double mutants have lost the expression of 22C10, indicating that the cells of the proneural clusters fail to differentiate neurone-specific traits. (E) Expression of a weak UASSu(H) line in the proneural clusters of Su(H) Psn double mutant wing imaginal discs with scaGal4. The expression of UASSu(H) re-establishes the expression of Sens and 22C10 in the cells of the proneural clusters.

 


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  		Fig. 5. Expression of Dl, the SOP-E and E(spl)m8-lacZ in Su(H){Delta}47 mutant wing imaginal disc of the late third larval instar stage. Anterior is towards the left, ventral towards the bottom. (A) Expression of Dl, revealed by anti Dl antibody staining, is elevated in the cells of the proneural clusters (highlighted by the arrows). (B) Expression of the SOP-E in Su(H){Delta}47 mutant discs indicates that most of the cells of the proneural clusters express this enhancer (arrows). (C) The same Su(H){Delta}47 mutant wing disc as shown in A and B showing expression of Dl (red) and anti ß-Gal antibody (green) to visualize the expression of E(spl)m8. Expression of E(spl)m8-lacZ is detectable in the cells of the proneural clusters. (D,E) Expression of E(spl)m8-lacZ in the notal region of a wild-type wing imaginal discs. (D) Expression of E(spl)m8-lacZ, (E) Expression of Sens (red) and E(spl)m8-lacZ (green). The comparison between D and E reveals that expression of E(spl)m8-lacZ is switched off in SOPs at the time when they initiate expression of Sens (Nolo et al., 2000Go). The arrows in D and E indicate the developing SOPs. (F) Results summary. Cells of the proneural cluster in Su(H) mutant notae are present but arrest their development before expression of sens and hnt is initiated.

 


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  		Fig. 6. Forced expression of UAS sens in Su(H){Delta}47 mutant wing imaginal discs re-establishes the expression of the SOP-E, hnt, 22C10 and elav in cells of the proneural cluster. Anterior is towards the left, ventral is towards the bottom. (A-G) Expression of UAS sens with dppGal4 in Su(H) mutant wing imaginal discs. (A) Expression of the SOP-E is activated in a stripe of cells (arrow) in the notum that corresponds to the dppGal4 expression domain. (B) Expression of 22C10 (arrow). (C) The same disc as in B showing expression of UASGFP in green and 22C10 in red. The double staining reveals that most of the cells in the notum that express Sens initiate expression of the neurone-specific marker 22C10 (arrow). The arrowhead in A-C indicates the boundary between the notum and the wing and highlights the fact that the ability of Sens to activate the SOP-E and 22C10 is restricted to cells of the notum. (D) Expression of elav revealed by anti Elav staining. (E) The same disc as in D showing the expression of UAS GFP in green and that of Elav in red. The double staining reveals that although Sens is expressed in a broad stripe in the notum, elav expression is activated only in clusters of cells that are located at positions of the proneural clusters (arrows). This observation suggests that other, locally restricted factors are in addition required to initiate the expression of elav. As in the case of 22C10, Sens can activate expression of Hnt in all cells of the notum where it is expressed (see F,G). (F) Expression of Hnt. (G) Expression of UAS GFP (green) and Hnt (red) in the same disc as shown in F. Arrowheads in F and G indicate the wing/notum boundary and that the ability of Sens to activate hnt is again restricted to cells of the notum. (H) Results summary. Sens seems to be required to activate the expression of hnt, and the neurone-specific genes 22C10 and elav in the developing SOP. This suggests that Sens coordinates the development and differentiation of the SOP. Furthermore, Sens seems to be required for the maintenance of the expression of the SOP-E and thus, the maintenance of high proneural gene activity in the SOP. Hence, the results suggest that loss of Sens activity is the cause for the observed arrest in development in Su(H) mutants.

 


