To answer the questions whether the residual activity of DlK2R is observed only under conditions of over-expression and, if not, whether it could provide sufficient activity for the complete development of Drosophila, we made use of a fly line where exon 6 of genomic Dl is replaced by an attP landing site (DlattP) [28]. Exon 6 encodes most of Dl, including its ICD (Fig. 1A). DlattP is a true null allele, causing the death of homozygous flies during embryogenesis. They display the neurogenic cuticle phenotype characteristic for loss of function of genes encoding members of the Notch signalling pathway [19, 28] (Fig. 1B, B’). DlattP allows to generate knock-in alleles encoding variants, such as DlK2R, expressed under the endogenous Dl-promoter by insertion of a modified exon 6 into the DlattP landing site (Fig. 1A). We initially generated two variants, DlattP-Dl-HA (control) and DlattP-DlK2R-HA. The HA-tag allowed us to monitor the expression pattern of these variants by anti-HA antibody staining. To judge the correctness of their expression, we used the expression of Dl::GFP, a genome edited functional version of Dl fused to GFP, as a reference [29]. The comparison of the expression with Dl::GFP in the wing, leg and haltere imaginal discs, the larval brain, the embryo, as well as in the adult gut revealed that both DlattP-variants are expressed in the correct pattern (Fig. 1C–D’’, Additional file 1: Fig. S1). To test whether the control DlattP-Dl-HA allele is expressed at the correct levels, we compared its expression with endogenous Dl. To do so, we induced Dlattp-Dl-HA homozygous clones adjacent to clones homozygous  for endogenous (wildtype) Dl and performed anti-Dl antibody staining. We did not observe any differences in the levels of Dl in the two types of clones, indicating that the knock-in Dl-HA allele is correctly expressed. The result also indicates that the addition of the HA tag has no obvious effect on expression or subcellular localisation (Fig. 1E, E’).

Fig. 1

Generation of the DlattP alleles. A Generation and use of DlattP. Exon 6 of Dl was replaced by an attP landing site. This replacement creates a Dl null mutant allele DlattP. The attP site allows the insertion of modified exon6-variants. B, B’ Cuticle preparations of wt (B) and DlattP (B’) embryos. The DlattP mutant flies possess only a small patch of dorsal cuticle (arrowhead in B’). This neurogenic phenotype is characteristic for mutants of genes encoding Notch pathway components. C–D’’ Expression of DlattP-Dl-HA (C–C’’) and DlattP-DlK2R-HA (D–D’’) in comparison to Dl::GFP. E, E’ The wing region of a wing disc bearing Dl-HA homozygous clones (bright green, Dl-HA). The clone is outlined in white and yellow. The clone is next to a clone homozygous for endogenous Dl (Dl, no green). E’ The expression levels of both Dl-variants is indistinguishable, indicating that the knock-in of a HA-modified exon 6 does not affect the levels of expression

One copy of endogenously expressed DlK2R-HA produces sufficient activity to allow complete development of Drosophila

We first tested the effect of only one copy of the two DlattP-variants on development by analysing them over the chromosomal deficiencies DlBSC850, or Dlrev10 (Df), which both include the Dl locus. As expected, DlattP-Dl-HA/Df control flies developed to adulthood and displayed the dominant haplo-insufficient phenotype characteristic for Dl-heterozygous flies (Fig. 2A, B, D–F). Thus, similar to endogenous Dl, one copy of DlattP-Dl-HA is sufficient to allow the complete development, giving rise to flies with the expected phenotype.

Fig. 2

Analysis of the phenotype of adult DlattP-Dl-HA and DlattP-DlK2R-HA flies. A–G The phenotype of flies with one copy of DlattP-Dl-HA (B, F) and DlattP-DlK2R-HA (C, G) over the deficiency DlBSC850 (Df). Compare with the wildtype control, shown in A, D. In contrast to the wildtype and DlattP-Dl-HA/Df flies, the tarsal segments 4 and 3 are fused in DlattP-DlK2R-HA/Df flies.E, FDlattP-Dl-HA/Df flies show the typical haplo-insufficient wing vein broadening of Dl. The vein broadening is only slightly enhanced in DlattP-DlK2R-HA/Df flies. H, J The phenotype of flies homozygous for DlattP-Dl-HA resembles that of wildtype flies (compare with A, D). I, K In contrast, homozygous DlattP-DlK2R-HA displays enhanced broadening of the wing veins and nicks in the wing margin (I, arrowheads). Moreover, tarsal segments 1 and 2 are fused in addition to 3 and 4 (K, arrowheads). L–N The phenotype of DlattP-DlK2R-HA homozygous flies is suppressed if two additional copies of Notch are present

To our surprise, DlattP-DlK2R-HA/Df flies also developed to the adult stage and hatched. The adult flies displayed a phenotype that was only slightly stronger than the haplo-insufficient Dl phenotype. They had fusions of tarsal segments 3 and 4 and slightly enhanced broadening of the wing veins in comparison to DlattP-Dl-HA/Df and Df/ + flies (Fig. 2A, C, D, G). Moreover, the DlattP-DlK2R-HA/Df females produced only very few eggs, revealing a strong decrease in fertility.

