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Development 120, 1959-1969 (1994)
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Confrontation of scabrous expressing and non-expressing cells is essential
for normal ommatidial spacing in the Drosophila eye
Michael C. Ellis*,†, Ursula Weber*, Volker Wiersdorff and Marek Mlodzik‡
Differentiation Programme, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
*Both authors contributed equally to this study
†Present address: Mercator Genetics Inc., 4040 Cambell Avenue, Menlo Park, CA 94025, USA
‡Author for correspondence
SUMMARY
The establishment of neural precursor cells in Drosophila
depends on cell-cell interactions and lateral inhibition.
Scabrous (sca) is involved in this process by preventing an
excess of cells from adopting a neural precursor fate.
Specifically in eye development, Sca protein function has
been implicated in the spacing pattern that is essential for
the ordered appearance of the ommatidial array. During
this process sca expression is restricted to neurogenic
groups of cells and later to the neural precursors. We
report that ectopic sca expression in the morphogenetic
furrow results in a rough eye phenotype with oversized and
fused ommatidia. These defects in adult eyes are due to the
generation of too many ommatidial preclusters in the morphogenetic furrow. Strikingly, sca loss-of-function mutants
have an almost identical phenotype. Our results suggest
that Sca plays a positive role in establishing the spacing
pattern within the furrow and that the quantitative difference in sca expression between neighboring groups of cells
is a determining factor in this process. Ectopic expression
of Sca also represses endogenous sca expression in the
furrow, suggesting that Sca is involved in a feedback loop
affecting its own transcription. Interestingly, sca shares
homology to a group of extracellular matrix proteins that
have been implicated in neuronal differentiation. We
present a model for sca function based on its phenotypic
and molecular features.
INTRODUCTION
Neural development in the eye imaginal disc is thought to
be similar to peripheral neurogenesis in other imaginal discs,
and mutations in most of the neurogenic genes also result in
neural hypertrophy in the eye (Dietrich and Campos-Ortega,
1984). Proneural genes and the extent of neurogenic regions in
the eye disc have not yet been characterized. Nevertheless, it
is thought that at least part of the region known as the morphogenetic furrow (MF; reviewed by Tomlinson, 1988; Ready,
1989) has the properties of a neurogenic region, since
reduction in the activity of the neurogenic locus Notch in the
MF leads to the formation of a large excess of neural precursors (Cagan and Ready, 1989; Baker et al., 1990).
In a similar manner to neurogenic genes, scabrous (sca) is
thought to be required for the restriction of neural potential
within each neurogenic group of cells. Although sca is first
expressed during embryonic neurogenesis, its function is dispensable for normal embryonic development. Sca mutant flies
have a visible phenotype in adults, which consists of duplicated
bristles and a rough eye due to the irregular spacing of too
many ommatidial preclusters forming in the MF (Baker et al.,
1990; Mlodzik et al., 1990a). In both tissues affected, the
observed phenotype of null alleles suggests a partially
redundant function for sca: less than half of the bristles are
affected in any single fly, and in the eye the number of cells
The development of the adult peripheral nervous system (PNS)
of Drosophila is initiated in the third larval instar when regions
within each imaginal disc are defined that will give rise to
sensory organ precursors. Subsequently, within each of these
neurogenic regions some cells become selected as neural precursors, whereas other cells develop as supporting epidermal
cells. In general, a process of lateral inhibition and cell-cell
interactions restricts the expression of ‘proneural’ genes to
those cells that will become neural precursors and subsequently
undergo neural differentiation (reviewed by Simpson, 1990;
Artavanis-Tsakonas and Simpson, 1991; Campuzano and
Modolell, 1992; Ghysen et al., 1993). The so-called ‘neurogenic’ genes are required in the lateral inhibition process
leading to the restriction of neural cell fate. Loss-of-function
mutations in the neurogenic genes usually cause all or most of
the cells of such a region to become neural precursors (Dietrich
and Campos-Ortega, 1984; Simpson and Carteret, 1989;
Heitzler and Simpson, 1991). In contrast, loss-of-function
mutations in the proneural genes lead to the absence of neural
precursors and sensory organs (Romani et al., 1989; reviewed
by Ghysen and Dambly-Chaudiere, 1988; Campuzano and
Modolell, 1992).