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  		Fig. 7. Clonal analysis of Su(H) during SOP development of the machrochaete. (A-E) An example of a large clone that encompasses part of the PPA, tr2/APA+tr1 and DC clusters. (A) Overview. Expression of the SOP-E is shown in blue and that of Hnt in red. The clones are labelled by the absence of the GFP marker. The arrowhead indicates the region that is shown at higher magnification in B-E. (B) Expression of Hnt is detected in single cells highlighted by the arrowhead and arrows. The cell highlighted by the arrowhead expresses high levels of Hnt. (C) Channel revealing the clone area by the absence of GFP. Arrowhead indicates a large GFP-positive cell that is located in the mutant territory. (D) Expression of the SOP-E is detected in groups of cells. (E) The pseudo-colour composite of the single channels shown in B-D. Green, GFP expression; red, Hnt expression; blue, SOP-E expression. The picture reveals that the Su(H) mutant cells of the DC cluster do express the SOP-E but not Hnt. Hnt is restricted to the green wild-type cell at the boundary of the clone. In the two other clusters, only one mutant cell (arrows in B,E) weakly express Hnt. This shows that the activity of Su(H) is required for the SOP to express Hnt. However, the requirement for Su(H) among cells of a proneural cluster seems to vary, as indicated by the weak expression of Hnt in a single mutant cell in the two cluster labelled by the arrows. Another important observation revealed by this figure is that wild-type cells can develop as SOP, even if located adjacent to Su(H) mutant cells. The arrowheads in B-E label a wild-type cell that, as the SOP-E expression reveals, is part of the otherwise Su(H) mutant DC cluster. This cell is the only cell that expresses Hnt, indicating that it has chosen the SOP fate, although surrounded by mutant cells. Hence, the Su(H) mutant cells are not able to inhibit the cells from adopting the SOP fate. The expression of Hnt in one cells of each of the two mutant clusters (arrows) also suggest that cells at specific positions in the clusters have a higher inclination to adopt the SOP fate. (F,G) Cells at specific positions within a proneural cluster are determined to adopt the SOP fate. (F) Expression of Hnt in cells of the DC cluster of the late third larval instar stage, where the posterior part consists of Su(H) mutant cells. The expression of SOP-E in this disc is shown in the insert. (G) Expression of the SOP-E (blue), hnt (red) in the same disc shown in F. The Su(H) mutant area is labelled by the absence of GFP (green). Two hnt-positive cells are detectable at the aDC position. These cells have switched off the SOP-E, indicating that these cells are the second order precursors of the aDC. In the mutant territory a single hnt-expressing cell is detectable. This cell is located at the position of the pDC and still expresses the SOP-E. The pDC develops earlier than aDC during normal development. Hence, it appears that the Su(H) mutant SOP has arrested its development at an early stage. For further information, see text. The phenotype of Su(H) mutant clones varies also among proneural clusters. An example is the SC cluster shown in H,I. (H) Expression of neurA101 in a Su(H) mutant clone that includes the SC cluster. Many, if not all cells of the cluster express this early marker. (I) By contrast, only a fraction of these cells also express Hnt, often at low levels. (J) Expression of Hnt in kuzES24 mutant proneural clusters. Expression of Hnt is shown in red, the clones are revealed by the absence of the green GFP marker. Hnt is expressed in probably all mutant cells of the proneural clusters. This suggests that, as in the case of homozygous kuz mutants, the cells of kuz mutant clusters do not arrest their development as SOP.

 


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  		Fig. 8. (A-D) Cells of Su(H) mutant proneural cluster express high levels of Dl. (A) Expression of Dl (red) and the SOP-E (blue) in a disc, bearing Su(H) mutant clones labelled by the absence of the green GFP marker. The arrow indicates the region shown in higher magnification in B-D. (B) Expression of Dl. (C) Expression of the SOP-E. (D) Pseudo-colour composite of the disc shown in A-C, showing expression of Dl in red, of the SOP-E in blue. The Su(H) mutant territory is revealed by the absence of GFP. The picture reveals that the cells that express the SOP-E and thus belong to a proneural cluster, also express high levels of Dl. (E-J) Expression of the Gbe+Su(H) construct [Gbe+Su(H)], Dl and Hnt in the notum of a wild-type wing imaginal disc of the late third larval instar stage. (E) Expression of Dl (red) and Gbe+Su(H) (green) occurs in similar domains. Hnt expression (also in red) reveals some of the SOPs present at this time. Arrow indicates the region of the pDC SOP. This region is shown at higher magnification in H-J. (F) Expression of Gbe+Su(H) in the notum of a wild-type disc. (G) Expression of Dl in the same disc as shown in F. The comparison of F and G further reveals the similarity of the expression pattern of Gbe+Su(H) and Dl. (H-J) Higher magnification of the region of the DC cluster, highlighted by the arrow in E. (H) Expression of Gbe+Su(H). (I) Expression of Dl and Hnt. At this focal plane expression of Hnt, but not Dl is visible. Dl is located on the apical side of the cell, which is out of focus. (J) Pseudo-colour image showing the expression of nuclear GFP in green to reveal all cells, of Hnt/Dl in red and the Gbe+Su(H) in blue. Comparison with I,J reveals that the expression of the Gbe+Su(H) is elevated in cells that are immediate neighbours of the SOP (arrowheads in H,J). The SOP itself does not express Gbe+Su(H). This suggests that the SOP sends a signal that activates the Notch pathway in its neighbours. (K-O) Su(H) mutant cells can activate the expression of Gbe+Su(H) in their wild-type neighbours. (K) Expression of Dl/hnt (red) and the Gbe+Su(H) (blue) in a wing disc, bearing Su(H) mutant clones, labelled by the absence of the green GFP marker. The arrow indicates the region shown at higher magnification in L-O. (L) Expression of Dl and Hnt. A group of cells express Dl in its membrane. One cell of this group expresses in addition Hnt, which is located in the nucleus (arrow). (M) Su(H) mutant area, revealed by the absence of GFP. Arrow indicates a single wild-type cell that is located in the Su(H) mutant territory. (N) Expression of Gbe+Su(H). The expression is lost in the mutant territory, indicating that it is dependent on the activity of Su(H). Arrowhead indicates the stripe of elevated expression of the Gbe+Su(H) in the wild-type cells at the clone boundary. (O) Pseudo-colour composite picture of the region shown in L-N. Expression of Dl and Hnt is shown in red, expression of Gbe+Su(H) is shown in blue. The clone is labelled by the absence of GFP. The arrow highlights the Hnt-expressing cell, which expresses GFP and is therefore Su(H) positive. Comparison with N reveals that this cell does not express the Gbe+Su(H), although it is surrounded by Su(H) mutant cells that strongly express Dl. At the left clone boundary, where the GFP positive wild-type cells are adjacent to the mutant cells (arrowhead) the expression of Gbe+Su(H) is elevated (revealed by the arrowheads in N,O). This suggests that the Su(H) mutant cells can activate the Notch pathway in their wild-type neighbours. Thus, Dl expressed in Su(H) mutant cells appears to be active. Nevertheless, the Notch pathway is not active in the wild-type SOP, suggesting that it is insensitive to Dl.