Altogether, these results indicate that the sole presence of a single copy of DlattP-DlK2R-HA in the genome allows the complete development of Drosophila, giving rise to vital, but nearly sterile flies, which display a surprisingly weak Notch loss of function phenotype. Complete loss of function of Dl results in embryonal lethality. Thus, DlattP-DlK2R-HA can provide a significant amount of Dl-activity.

The Ks in the ICD of Dl are required to suppress CI

As expected for a wildtype allele, homozygous DlattP-Dl-HA flies resembled wildtype flies in all aspects monitored, indicating that an increase in copy number leads to an increase in activity of the Notch pathway and the suppression of the haplo-insufficient phenotype of Dl. (Fig. 2D, H, J). In contrast, homozygous DlattP-DlK2R-HA flies displayed a more severe phenotype than DlattP-DlK2R-HA/Df flies. The broadening of wing veins was significantly more enhanced and nicks occurred in the wing margin (Fig. 2K arrowheads, compare with G). Moreover, the tarsal segments 1 and 2 were fused in addition to 3 and 4 (Fig. 2K, compare with C). Hence, the increase in copy number of DlattP-DlK2R-HA (homozygosity) caused a more severe, instead of the expected wildtype phenotype, indicating a reduction in Notch pathway activity.

Concentration-dependent suppression of Notch activity by the ligands is the hallmark of CI, as it depends on the ratio between the levels of Notch and its ligand [24, 30]. We have previously shown that Gal4 over-expressed DlK2R had increased cis-inhibitory abilities, which could be suppressed by an increase of the levels of Notch [27]. To test whether an increase in CI causes the enhancement of the phenotype of homozygous DlattP-DlK2R flies, we increased the copy number of Notch by introducing a BAC that contains the genomic region of Notch. The BAC encodes a fully active NotchiGFP fusion protein (NiGFP) [31]. We found that the presence of two copies of NiGFP in the genome in addition to the endogenous copies, suppressed the phenotype of DlK2R-HA homozygous flies. The original number of five tarsal segments was restored and the broadening of the veins suppressed (Fig. 2L, M, O). In addition, no nicks in the margin were observed (Fig. 2L, I). Note that the combination mutually repressed the wing phenotypes of each individual genotype. Both individual genotypes displayed broadening of the wing veins (compare Fig. 2I, L, N). This finding confirms the finding of Berndt et al. [27] that DlattP-DlK2R-HA has increased cis-inhibitory abilities.

The Ks are required for effective endocytosis of Dl

Upon comparison of the expression of both DlattP-variants, we had the impression that DlK2R-HA is more abundant in the plasma membrane than Dl-HA and Dl::GFP. To validate this impression, we used clonal analysis to directly compare the expression of DlK2R with that of endogenous untagged Dl in adjacent homozygous clones, as we have done for an Dl-HA (see Fig. 1D, D’). We found higher levels of Dl in DlattP-DlK2R-HA compared to wt clones (endogenous Dl) in the apical plasma membrane (Fig. 3A-A’’’’). The higher abundance of DlK2R in the apical membrane was also observed when we directly compared the expression of DlattP-DlK2R-HA with DlattP-Dl-HA using clonal analysis (Additional file 1: Fig. S2A-B’’). Western Blot analysis revealed that both DlattP-variants are expressed at similar levels in the cell (Fig. 3B). Therefore, the difference between DlK2R-HA and Dl-HA must be founded in different subcellular localisation and DlK2R is more abundant in the plasma membrane. To further confirm this notion, we performed antibody staining on discs where the cells were not permeabilised with detergents during the incubation with a primary antibody directed to the ECD of Dl. In this set up, only Dl inserted in the plasma membrane (surface Dl) is detected. The staining confirmed the higher abundance of DlK2R in the plasma membrane (Fig. 3C, C’, arrow and arrowhead). These results suggest that the loss of the Ks in the ICD results in an accumulation of Dl in the apical membrane.