Key words: Drosophila, spacing pattern, scabrous, neurogenic
genes, extracellular matrix
1960 M. C. Ellis and others
that initiate photoreceptor development in the MF is only
approx. 20% of those that have the potential to do so in a Notch
mutant furrow (Cagan and Ready, 1989; Baker et al., 1990).
Interestingly, during imaginal disc development sca
expression follows the dynamic expression pattern typical of
the proneural genes: after initial expression in all cells of a neurogenic cluster it becomes restricted to the neural precursor(s)
within each group. In the eye imaginal disc, sca expression can
first be detected in groups of cells at the anterior edge of the
MF before it becomes confined to the first photoreceptor
precursor to be determined, the R8 cell (Mlodzik et al., 1990a).
Molecular analysis revealed that sca encodes a putative
secreted protein (Baker et al., 1990). Based on its expression
pattern and molecular features it has been proposed that Sca
could function as a lateral inhibitor during the process of
restriction of neural potential (Baker et al., 1990). Furthermore,
sca has been found to interact genetically with the neurogenic
gene Notch (Brand and Campos-Ortega, 1990; Rabinow and
Birchler, 1990), which encodes a transmembrane receptor
protein (Wharton et al., 1985; Heitzler and Simpson, 1991).
However, the significance of the sca expression pattern and the
function of sca during the process of lateral inhibition remain
unclear.
We report the effects of ectopic expression of sca in all cells
just anterior to or within the morphogenetic furrow. Flies displaying such an aberrant expression pattern have irregularly
spaced preclusters in the MF and subsequently R8 precursors
that are often too closely spaced, leading to fusions of
ommatidia and rough eyes. Interestingly, this phenotype is
reminiscent of the loss-of-function sca phenotype and indicates
that the correct restriction of sca expression is essential for its
function. Our results suggest that Sca plays a positive role in
establishing and/or maintaining the spacing pattern within the
furrow. Furthermore, we show that Sca shares homology with
several proteins of the vertebrate neuronal extracellular matrix.
We discuss the possible function(s) of sca with respect to its
loss-of-function and overexpression phenotypes and its
molecular features.
MATERIALS AND METHODS
Plasmid construction and generation of P-element lines
The heat-shock sca (HS-sca) P-element construct was made by
cloning a 2.6 kb XhoI/XbaI fragment (blunt ended at the XhoI site)
from the sca cDNA, pcsca6A (Baker et al., 1990) into the HpaI and
XbaI sites of the polylinker region of the CaSpeRhs vector (Thummel
and Pirotta, 1991; Fig. 1A).
The rough enhancer fragment (a kind gift from Ulrike Heberlein)
that drives expression broadly in the MF and later in R2/5 and R3/4
as described for rough expression (Kimmel et al., 1990), was cloned
as a 2 kb NotI fragment into the NotI site of the HS-sca plasmid downstream of the sca ORF (roE-sca; Fig. 1C,D). This orientation is
identical to the situation in the wild-type rough gene itself where this
fragment is localized within an intron (U. Heberlein unpublished
data).
Sca expression ahead of the MF was generated using the GAL4
enhancer detector system (Brand and Perrimon, 1993). The 2.6 kb
XhoI/XbaI fragment from the sca cDNA (see above) was cloned into
the respective sites of pUAST, which contains GAL4-UAS sequences
and an hsp70 TATA box. This was genetically combined with an
insertion of pGawB, a GAL4 enhancer detector construct, in the hairy
gene (Brand and Perrimon, 1993) to drive hairy-dependent sca
expression (hG-sca; Fig. 1E,F). All constructs contain the hsp70 transcription terminator, except pUAST, which has the SV40 terminator.
