The meaning of ubiquitylation of the DSL ligand Delta for the development of Drosophila

Abstract

Background: Ubiquitylation (ubi) of the intracellular domain of the Notch ligand Delta (Dl) by the E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for the its signalling activity. Nevertheless, we have previously shown that DlK2R-HA, a Dl variant where all Ks in its intracellular domain (ICD) are replaced by the structurally similar arginine (R), still possess weak activity if over-expressed. This suggests that ubi is not absolutely required for Dl signalling. However, it is not known whether the residual activity of DlK2R-HA is an effect of over-expression and, if not, whether DlK2R can provide sufficient activity for the whole development of Drosophila.

Results: To clarify these issues, we generated and analysed Dl^attP-DlK2R-HA, a knock-in allele into the Dl locus. Our analysis of this allele reveals that the sole presence of one copy of Dl^attP-DlK2R-HA can provide sufficient activity for completion of development. It further indicates that while ubi is required for the full activity of Dl in Mib1-dependent processes, it is not essential for Neur-controlled neural development. We identify three modes of Dl signalling that are either dependent or independent of ubi. Importantly, all modes depend on the presence of the endocytic adapter Epsin. During activation of Dl, direct binding of Epsin appears not to be an essential requirement. In addition, our analysis further reveals that the Ks are required to tune down the cis-inhibitory interaction of Dl with Notch.

Conclusions: Our results indicate that Dl can activate the Notch pathway without ubi of its ICD. It signals via three modes. Ubi is specifically required for the Mib1-dependent processes and the adjustment of cis-inhibition. In contrast to Mib1, Neur can efficiently activate Dl without ubi. Neur probably acts as an endocytic co-adapter in addition to its role as E3 ligase. Endocytosis, regulated in a ubi-dependent or ubi-independent manner is required for signalling and also suppression of cis-inhibition. The findings clarify the role of ubi of the ligands during Notch signalling.

Keywords: Cell communication; DSL-ligands; Delta; E3-ligase; Endocytosis; Mindbomb1; Neuralized; Notch; Ubiquitylation.

Fig. 1

Generation of the Dl^attP alleles. A Generation and use of Dl^attP. Exon 6 of Dl was replaced by an attP landing site. This replacement creates a Dl null mutant allele Dl^attP. The attP site allows the insertion of modified exon6-variants. B, B’ Cuticle preparations of wt (B) and Dl^attP (B’) embryos. The Dl^attP 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 Dl^attP-Dl-HA (C–C’’) and Dl^attP-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

Fig. 2

Analysis of the phenotype of adult Dl^attP-Dl-HA and Dl^attP-DlK2R-HA flies. A–G The phenotype of flies with one copy of Dl^attP-Dl-HA (B, F) and Dl^attP-DlK2R-HA (C, G) over the deficiency Dl^BSC850 (Df). Compare with the wildtype control, shown in A, D. In contrast to the wildtype and Dl^attP-Dl-HA/Df flies, the tarsal segments 4 and 3 are fused in Dl^attP-DlK2R-HA/Df flies.E, FDl^attP-Dl-HA/Df flies show the typical haplo-insufficient wing vein broadening of Dl. The vein broadening is only slightly enhanced in Dl^attP-DlK2R-HA/Df flies. H, J The phenotype of flies homozygous for Dl^attP-Dl-HA resembles that of wildtype flies (compare with A, D). I, K In contrast, homozygous Dl^attP-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 Dl^attP-DlK2R-HA homozygous flies is suppressed if two additional copies of Notch are present

Fig. 3

The Ks in the ICD of Dl are required for efficient endocytosis. A–A’’ Clonal analysis of Dl^attP-DlK2R-HA. The homozygous Dl^attP-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 Dl^attP-Dl-HA clones (dark green, arrowhead) adjacent to homozygous Dl^attP-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 Dl^attP-Dl-HA cells have less Dl on its surface than homozygous or heterozygous Dl^attP-DlK2R-HA cells. D–E’’’’ Wing discs where Dmon1 and Dl^attP-Dl-HA- (D–D’’’’) or Dmon1 and Dl^attP-DlK2R-HA- (E–E’’’’) clones were induced. D, E Overview of the disc bearing the clones. The homozygous Dmon1Dl^attP-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