Fig. 3

The Ks in the ICD of Dl are required for efficient endocytosis. A–A’’ Clonal analysis of DlattP-DlK2R-HA. The homozygous DlattP-DlK2R-HA clones are labelled by loss of GFP and highlighted by the arrows. The comparison with the clone homozygous for endogenous Dl (arrowhead), reveals the higher abundance of DlK2R-HA in the apical plasma membrane of homozygous cells (arrows). See also the z-sections in A’, A’’’’, asterisk. A’’ Pixel density measurement of the apical region highlighted in A with the rectangle (s: start-point of measurement). It shows the higher abundance of DlK2R-HA in the apical membrane. B–B’’ Western-blot analysis of the Dl variants revealed that they are similarly expressed (n = 3). C, C’ Detection of surface Dl by applying the primary antibody in the absence of detergence. In this clonal analysis, homozygous DlattP-Dl-HA clones (dark green, arrowhead) adjacent to homozygous DlattP-DlK2R-HA clones (arrow) were induced and the Dl-variants detected with anti-Dl antibody the binds to the ECD. The staining reveals that homozygous DlattP-Dl-HA cells have less Dl on its surface than homozygous or heterozygous DlattP-DlK2R-HA cells. D–E’’’’ Wing discs where Dmon1 and DlattP-Dl-HA- (D–D’’’’) or Dmon1 and DlattP-DlK2R-HA- (E–E’’’’) clones were induced. D, E Overview of the disc bearing the clones. The homozygous Dmon1DlattP-variants clones are labelled by the loss of GFP and highlighted by the arrow. D’–D’’’’, E’–E’’’’ z-section of the regions highlighted in D, E by the rectangle. A double mutant clone is outlined in white or yellow. F Quantification of the association of the enlarged Notch-positive endosomes of Dmon1 cells with the HA-signal (Dl-variants, n = 3 for each genotype, see M&M for details). It confirms less association of DlK2R-HA with the enlarged Notch positive endosomes compared to Dl-HA

In our clonal analysis shown in Fig. 3A-A’’’, the differences in the abundance of Dl were less significant if we compared heterozygous DlK2R/ + with homozygous DlK2R clone cells. We have previously shown that DlK2R is less efficiently endocytosed and degraded under over-expression conditions [27]. Thus, it is likely that the observed accumulation of DlattP-DlK2R-HA in the plasma membrane is caused by its inefficient endocytosis. This accumulation of DlattP-DlK2R-HA could explain the observed smaller difference between heterozygous and homozygous DlattP-DlK2R-HA cells. To further confirm a defect in endocytosis, we compared the abundance of the variants in endosomes of Dmon1 mutant cells. The loss of Dmon1 function results in a failure of fusion of the endosomes with the lysosome [32, 33]. As a result, the lifetime of the endosomes is extended, they enlarge dramatically and accumulate cargo, such as Notch and Dl [33]. We found that fewer of the enlarged Dmon1-mutant endosomes contained DlattP-DlK2R-HA compared to DlattP-Dl-HA and that the signal associated with the endosomes is weaker, suggesting that DlK2R-HA does enter the endosomal pathway with less efficiency (Fig. 3D–F’’’).

Formally, the higher abundance of DlK2R in the apical membrane could also be caused by enhanced recycling after endocytosis. The main recycling routes are controlled by the small GTPases Rab11 and Rab4 (slow and fast recycling, respectively) [34]. We found that the concomitant efficient co-depletion of Rab11 and Rab4 did not affect the level of DlK2R in the plasma membrane of wing disc cells (Additional file 1: Fig. S2C-D’). Thus, it appears that the higher abundance of DlK2R in the plasma membrane is not caused by enhanced recycling. Our results are in line with previous work by Banks et al. [35]. Altogether, these findings suggest that the accumulation of DlK2R in the plasma membrane is a result of reduced endocytosis in comparison to Dl. Thus, the Ks of the ICD appear to contribute to the efficiency of endocytosis of Dl. However, it is worth to highlight that DlattP-DlK2R-HA can still be detected in endosomes, confirming previous work that Dl is also endocytosed in a K- and ubi-independent manner [14, 21, 27].

Signalling activity of the DlattP-variants

We next compared the signalling abilities of the two DlattP-variants. We first monitored the global expression of the Notch activity reporter Gbe + Su(H) in the notum of wing imaginal discs. In this region, Gbe + Su(H) is expressed in four stripes [36] (Fig. 4A).