 


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  		Fig. 9. (A-C) Analysis of SOP development in the notum of PsnC1 HE31 double mutant wing imaginal discs. Anterior is towards the left, ventral is to the bottom. Expression of Sens is revealed by antibody staining. (A) Expression of Sens is strongly reduced or absent in the PsnC1 HE31 double mutant proneural clusters. (B) The cells of the double mutant clusters still express the SOP-E (arrows), indicating that they are present. (C) The same disc as in A and B, showing expression of the SOP-E in green and of Sens in red. The analysis indicates that, in the double mutant discs, SOP development arrests in a similar manner as in Su(H) mutants. (D,E) Expression of UAS Su(H)VP16 with dppGal4 in wing imaginal discs of the late third larval instar stage. (D) Expression of sens. Expression of Sens is lost in most SOPs of the notum. Compare with Fig. 2E for normal expression of sens. The arrow indicates ectopic expression of Sens in the wing area, which is a result of the ectopic induction of the wing margin and bristles by UAS Su(H)VP16 (Klein et al., 2000Go; Furriols et al., 2000). (E) Expression of UAS GFP in a disc of the same genotype as in D, showing that the expression of dppGal4 has expanded over most of the notum. (F) A PsnC1 mutant wing imaginal disc where UAS Su(H){Delta}H is expressed with scaGal4. The discs is stained by anti Hnt antibody staining. No expression of Hnt is observed in the region of the notum. (G) The same disc as in F showing expression of Hnt in red and of GFP in green. The expression of the GFP marks the cells of the proneural clusters. This result shows that although the cells of the clusters are present, they fail to develop to the stage at which Hnt expression is initiated. Thus, the expression of UAS Su(H){Delta}H result in an arrest of SOP development in a similar manner as in Psn H double mutants. (H) Summary of the results shown in this picture. The results suggest that Su(H) suppresses the expression of a negative regulator of sens. For this function, Su(H) requires the presence of H. Recent evidence by Barolo et al. (Barolo et al., 2002Go) indicates that H acts as a bridge between Su(H) and its co-repressors Groucho (Gro) and CtBP. It is likely that Gro and CtBP are also part of the repressor complex required for the proper expression of sens.

 


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  		Fig. 10. One or more members of the E(spl)-C encode for the repressor activity that arrests SOP development in cells of Su(H) mutant proneural clusters. (A) Organization of the E(spl)-C. The diagram is based on previous work (Schrons et al., 1992Go; Bailey and Posakony, 1995Go; deCelis et al., 1996Go; Lai et al., 2000aGo; Lai et al., 2000bGo). Blue boxes label the genes that encode bearded-like genes; red labels the genes that encode bHLH repressors. The Df(3R)E(spl)b32.2 uncovers all genes of the complex. The diagram reveals that six genes of the complex are expressed in the notal region of the wing imaginal disc. The expression of three of these genes [m{alpha}, m4 and E(spl)m8] is upregulated in cells of Su(H) mutant proneural clusters. Overexpression of m{alpha} and m4 causes neurogenic and E(spl)m8 anti-neurogenic phenotypes. (B,C) Expression of Sens (B) and Hnt (C) in Su(H) mutant wing imaginal discs, which carry one copy of the deficiency Df(3R)E(spl)b32.2 in their genome. The expression of the markers is revealed by antibody staining. (B,C) A reduction of the number of the genes of the E(spl)-C by half is sufficient to regain expression of hnt and sens in cells of Su(H) mutant proneural clusters. This suggests that the repressor function that is repressed by Su(H) is encoded by one or more members of the complex. (D) Expression of E(spl)m8-lacZ in PsnC1 mutant wing imaginal discs. Arrows indicate the proneural clusters. The cells of the clusters express high levels of E(spl)m8-lacZ. (D,E) Expression of E(spl)mß-CD2 in wild-type (D), Su(H){Delta}47 mutant wing imaginal discs (E). Expression of E(spl)mß-CD2 is reduced in the Su(H) mutant wing imaginal discs. A similar reduction is observed in Psn mutant discs (not shown)

 




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