Germ-line transformation was performed by standard procedures as
described by Spradling and Rubin (1982) using w1118 as host strain
and pUCHSπ∆2-3 (Mullins et al., 1990) as helper plasmid. Plasmid
DNA for injections was purified over Qiagen columns (Qiagen). For
each construct several independent insertions were obtained and
analyzed:
roE-sca. 3 primary insertions and 7 additional insertions were
generated by secondary jumps as described by Robertson et al. (1988).
Four insertions show the rough eye phenotype (see Results) with
complete penetrance when homozygous, the others show a weakly
rough eye with reduced frequency. 4-copy flies were generated by
standard meiotic recombination.
hG-sca. 14 independent insertion lines were established together
with the GawB hairy insertion. All combinations display the rough
eye phenotype when homozygous. Other adult structures and the
viability and fertility of these flies are also affected (U. W. and M.
M., unpublished data).
HS-sca. 6 independent insertions. The heat shock regime consisted
of up to six periods of 1 hour at 37°C followed by 1 hour at 25°C,
during the wandering 3rd instar larval stage when sca is usually
expressed and required, or several pulses of 20 minutes at the respective temperatures during embryogenesis.
Antibodies and marker strains
The following antibodies were used as cell type specific markers: αElav, a monoclonal antibody against Elav, specific for neuronal cells,
in this case photoreceptor neurons (kind gift from the Rubin lab;
expression pattern described by Robinow and White, 1991); monoclonal antibody α-Boss1 (specifically staining R8; Krämer et al.,
1991; a gift from the Zipursky lab); α-BarH1/H2, affinity-purified
rabbit antiserum (specific forR1/6; Higashijima et al., 1992; gift from
K. Saigo); α-Sca, a mouse polyclonal serum raised against a trpE-sca
fusion protein (Baker et al., 1990).
The following enhancer trap lines providing nuclear β-galactosidase expression were used as cell-type specific markers: A2-6
(Mlodzik et al., 1990a) for endogenous sca expression and as early
R8 marker; BB02 (Hart et al., 1990) and rO156 (U. Gaul, unpublished
data) for R8 differentiation; 3-5138, an insertion in svp, for R1/6 and
R3/4 (Mlodzik et al., 1990b and unpublished data); X81, an insertion
in rhomboid, and AE33 (Freeman et al., 1992) for R8 and R2/5; H214
for R7 (Mlodzik et al., 1992). β-galactosidase expression in the
respective cell types in all these lines was detected with a monoclonal
antibody purchased from Promega.
Histology
Antibody stainings of eye imaginal discs and sectioning of adult
retinae were performed as described by Tomlinson and Ready (1987).
For scanning electron microscopy the eyes were prepared by critical
point drying and coated with 2 nm of gold. Images were taken on a
low-voltage prototype SEM, stored digitally and processed with NIHImage.
RESULTS
The first steps of pattern formation during eye development
take place in the morphogenetic furrow (MF) when groups of
cells form regularly spaced clusters described as rosettes
(Wolff and Ready, 1991). Subsequently, these groups become
rearranged to form the so-called preclusters. Each of these
preclusters consists of the future photoreceptor 8 (R8), which
is the first cell to initiate neural differentiation, precursor cells
of R2, R3, R4, R5 and the mystery cells (Tomlinson and
Ready, 1987; Wolff and Ready, 1991). The regular spacing of
sca and the morphogenetic furrow 1961
Fig. 1. Schematic structure and expression patterns of sca ectopic expression constructs. A, C and E show the expression constructs and B
wild-type sca expression for comparison. D and F illustrate the expression patterns in the eye disc for constructs shown in C and E respectively.