Fig. 4

The Ks in its ICD are required for the full activity of Dl. A, B Gbe + Su(H) expression in DlattP-Dl-HA and DlattP-DlK2R-HA/Df discs. Four stripes are recognisable in the wt disc in A. The arrow points to stripe3 which is decreased in DlattP-DlK2R-HA/Df discs. C–D’’ A wing disc bearing homozygous DlattP-Dl-HA (bright green outlined in white) andDlattP-DlK2R-HA (no green) twin clones.C Overview. D–D’’’’ Magnification of the notal area highlighted in C with the arrowhead. The homozygous Dl-HA clone is outlined in white or yellow. Although DlattP-Dl-HA accumulates to lower levels than DlattP-K2R-HA in the plasma membrane (D’), Gbe + Su(H) is stronger expressed in DlattP-Dl-HA homozygous territory (D’’), indicating that DlattP-Dl-HA can activate the Notch pathway more strongly and that also stripe2 is affected by the loss of the Ks in the ICD of Dl. D’’’, D’’’’ pixel density measurement reveals the Gbe + Su(H) is increased in the clone homozygous for Dl-HA, whereas the HA-signal drops. The converse is true in the adjacent DlK2R-HA homozygous clone. s: start of the measurement. See also Additional file 1: Fig. S3 for more examples. E–E’’’ Measurement in a control wing disc with the same genotype as in C–D'’’’ without clones for comparison. F, G’ Expression of Wg in DlattP-Dl-HA/DfandDlattP-DlK2R-HA/Df discs. The arrow points to the expression of Wg along the D/V-boundary. H–L induction of ectopic Wg expression by expression of Fng with ptcGal4. The length of the ectopic stripe of Wg expression is strongly reduced in DlattP-DlK2R-HA/Df compared to DlattP-Dl-HA/Df discs (arrow). L Quantification of the length of the ectopic stripe of Wg expression induced by Fng in the DlattP variants. The length is measured by the number of cells in the ectopic Wg stripe (rectangle, n = 12 for Dl-HA and n = 18 for DlK2R)

We observed a significant reduction in expression of stripe3 in DlattP-DlK2R-HA/Df, compared to DlattP-Dl-HA/Df notae (Fig. 4A, B, arrow). This reduction in Gbe + Su(H) expression indicates a significant reduction in the ability of DlattP-DlK2R-HA to activate the Notch pathway.

Next, we compared the expression of Gbe + Su(H) in the described twin clone experiment where the twin clones are homozygous for one of the DlattP-variants to test whether stripe2 expression of the Notch activity reporter Gbe + Su(H) is also reduced in DlattP-DlK2R-HA homozygous cells. The expression in stripe2 was reduced in homozygous DlattP-DlK2R-HA clones compared to the adjacent homozygous DlattP-Dl-HA twin clones, although DlattP-DlK2R-HA accumulated to higher levels in the plasma membrane than DlattP-Dl-HA (Fig. 4C–E’’’, see Additional file 1: Fig. S3 for more examples). This result shows that the increased presence of DlattP-DlK2R-HA in the plasma membrane correlates with reduced signalling and supports the notion that ubi-dependent endocytosis is required for normal levels of Dl-signalling and abundance in the apical membrane.

The development of the wing margin depends on continuous Notch signalling along the dorsoventral (D/V) compartment boundary [37]. The signalling results in the expression of Wg, which is crucial for margin formation and is initiated at the boundary of Fringe (Fng) expressing and non-expressing cells, by mutual signalling via Ser and Dl [38]. We found that the stripe of Wg expression along the D/V boundary was grossly normal in DlattP-DlK2R-HA/Df wing discs, indicating that DlK2R-HA can provide sufficient activity for expression of Wg and margin formation (Fig. 4F–G’, arrow). This is in line with the finding that the adult flies have a normal looking margin (Fig. 2G). To test whether signalling along the D/V boundary by DlattP-DlK2R-HA is weaker than by DlattP-Dl-HA, we monitored the ability of the DlattP variants to activate Notch at an ectopic boundary of Fng-expressing and non-expressing cells in the wing primordium. Ectopic expression of Fng with ptcGal4 induces an ectopic stripe of Wg expression in the ventral compartment of the wing anlage that straddles the anterior–posterior compartment boundary [39] (Fig. 4H, arrow). The stripe depends on mutual endogenous Dl/Ser signalling, but is initiated by Dl. Its length depends on the level of Dl signalling [39,40,41]. Consequently, the Fng-induced ectopic stripe is shorter in Dl heterozygous than wildtype wing discs [39, 41] (Fig. 4I, J, arrow). In DlattP-Dl-HA/Df flies, the expression of Fng induced a stripe of ectopic Wg expression of similar length than in Dl heterozygous flies, confirming the full functionality of DlattP-Dl-HA (Fig. 4J). In contrast, the ectopic stripe induced by Fng in DlattP-DlK2R-HA/Df discs was much shorter, confirming a substantial reduction of the signalling activity of DlattP-DlK2R-HA, compared to DlattP-Dl-HA or endogenous Dl (Fig. 4J, K, arrow, quantification in L).

