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Fig. 4. Ectopic expression of EDL/MAE antagonizes pnt function in vivo.
(A-I) The effect of misexpression of edl in all neurons. Scanning
electron micrographs of eyes of wild type (A) and elav-GAL4, UAS-edl
(B). Expression of edl in all neurons reduces the eye size. In the
apical tangential section (C) photoreceptor neurons are hardly detected and
most of the area is occupied by pigment cells. Neuronal marker ELAV expression
(D,E,H) and R8 marker BOSS expression (F,G,I) in wild type (D,F),
elav-GAL4, UAS-edl (E,G) discs, and discs with a clonal patch
(bracketed) of pnt 88 mutant
cells (H,I). As misexpressing EDL/MAE by elav-GAL4 could down
regulate the expression of elav-GAL4 driver itself, we examined GAL4
activity in elav-GAL4; UAS-edl animals using a
UAS-NLS-lacZ reporter gene. Although in normal embryos expression of
ß-galactosidase coincided with ELAV expression, upon EDL/MAE
misexpression many ELAV- ß-gal+ cells were present
in a basal focal plane, where undifferentiated cells are present. Continued
expression of GAL4 in these cells accounts for the strong effect of EDL/MAE
misexpression on neuronal recruitment. (J-Q) EDL/MAE suppresses neuronal
development even in the presence of activated Ras. The effect of strong (J-M)
and weak (N-Q) ectopic EDL/MAE expression on both photoreceptor and cone cell
development. Neuronal development was monitored by anti-ELAV staining of
imaginal discs (J-M) and in sections of the adult eye (N-Q). (J,N) Wild type,
(K,O) sevERasV12, (L,P) sevE-GAL4, UAS-edl, (M,Q)
sevE-RasV12, sevE-GAL4, UAS-edl. Although
expression of activated Ras transforms cone cells into R7 neurons (K,O),
expression of EDL/MAE has no such effect and suppresses differentiation of
endogenous neurons (L,P), even in the presence of activated Ras (M,Q). (R,S)
The effect of ectopic EDL/MAE is enhanced by halving the dose of pnt.
(R) sevE-GAL4, UAS-edl, (S) sevE-GAL4, UAS-edl,
pnt88/+. (T-W) Ectopic expression
of EDL/MAE phenocopies pnt loss of function phenotype. (U) EDL/MAE
expression in ovarian follicle cells using CY2-GAL4 line dorsalizes
the chorion, resulting in fused dorsal appendages. (W) Expression of EDL/MAE
in the posterior wing using engrailed-GAL4 duplicates the wing. (T,V)
Animals without transgenes.
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1.5
kb. N9 thus represents a minor product of edl. (C,D) edl
represents a novel class of Ets proteins. (C) Line diagram showing the
structures of EDL and examples of other Ets proteins (D, Drosophila;
H, Human). The ETS domain is shown by a blue box, Pointed domain by an orange
box. Essential MAPK phosphorylation sites are shown by triangles. Numbers on
the right indicate the amino acid length. (D) An alignment of the Pointed
domain. Amino acids that are conserved in all or most of the proteins (15-16
out of 17) analyzed in H are shaded yellow, and are shown with capital or
lower case letters, respectively. Other amino acids that match those of
EDL/MAE are shaded blue. (E,F) Expression of edl mRNA (E) and
enhancer trap line edlJS (F) in the eye imaginal
disc. Anterior is towards the left, the position of the morphogenetic furrow
is shown by an arrowhead. ß-galactosidase expression (magenta) is seen in
R8 (open circle) and R2/R5 (asterisk), whereas ELAV (green) is expressed in
all neurons. Overlap of magenta and green is white. edl-lacZ
reporter is also expressed in the subretinal glial cells, located below this
focal plane. (G) In the developing chordotonal organ, edl mRNA is
found in five COPs, C1 to C5 (arrowheads). (H) A phylogenic tree of the
Pointed domain. Sequences of all Ets proteins containing Pointed domain from
Drosophila (five sequences) and human (eleven sequences) are aligned
with EDL/MAE using Clustal W. Bootstrap value more than forty (based on 100
replicates) are shown. Notice that all of the Drosophila members
belong to different branches.