The MF is indicated by an arrow, posterior is up. (A) cDNA encoding the Sca ORF fused to the hsp70 promoter. (C,D) ro-enhancer construct
containing a hsp70 promoter (roE-sca) and its expression pattern close to MF, respectively. (E,F) GAL4 responsive UAS-containing promoter
fused to Sca ORF and GAL4 enhancer trap vector inserted in hairy (Brand and Perrimon, 1993) and the corresponding expression in eye discs
when both constructs are combined (hG-sca). All independent combinations tested show the same pattern. Besides the strong expression
anterior to the MF there is also diffuse weak staining in and posterior to the furrow, which is not seen with endogenous hairy. The same pattern
is observed when β-gal is used as a reporter for GAL4 driven expression in the same context (not shown). This could be due to either some
leakiness in the expression system or the stability of GAL4. The hG-sca derived expression is, nevertheless, largely restricted to the stripe just
anterior to the furrow.
preclusters and R8 cells is essential for the precise arrangement
of ommatidia during later development. The other photoreceptor cells are then determined and begin differentiation
following an invariant sequence in which neural differentiation
of R8 is followed by the pairwise addition of R2/5 and R3/4.
After a wave of cell division amongst the yet uncommitted
cells, R1/6 and finally R7 join the cluster. These consecutive
steps of spacing, recruitment and differentiation occur in a spatiotemporal sequence of columns that follow each other in
intervals of about 90 minutes (Tomlinson and Ready, 1987; for
reviews see Tomlinson, 1988; Ready, 1989; Banerjee and
Zipursky, 1990; Wolff and Ready, 1993).
The sca eye phenotype and expression
The rough eye phenotype of homozygous mutant sca− flies is
characterized by oversized facets that contain either too many
photoreceptors, or 2 or more normal R-cell complements
enclosed by only one set of pigment cells (Fig. 2I,J; Baker et
al., 1990). Other defects seen in sca− eyes are ommatidia with
too few photoreceptor cells, or ommatidia with abnormal orientation with respect to each other (Fig. 2I,J). Analysis of sca−
eye discs revealed that these defects are due to irregular
ommatidial spacing manifested already in the MF (Baker et
al., 1990; Wolff and Ready, 1991). From their first
appearence, the rosettes and preclusters are too crowded and
1962 M. C. Ellis and others
Fig. 2. Adult eye phenotypes of roE-sca flies. Eyes shown are from female flies reared at 25°C. The respective genotypes are indicated on the
left. A, C, E, G and I show SEM pictures of parts of the eyes and B, D, F, H and J show tangential eye sections of same genotypes. (A,B) Wildtype eyes. (C,D) Eyes from flies carrying 2×roE-sca. (E,F) 4×roE-sca. (G,H) 4×roE-sca in the null allele scaBP2. (I,J) scaBP2 eyes.
Magnification in all panels is 800×. Note that the roE-sca phenotype is temperature sensitive, similar to hypomorphic sca alleles, so that flies
reared at 25°C are more strongly affected than those at 18°C.
sca and the morphogenetic furrow 1963
wt
roE-sca
A2-6
Elav
R3/4
R1/6
R8
R2/5
irregularly arranged, resulting in fused preclusters and
ommatidia with abnormal complements of photoreceptor
cells. In addition, in sca− more than one cell can adopt the R8
fate within each precluster, resulting in ommatidia with
multiple R8 cells (Fig. 4B; Baker et al., 1990). The recruitment steps following R8 specification appear not to be
affected (Baker et al., 1990), although in sca− adult eyes there
are also ommatidia present with fewer than the wild-type photoreceptor number (Fig. 2B; Mlodzik et al., 1990a). The
presence of too many preclusters in the MF in sca− and the
limited pool of cells available for later recruitment could lead
Fig. 3. Phenotypic analysis of
roE-sca eye discs. Expression of
cell type specific enhancer
detector lines or the nuclear
neuronal Elav antigen is shown.
The MF is indicated by open
arrows, posterior is to the right.