Altogether, the results extend our previously reported ones, achieved with over-expression, by showing that the loss of the Ks in the ICD causes a significant reduction in the activity of Dl in Mib1-dependent processes.

Ubi is required for efficient trans-endocytosis of the extracellular domain of Notch during Dl signalling

Notch signalling initiated by Dl plays an important role in the female germline. DlattP-DlK2R-HA/Df and homozygous DlattP-DlK2R-HA females deposited only few eggs. This factual sterility is probably caused by non-penetrant defects that are characteristic for Notch loss of function, such as fused egg chambers (Fig. 5A, D, F). We therefore monitored the activity of Notch in the female germline of the DlattP-DlK2R-HA/Df, and DlattP-Dl-HA/Df flies during a prominent Dl-signalling event from the germline cells to the cells of the follicle epithelium that occurs during stages 5–7 of oogenesis to induce their differentiation [42, 43]. The signalling is achieved by the strong up-regulation of Dl expression during these stages in the germline cells and results in the termination of expression of Cut and the induction of expression of Gbe + Su(H) ([42,43,44], Fig. 5A, B, red arrows). The expression of Gbe + Su(H) is especially strong in follicle cells at the posterior pole, close to the oocyte (Fig. 5C, white arrows). The exceptional strong signalling at this site is probably induced by the observed strong expression of Epsin (encoded in Drosophila by lqf) in the oocyte (Fig. 5A, white arrows). We found that during the described stages, Gbe + Su(H) expression is strongly reduced in DlattP-DlK2R-HA/ Df, compared to DlattP-Dl-HA/Df flies, indicating a reduced activity of DlattP-DlK2R-HA also in this process (Fig. 5C, D, arrows). The weaker signalling probably causes the observed oogenesis defects observed in DlattP-DlK2R-HA/Df and DlattP-DlK2R-HA homozygous flies and explains their strongly reduced fertility.

Fig. 5

Analysis of oogenesis of DlattP-DlK2R-HA/Df flies. A, B Expression of Lqf-GFP, Gbe + Su(H) and Dl::mCherry in wildtype ovarioles. The red arrows point to egg chambers in stages 5–7 where Dl is up-regulated. The white arrows in A highlight the up-regulation of expression of Lqf/Epsin in the oocyte of maturing egg chambers. The arrowhead in A points to the constant expression of Gbe + Su(H) in the follicle epithelium, most prominent close to the oocyte. C, D Expression of Gbe + Su(H) in the follicle epithelium of egg chambers of DlattP-Dl-HA/Df (C) andDlattP-DlK2R-HA/Df (D) flies. The arrows points to the expression of Gbe + Su(H), which is strongly reduced in the DlattP-DlK2R-HA/Df egg chambers. Note the fusion of the egg chambers in the DlattP-DlK2R-HA/Df ovariole highlighted with the arrowhead in D. E, F Trans-endocytosis of the YFP-Notch ECD in the egg chambers of DlattP-Dl-HA/Df (E) and DlattP-DlK2R-HA/Df (F) ovarioles. It is best seen in the developing oocyte of egg chambers, highlighted with the arrows in (E). The trans-endocytosis of the ECD is dramatically reduced or absent in the DlattP-DlK2R-HA/Df oocytes (F, arrows). G, H Clonal analysis of the function of lqf in the female germline. The loss of lqf function dramatically reduces the trans-endocytosis of YFP-NECD in the oocyte. The arrowheads point to the oocyte of the egg chambers. H Magnification of the egg chambers of the region highlighted with the arrow in G. Note that the oocyte of the younger wildtype (smaller) egg chamber already accumulated YFP-NECD, while no YFP-NECD can be detected in the lqf-mutant oocyte of the adjacent, older chamber. I–J’mib1 function is not required during signalling of Dl from the germline to the follicular epithelium. Clonal analysis of the function of mib1 in the female germline. Expression of Cut in a wt ovariole (I, I’) and an ovariole carrying egg chambers with a mib1 mutant germline (J, J’). I, I’ Expression of Cut is down-regulated during stages 5–7 as a consequence of Dl signalling from the germline. The arrow points to an egg chamber in stage 6/7 that expresses Cut in the follicle epithelium, the arrowhead to an adjacent older egg chamber in stage 8/9 where Cut expression has been terminated because of the activation of the Notch pathway. J, J’ The arrow in J points to an egg chamber with a mib1 mutant germline expressing Cut, the arrowhead highlights an mib1 egg chamber in stage 8/9, which has terminated Cut expression despite the lack of mib1 function in the germline. This indicates that Dl can sufficiently signal to the epithelium despite the lack of mib1 function. In contrast, the ectopic stripe induced by Fng in DlattP-DlK2R-HA/Df discs was much shorter, confirming a substantial reduction of the signalling activity of DlattP-DlK2R-HA, compared to DlattP-Dl-HA or endogenous Dl (Fig. 4J, K, arrow, quantification in L)