Fig. 4

The Ks in its ICD are required for the full activity of Dl. A, B Gbe + Su(H) expression in Dl^attP-Dl-HA and Dl^attP-DlK2R-HA/Df discs. Four stripes are recognisable in the wt disc in A. The arrow points to stripe3 which is decreased in Dl^attP-DlK2R-HA/Df discs. C–D’’ A wing disc bearing homozygous Dl^attP-Dl-HA (bright green outlined in white) andDl^attP-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 Dl^attP-Dl-HA accumulates to lower levels than Dl^attP-K2R-HA in the plasma membrane (D’), Gbe + Su(H) is stronger expressed in Dl^attP-Dl-HA homozygous territory (D’’), indicating that Dl^attP-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 Dl^attP-Dl-HA/DfandDl^attP-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 Dl^attP-DlK2R-HA/Df compared to Dl^attP-Dl-HA/Df discs (arrow). L Quantification of the length of the ectopic stripe of Wg expression induced by Fng in the Dl^attP 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)

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 Dl^attP-Dl-HA/Df (C) andDl^attP-DlK2R-HA/Df (D) flies. The arrows points to the expression of Gbe + Su(H), which is strongly reduced in the Dl^attP-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 Dl^attP-Dl-HA/Df (E) and Dl^attP-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 Dl^attP-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 Dl^attP-DlK2R-HA/Df discs was much shorter, confirming a substantial reduction of the signalling activity of Dl^attP-DlK2R-HA, compared to Dl^attP-Dl-HA or endogenous Dl (Fig. 4J, K, arrow, quantification in L)

Fig. 6

Neur-dependent Dl-signalling prevents a neurogenic phenotype of homozygous Dl^attP-DlK2R-HA flies. A–C Analysis of Dl^attP-DlK2R-NEQN2A-HA. A Cuticle preparation of a homozygous Dl^attP-DlK2R-NEQN2A-HA embryo. It shows the severe reduction of cuticle (arrow) that is characteristic for the neurogenic phenotype. B–C’ Clonal analysis of Dl^attP-DlK2R-NEQN2A-HA in wing discs. Clones are labelled by the loss of GFP. Supernumerary SOPs, labelled by Hnt-expression, develop in the homozygous Dl^attP-DlK2R-NEQN2A-HA clones in the notum (arrows) and the anterior wing margin (arrowhead), indicating that Dl^attP-DlK2R-NEQN2A cannot mediate the Neur-dependent selection of the SOP. C, C’ Magnification of the notal region highlighted in A with the arrows. It reveals that the developing SOPs accumulate DlK2R- NEQN2A-HA in their plasma membranes to high levels (arrows). D, D’ A wing disc bearing large homozygous DlattP-DlK2R-HA clones, labelled by loss of GFP and outlined in white. The arrow points to the SOP at the prominent halo of Gbe + Su(H) expression surrounding the emerging SOP at the tr1/APA position in the homozygous clones. The halo reveals the presence of Neur-mediated signalling of the ligands. E–E’’’ A wing disc bearing Neur-expressing MARCM clones which are also homozygous for Dl^attP-DlK2R-HA (genotype: hsFlp tubGal4 UAS GFPnls; + / UAS neur; FRT 82B tubGal80/ FRT82B Dl^attP-DlK2R-HA). The arrow in E, E’ points to a Dl^attP-DlK2R-HA homozygous clone shown at higher magnification in the z-section in E’’. In contrast to Dl^attP-DlK2R-HA clones, the abundance of DlK2R in the plasma membrane (E’) is strongly reduced if Neur is additionally expressed. E’’’ Pixel intensity measurement of the apical region including the clone shown in E’’. The levels of apical Dl are reduced in the GFP-positive MARCM clone (high GFP). Note, that an increase in the abundance of DlK2R-HA is normally observed in the homozygous cells (see Fig. 3A–A’’’)