(A,B) β-galactosidase
expression in A2-6 enhancer
detector in wild-type and 4×roEsca discs, respectively. Note
abnormal R8 spacing behind the
MF and reduced staining in the
furrow in roE-sca discs. Owing
to its stability, β-galactosidase is
detected throughout the
posterior part of disc.
(C,D) Elav expression in
differentiating R-cells following
the invariant sequence starting
with R8, followed by R2/5,
R3/4, R1/6 and R7. Note the
irregular arrangement in roE-sca
discs (D), where R-cell clusters
are often too close and touching
each other. (E,F) lacZ enhancer
detector insertion in seven-up
with R3/R4 and R1/R6 specific
staining pattern in wild-type and
2×roE-sca discs, respectively.
There is no apparent change in
cell fate or missing cell types in
roE-sca. (G,H) R8 and R2/5
specific lacZ staining in the X81
line in wild-type and 2×roE-sca
discs, respectively. Note
reduced staining in R2 and R5 in
roE-sca disc. The cells are
numbered accordingly in one
cluster each in E and G.
to ommatidia with some R-cells missing. Generally, the
spacing defects observed in sca− discs are more severe in and
close to the MF than in more mature parts of the eye disc
(Wolff and Ready, 1991 and our unpublished observation),
suggesting that a secondary mechanism is partly compensating for the early furrow defects.
During the early spacing process, sca is first expressed in
groups of about 7-10 cells, equivalent to one column of
ommatidia and ahead of any visible pattern formation at the
anterior edge of the MF (Fig. 1B). Expression within these
regularly spaced groups is fairly uniform except that it is
1964 M. C. Ellis and others
weaker anteriorly; no Sca protein can be detected between
them. They might represent cells that slightly later are part of
the so-called rosettes. In the next column, sca expression
becomes restricted to one cell in each group. The analysis of
lacZ expression in sca enhancer trap lines and double label
experiments have identified this cell as the R8 precursor.
Expression persists in R8 for 6-8 hours or about four columns
of ommatidial development (Fig. 1B; Mlodzik et al., 1990a,
Baker et al., 1990).
Constructs employed for ectopic expression of Sca
We have ectopically expressed Sca to analyze the functional
significance of the sca expression pattern and to gain insights
into its role during the generation of the spacing pattern. Three
different promoter/enhancer constructs were employed: (1) the
heat-inducible hsp70 promoter (HS-sca; Fig. 1A), (2) an
enhancer fragment from the rough gene that drives expression
in all cells of the MF (roE-sca; Fig. 1C,D), and (3) a GAL4
enhancer detector insertion in the hairy gene (Brand and
Perrimon, 1993) combined with different UAS-sca insertions
driving expression during eye development in a stripe of cells
just ahead of the MF (hG-sca; Fig. 1E,F; for details and
features of misexpression constructs see Materials and
Methods).
Flies carrying the HS-sca construct do not show phenotypic
aberrations even when repeated heat pulses are applied at
different developmental stages (Materials and Methods). Sca
protein is produced in all cells from the hsp70 promoter upon
heat shock, as determined by antibody detection in imaginal
discs (not shown), but the half-life of Sca protein in vivo
appears to be very short in pulse-chase experiments (about 15
min; Materials and Methods). When discs were allowed to
recover for 30 minutes prior to fixation and antibody staining,
Sca distribution in HS-sca flies was virtually identical to wild
type (Fig. 1B) except for a slight reduction in staining intensity
in the MF (not shown). The short half-life of Sca, its requirement to be secreted and the exceptional state of the cell during
heat shock may explain the lack of effect of heat shock induced
Sca during imaginal disc development.