Previous work revealed that trans-endocytosis of the NECD from follicle cells into the germline, where Notch is not expressed, can be observed with exceptional clarity during the described Dl signalling event at stages 5–7 of oogenesis [42, 43]. It is best observed in the oocyte (Fig. 5E, arrows). To monitor this trans-endocytosis event in DlattP-DlK2R-HA/Df flies, we used YFP-Notch, a functional Notch protein trap with YFP inserted in its ECD [45]. In contrast to DlattP-Dl-HA/Df flies, YFP-NECD was dramatically reduced in DlattP-DlK2R-HA/Df oocytes, indicating a strong reduction of trans-endocytosis of the NECD from the follicle cells (Fig. 5E, F, arrows). This is compatible with the notion that the Ks in the ICD enable Dl to exert a strong pulling force on Notch via endocytosis to efficiently activate the pathway.

In agreement with the notion that Epsin is required for the trans-endocytosis of NECD and the generation of pulling force by Dl, we found that the YFP-NECD signal was also strongly reduced in oocytes of lqf mutant egg chambers (Fig. 5G, H, [5,6,7]).

Altogether, these results indicate that ubi of Dl and its subsequent Epsin-mediated endocytosis are required for trans-endocytosis of NECD during activation of the Notch pathway in the female germline. Since trans-endocytosis of NECD is a result of successful pulling of Dl on Notch in trans and Epsin is required for the generation of a strong pulling force, the result support a requirement of ubi during pulling force generation.

Interestingly, we found that loss of mib1 function in the germline has no detectable effect on Dl-mediated down-regulation of expression of Cut in the follicle epithelium during stages 5–7, suggesting that in this signalling event, either Neur-mediates Dl-signalling or none of the two E3-ligases is required (Fig. 5I–J’).

Neur-induced ubi-independent signalling prevents embryonic lethality of DlattP-DlK2R flies

Previous work showed that the E3-ligase Neur plays an important role in Dl-induced Notch signalling during neurogenesis [19, 46]. Similar to the absence of Dl, absence of Neur results in embryonic lethality caused by the development of a neurogenic phenotype [19]. Our previous work showed that Neur can strongly activate Dl in a K-independent manner in over-expression experiments [27]. Combined, these findings suggest that the embryonic lethality of DlattP-DlK2R/Df or homozygous flies is prevented by the ability of Neur to activate DlK2R via the ubi-independent pathway, also under endogenous DlK2R-expression conditions. To rigorously test this assumption, we first analysed the Neur-mediated selection of the SOP in DlattP-DlK2R/Df wing discs.

The mechano-sensory bristles are a major part of the adult peripheral nervous system. Their formation depends on Neur- and Dl-mediated Notch signalling [47]. Each bristle is made of four cells, which are the progenies of the sensory organ precursor cell (SOP). Notch signalling is required to select the SOP from an equivalence group, the proneural cluster. The loss of Notch activity causes the formation of an excess of SOPs, since all proneural cluster cells adopt the SOP fate (neurogenic phenotype). We analysed the emergence of the SOP of the large bristles in the notal region of the wing imaginal discs, which arise during the third larval instar stage. To do so, we used the expression of sca-lacZ, which detects the whole proneural cluster, of E(spl)m8-SM-GFP, which reports only high proneural activity, and of neur-RFP, a marker for the emerging SOP (Additional file 1: Fig. S4A-A’’’) [36]. We found no obvious differences in the expression of these markers between the wildtype and the two DlattP