Fig. 7

A–D’ Ubi-independent Dl-signalling at the stripe3 of Gbe + Su(H) expression domain. A Expression of Gbe + Su(H) in a wildtype wing disc. The arrowhead points to stripe3. B Residual expression of stripe3 is observed in mib1 mutant wing discs (arrowhead). C–D’ Stripe3 is also present in mib1-mutant Dl^attP-Dl-HA/Df (C, C’) and mib1-mutant Dl^attP-DlK2R-HA/Df (D, D’) discs (arrowhead). E,E’ A disc bearing a large lqf-mutant clone covering the region of stripe3 expression (arrowhead). The clone is labelled by absence of GFP. The expression of stripe3 is abolished. F, G The wing phenotype of Dl^attP-Dl-HA/Dfandmib1Dl^attP-DlK2R-HA/Df flies upon reduction of lqf activity. The wing of mib1Dl^attP-DlK2R-HA/Dflqf flies is much more severe than that of DlK2R-HA/Df flies (compare with Fig. 2G).H, I Suppression of the phenotype of homozygous DlattP-DlK2R-HA flies by elevation of the copy number of lqf. H The original number of tarsal segments is re-established if two additional copies are present. The arrow points to the incomplete formation of the joint between tarsal segments 3 and 4. Note, the homozygous flies normally had only three discernible segments due to the fusion of segments 3 + 4 and 1 + 2 (see Fig. 2K). I The wing phenotype of homozygous DlattP-DlK2R-HA is strongly suppressed by elevation of the levels of Lqf (compare with Fig. 2I)

Fig. 8

RCI in LqfUIM1^3E/3A-∆UIM2-GFP wing discs. A, A’ Gbe + Su(H) expression in LqfUIM1^3E/3A-∆UIM2-GFP wing discs. The arrow points to the residual expression of stripe3. Compare with Fig. 7A, B. B, B’ Detection of RCI in the notal area of wing discs by induction of Dl/Ser-double-mutant clones. Ectopic expression of Gbe + Su(H) is activated in mutant cells at the clone boundary (arrowheads). C–D’’’’ A wing disc bearing lqf- and Dl/Ser mutant clones. The analysed disc is lqf-mutant, rescued by a GFP-tagged lqf construct, expressed under control of the endogenous lqf-promoter. In the rescued discs, the induced Dl/Ser double-mutant clones are labelled by the absence of RFP. The lqf-mutant clones are labelled by absence of the GFP signal caused by the loss of Lqf-GFP. The activity of Notch is revealed by the expression of Gbe + Su(H). The arrowheads highlight regions with strong ectopic induction of Notch activity (Gbe + Su(H) expression) in Dl/Ser-mutant cells at the clone boundary. B–B’’’’’ Independent induction of lqf- and Dl/Ser-clones in discs generates regions devoid of lqf functions adjacent to Dl/Ser-mutant lqf + clones. The clones are outlined in yellow in D’’’, D’’’’. The expected induction of Notch activity (Gbe + Su(H) expression) upon RCI is absent (arrow), indicating that the Dl/Ser expressing cells require the function of lqf. The arrowhead points to a lqf + region adjacent to a Dl/Ser-mutant clone. As expected the Dl/Ser-mutant cells initiate ectopic expression of Gbe + Su(H). The experiment confirms that Lqf is required in the signalling cells to induce Notch activity in the mutant boundary cells. E–F’Dl/Ser-clones in LqfUIM1^3E/3A-∆UIM2-GFP wing discs. E, E’ Gbe + Su(H) is also activated in the Dl/Ser-double-mutant boundary cells, indicating RCI occurs in cells with a Lqf variant without functional UIMs (arrowheads). Thus, Dl/Ser can signal despite the absence of functional UIMs in Lqf. F, F’ As expected, no induction of Notch activity is observed if the Dl/Ser- mutant clone incudes the whole notum, because no boundary of ligand-expressing and non-expressing-cells are present (asterisk in (F)). G The loss of Dl/Ser-function in LqfUIM1^3E/3A-∆UIM2-GFP wing discs results in a strong neurogenic phenotype, confirming that the ligands are required for SOP selection in this genetic background

Fig. 9

Gut homeostasis in D^lattP-DlK2R-HA/Df flies. A Schematic of the ISC lineage, giving rise to the EC and EE cells. B–C’ A representative picture of the R5 region of the gut of Dl^attP-DlK2R-HA/Df and D^lattP-Dl-HA/Df flies. The expression of Pros and Esg-GFP are shown. The insert C’ shows the rare Pros and Esg-GFP double positive EEP precursor that differentiate into EEs. D Quantification of the number of ISCs, EEs and EEPs in the two genotypes revealed no significant differences (n = 11, 15). Error bars are standard error of the mean (SEM). A two-tailed student´s t-test was used for statistical analysis (*p < 0.05, **p < 0.01; ***p < 0,001)