Precluster and R8 spacing are affected in roE-sca
flies
Rough (ro) is expressed broadly in the MF in a stripe that is
about equivalent to one column of ommatidia (Kimmel et al.,
1990). We have used an enhancer fragment from the rough
gene to ectopically express Sca within the furrow (roE-sca:
Fig. 1C,D). These roE-sca flies display a rough eye phenotype
that consists of oversized, fused and often irregularly oriented
ommatidia reminiscent of the phenotype observed in sca− adult
eyes (Fig. 2C-F). The qualitative characteristics of this
phenotype are similar in flies carrying either two or four copies
of the construct (Fig. 2C-F). However, quantitative differences
can be observed: while 4-copy flies always display the rough
eye phenotype in the whole eye, often only part of the eye is
affected in flies carrying 2 copies. Unless otherwise stated, 4copy flies have been used for further analysis.
To analyze the cause of the adult eye phenotype observed in
roE-sca, we have used different molecular markers to visualize
potential defects as early as in the MF. Early steps of ommatidial assembly and development (see above) can be visualized
with neuron-specific antibodies and other markers specific for
the individual cell types (see Materials and Methods). A monoclonal antibody that recognizes the neuronal nuclear Elav
antigen (Robinow and White, 1991) was used to monitor
general neural differentiation. In wild type, expression in R8
and R2/5 is detected as early as in the first and third columns
of ommatidial assembly, respectively, revealing the regular
arrangement of the clusters and the stepwise recruitment of
additional R-cells (Fig. 3C). The earliest markers available for
R8 development are enhancer detector P-insertions in sca
itself. Nuclear lacZ expression in such an enhancer detector
line (A2-6) faithfully reflects endogenous sca expression: after
initial detection in the characteristic groups of cells at the
anterior edge of the MF, β-gal is seen in R8 precursors leaving
the furrow (Fig. 3A). Other R8-specific markers that highlight
the regular ommatidial arrangement are the Boss gene product,
which is first detected in the third column of ommatidial
assembly (Krämer et al., 1991; Fig. 4A), and enhancer detector
insertions rO156 and BB02 (not shown).
In roE-sca discs the expression patterns of all the markers
monitoring precluster and R8 spacing are abnormal. The β-gal
expression pattern in the A2-6 line is affected in two ways.
First, the expression within the MF is reduced (see below), and
as soon as it becomes restricted to R8 the irregular, overcrowded spacing of these cells is apparent (Fig. 3B). Consistent with this, early clusters showing Elav expression in R8 and
R2/5 form too close to each other (arrowheads in Fig. 3D) and
some older clusters appear fused. The same defects can also be
visualized with anti-Boss staining (Fig. 4C,E) and by R8specific β-gal expression in the lines rO156 and BB02 (not
shown).
Expression of markers that monitor the recruitment of other
R-cell types during later stages of ommatidial assembly is very
similar to wild type, indicating that the recruitment of photoreceptors is largely not affected. The abnormalities observed
can be attributed to the spacing defects described above,
leading to ommatidial fusions and thus abnormal photoreceptor complements (as exemplified by the R3/4 and R1/6 specific
svp expression: Fig. 3E,F). Interestingly, expression of R2/5
markers (X81 and AE33) is delayed. Both markers used are
expressed in R2/5 several hours later than in wild type, whereas
their expression in R8 is not affected (X81 is shown in Fig.
3G,H). However, this delay does not seem to impair the recruitment of R3/4, R1/6 and R7 or the expression of Elav in R2/5
(see above), suggesting that only some aspects of R2/5 differentiation are affected.
In summary, the defects observed in adult eyes in roE-sca
flies are the consequence of the irregular, often too close
spacing of preclusters and R8 precursors within the MF. Strikingly, almost all features of the phenotype described above are
also observed in sca− mutants.
Ectopic Sca interferes with endogenous sca
expression
The A2-6 enhancer detector insertion in sca is a faithful marker
of endogenous sca expression (Mlodzik et al., 1990a; Fig. 3A).
Analysis of A2-6 reveals that in roE-sca eye discs β-gal
expression is clearly reduced in the early clusters of scaexpressing cells within the MF as compared to wild type (note
the difference in intensity close to the arrow in Fig. 3A and B).
