Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila

In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H)MAPK-ko and a phospho-mimetic Su(H)MAPK-ac isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vgBE-lacZ). In general, Su(H)MAPK-ko induced a stronger response than wild-type Su(H), whereas the response to Su(H)MAPK-ac was very weak. Notch target genes cut, wingless and vgBE-lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DERact) or the MAPK (rlSEM) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones.


a b s t r a c t
In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes.
Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wildtype Su(H), a phospho-deficient Su(H) MAPK-ko and a phosphomimetic Su(H) MAPK-ac isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vg BE -lacZ). In general, Su(H) MAPK-ko induced a stronger response than wildtype Su(H), whereas the response to Su(H) MAPK-ac was very weak. Notch target genes cut, wingless and vg BE -lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DER act ) or the MAPK (rl SEM ) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones.
& Imaginal discs were dissected, fixed, washed and blocked before adding antibodies.

Experimental features
Tissue-specific expression of respective transgenes was induced with the Gal4:: UAS-system. Gene expression was monitored directly or from reporter genes by antibody staining of the protein products. Data source location n.a.

Data accessibility
The data is with this article

Value of the data
This data shows the responses of several Notch target genes to modulations of Su(H) activity by the EGFR pathway.
The data allow for a visual comparison of the spectrum of Notch target gene responses to Su (H) overexpression.
Overexpression of activated components of the EGFR pathway and Su(H) variants, alone or in combination, can be compared in various Drosophila tissues.
This data may be extended by analyses on DER act activity during Drosophila wing development.

Data
Suppressor of Hairless [Su(H)] is the transcription factor that regulates the expression of the target genes of the Notch signalling pathway [1,2]. Su(H) protein may be phosphorylated by MAPK as a result of Epidermal Growth Factor Receptor (EGFR) activation, providing a means of a direct cross-talk between these two pathways [3][4][5]. The response of several Notch target genes to the modulations of Su(H) by EGFR signalling activity was analysed by the local overexpression of either wild-type Su(H), a phospho-deficient Su(H) MAPK-ko and a phospho-mimetic Su(H) MAPK-ac variant [3] using the Gal4::UAS system [6], and staining of the tissues with respective antibodies. Moreover, activated components of the EGFR pathway (DER act , rl SEM ) were overexpressed alone or in combination with individual Su(H) variants. The response of the Notch target gene cut was observed in cell clones of wing imaginal discs, and the resultant phenotypes on thorax, wings and eyes were recorded in adult flies.

Response of cut expression to the combined induction of Su(H) variants and activated components of the EGFR pathway during wing development
The expression of the Notch target gene cut was analysed in cell clones overexpressing either of the three Su(H) isoforms alone or in combination with the activated EGF-receptor (DER act ) or the activated MAPK (rl SEM ) [14,15] (Fig. 5). Overexpression clones were induced in wing imaginal discs [16]. Su(H) overexpression induced cut expression, whereas it repressed it when simultaneously overexpressed with rl SEM (Fig. 5A-A‴ and C-C‴) [3]. Likewise repression was observed with Su (H) MAPK-ko , but less with Su(H) MAPK-ac ( Fig. 5D and E‴). Cell clones overexpressing DER act were frequently distorted, and cut expression was induced at the boundary of DER act expressing and non-expressing cells independent of the overexpression of any Su(H) variant (arrowheads in Fig. 5F 0 -I‴).

Adult phenotypes resulting from the combined overexpression of Su(H) variants and activated components of the EGFR pathway
Overexpression of UAS-DER act in the thorax (Fig. 6) or the wing anlagen (Fig. 7A) using Bx-Gal4 [17] was fully epistatic to the Su(H) gain of function phenotypes. This was in contrast to the simultaneous overexpression of UAS-rl SEM with the UAS-Su(H) isoforms: in these experiments the Su(H) gain of function phenotypes prevailed (Figs. 6 and 7B). It has been described before that the overexpression of Su(H) in the developing sensory organs using sca-Gal4 causes a shaft to socket transformation [18], which we also observed upon overexpression of Su(H) MAPK-ko or Su(H) MAPK-ac (Fig. 8). Whereas sca:: rl SEM was similar to the control, sca:: DER act animals developed tufts of macrochaetae on the posterior thorax (Fig. 8). Interestingly, in combination with any of the Su(H) variants, the double socket phenotype resulting from Su(H) overexpression prevailed (Fig. 8). Finally, consequences of Su(H) overexpression in the developing eye using gmr-Gal4 were addressed (Fig. 9). As the Gal4::UAS system is temperature sensitive, phenotypes were strong at 29°C, revealing defects in the control as well [19]. At this temperature, Su(H) overexpression caused overgrowth of the eye, irregular facets and necrosis. At 25°C the phenotypes were much weaker resembling the control. A combination with rl SEM enhanced the irregular facet and necrotic phenotype, whilst gmr::rl SEM flies were very similar to the control (Fig. 9).

Fly stocks, husbandry and analyses
Flies were obtained from the Bloomington stock collection if not noted otherwise. Fly husbandry was according to standard protocols at 29°C, 25°C or 18°C as noted. y 1 w 1118 , UAS-lacZ and UAS-GFP served as control. For information on fly stocks we refer to http://flybase.bio.indiana.edu. Adult wings of female flies were dehydrated in ethanol and mounted in Euparal (Roth, Karlsruhe, Germany) and dried over night. Pictures of wings or adult flies were taken on a Zeiss Axiophot or a Wild 5M stereomicroscope, respectively, using an ES120 camera (Optronics, Goleta CA, USA) and Pixera Viewfinder software, version 2.0.

Immunohistochemistry
Imaginal discs were stained according to standard protocols using mouse monoclonal antibodies directed against Cut, Wingless or beta-Galactosidase (developed by G.M. Rubin, S.M. Cohen, and J.R. Sanes respectively, and obtained from DSHB or using a polyclonal antiserum directed against Su(H)) [22]. Secondary antibodies coupled to FITC, Cy3 or Cy5 (1:200) were obtained from Jackson Immuno-Research Laboratories (Dianova, Hamburg, Germany). Samples were mounted in Vectashield (Vector Lab) and examined on a Zeiss Axioskop coupled to a BioRad MRC1024 confocal microscope using LaserSharp 2000TM software (Carl Zeiss, Jena, Germany).   9. Overexpression consequences of Su(H), DER act and rl SEM in the developing eye. Co-overexpression of UAS-Su(H) variants together with UAS-lacZ (control) or UAS-rl SEM was driven in the developing eye using gmr-Gal4. At 29°C, gmr::lacZ flies have smaller eyes with irregular facets giving the eye a rough appearance. In contrast, overexpression of Su(H) variants at this temperature causes enlarged eyes that appear slightly bulgy. Both Su(H) and Su(H) MAPK-ko induced irregularities in the arrangement of the facets and necrosis (arrowhead), in contrast to Su(H) MAPK-ac . At 25°C the phenotypes are much milder, and eyes appear like wild type (Su(H) MAPK-ac ) or slightly rough (Su(H) and Su(H) MAPK-ko ). A similar rough eye phenotype was observed upon induction of rl SEM at 25°C. The combined overexpression of Su(H) and rl SEM gave a mixed phenotype, i.e. eyes were smaller, rough and necrotic (arrowhead). Similar necrotic patches (arrowhead) and size decrease were also observed in the eyes of gmr:: Su(H) MAPK-ko þ rl SEM animals, which in addition had a glossy appearance. In contrast, the eyes of the gmr:: Su (H) MAPK-ac þ rl SEM animals looked similar to gmr:: rl SEM . Typical representatives are shown in each case.