This is also observed with anti-Sca staining (Fig. 1B,D and not
shown). A similar repression effect on endogenous sca
sca and the morphogenetic furrow 1965
Table 1. Relative precluster density in sca− and roE-sca
eye discs
Genotype
wild type
2×roE-sca
4×roE-sca
sca−; 2×roE-sca
sca−; 4×roE-sca
sca−
Precluster density
1.00±0.03
1.09±0.06
1.39±0.13
1.41±0.11
1.40±0.08
1.40±0.11*
The relative precluster density as compared to wild type is indicated for
each genotype. An area of about 6000 µm2, containing 17 or 18 preclusters in
wild type, was analyzed in each disc. At least 4 discs from each genotype
were subjected to this quantitative analysis.
*Note that due to the presence of multiple R8 cells in single preclusters
(arrowheads in Fig. 4B), the number of R8 cells is significantly higher in sca−
discs. However, the overall precluster density is not affected by this feature of
the sca phenotype.
Fig. 4. Boss expression in roE-sca and scaBP2 discs. Eye discs with
R8 specific expression of Boss in apically localized multivesicular
bodies are shown in all panels. The MF is on the left edge of each
panel, posterior is to the right. (A) Wild type. (B) scaBP2. (C) 2×roEsca. (D) scaBP2; 2×roE-sca. (E) 4×roE-sca. (F) scaBP2; 4×roE-sca.
Arrowheads indicate duplicated neighboring R8 cells that are only
apparent in scaBP2 discs. Note that the overall precluster density,
irrespective of the R8 duplication feature in scaBP2, is very similar in
B, D, E and F (see also Table 1).
expression is observed when Sca is expressed ectopically just
ahead of the MF (see below, Fig. 1F). We conclude that ectopic
Sca in the vicinity of its normal expression domain leads to
repression of its early MF-specific endogenous expression,
probably at the level of transcription. Interestingly, this repression is not observed during the second phase of sca expression
in R8 cell precursors emerging from the furrow (compare
intensity in isolated R8 cells posterior to MF in Fig. 3A and
B).
Effects of endogenous sca on the roE-sca
phenotype
To test if endogenous sca plays a role in the roE-sca induced
phenotype or if ectopic Sca can partially rescue the sca−
phenotype, we have established fly lines carrying two or four
copies of roE-sca in the background of the null allele scaBP2.
Such sca−; 4×roE-sca flies have a phenotype similar to 4×roEsca in wild type (Fig. 2G,H; compare with Fig. 2C-F), which
is itself very similar to sca− (Fig. 2I,J). Although the adult eye
phenotypes are almost identical, the defects observed in the
developing eye discs are slightly more severe in sca−. This is
best seen in discs of the different genotypes stained for Boss
expression (Fig. 4). The overall precluster spacing defects and
density are very similar in sca−; 4×roE-sca, 4×roE-sca or sca−
eye discs as judged by their relative density compared to wild
type (Table 1). However, in sca− discs more R8 cells are
present than in discs of the other genotypes, because many of
the preclusters contain more than one R8 cell (arrowheads in
Fig. 4B), a feature that is not found in 4×roE-sca discs. This
aspect of the sca− phenotype is almost completely rescued by
ectopic Sca expression in sca−; 2× or 4×roE-sca discs.
When only two copies of roE-sca are present, endogenous
sca has a significant influence on precluster density and the
resulting phenotype (Table 1 and Fig. 5). The defects observed
with 2×roE-sca are quantitatively enhanced when one copy of
endogenous sca is removed. This enhancement is not only
evident from the external eye morphology (Fig. 5D-F), but also
from Sca distribution itself (Fig. 5A-C). In a weak 2×roE-sca
genotype, Sca expression is similar to that in wild type (Fig.
5A). When one copy of the endogenous gene is removed (sca−
/+) in the same 2×roE-sca background, Sca protein distribution in the MF is more irregular (Fig. 5B). This dominant
enhancer effect could be explained in terms of the repression
of endogenous sca (see above). In 2×roE-sca the amount of
ectopic protein appears not to be sufficient to reduce endogenous expression significantly and thus Sca distribution in the
MF is not severely affected. When one copy of sca is deleted,
however, the ectopic Sca protein seems to be able to repress
the remaining endogenous expression. In a homozygous sca−
background both 2× and 4×roE-sca have identical phenotypes,
similar to sca− itself (see above). Thus, ectopic Sca in the MF
does not rescue the abnormal spacing feature of the sca
phenotype. Taken together, these results suggest that the
absolute levels of Sca within the MF are less important than
its distribution (see Discussion).
Misexpression of Sca anterior to the morphogenetic
furrow
To test if ectopic Sca expression anterior to the MF and its
normal expression domain have an effect on the spacing
pattern, we have used the GAL4 expression system (Brand and
Perrimon, 1993). To this end, flies carrying a GAL4 enhancer
detector insertion in hairy (h) were employed (Brand and
Perrimon, 1993). h is expressed in a stripe in front of the MF
just anterior to the sca domain, without any apparent function
at this stage of eye development (Brown et al., 1992). UAS-sca
combined with the GAL4 insertion in h (subsequently referred
1966 M. C. Ellis and others
Fig. 5. Interactions of 2×roE-sca
and endogenous sca. The
enhancement of the 2×roE-sca
phenotype by removal of one copy
of the endogenous sca gene is
shown. A-C are eye discs stained
with anti-Sca antiserum visualizing
Sca distribution in the MF. D-F are
SEMs of adult eyes corresponding
to the respective Sca protein
distribution. (A,D) Weak 2×roEsca
phenotype in a wild-type
background. Note the remaining
periodicity of Sca protein levels
(highlighted by arrowheads) in the
MF. (B,E) The same 2×roE-sca
genotype in scaBP2/+. Note the loss
of periodicity and even Sca
distribution in the furrow as
compared with A. (C,F) The
4×roE-sca phenotype (in wild-type
background) for comparison. The
MF is indicated by open arrows.
Due to the depression in the furrow
not all Sca-expressing cells are in
focus.
to as hG-sca; Fig. 1E) result in Sca expression in a band of
cells located anterior to the MF (Fig. 1F). Note that there is
reduced endogenous sca expression in the furrow, suggesting
that as in roE-sca the endogenous gene is repressed (see
above).
Flies from all hG-sca combinations with independent UASsca insertions display a rough eye phenotype. In such eyes,
ommatidia are irregularly arranged, often as fusions or
oversized facets (Fig. 6A,B), similar to the defects observed in
sca− and roE-sca flies (see above). As in the other genotypes,
the cause of this phenotype is the increased density of preclusters within the MF resulting in fused ommatidia at later stages
(Elav and Boss expression patterns are shown in Fig. 6C,D;
compare with Figs 3 and 4). Thus, ectopic Sca expression
anterior to the MF causes a phenotype which is very similar to
that resulting from ectopic expression within the furrow.
DISCUSSION
The extent of neurogenic regions in the eye imaginal disc and
the MF is not well defined, although temperature shift experiments with the Notchts allele suggest that most of the furrow
has the potential to give rise to R8 neurons (Cagan and Ready,
1989; Baker et al., 1990). Sca expression is the first molecular
marker that visualizes a regionalization within the MF. Based
on its predicted structure as a secreted molecule and its mutant
phenotype it has been proposed that Sca could act as a diffusible lateral inhibitor in the generation of the spacing pattern
in the MF (Baker et al., 1990). The experiments described here
address the function of Sca and the significance of its restricted
expression for the precluster and R8 spacing pattern. Our
results indicate that sca expression in evenly spaced groups of
cells at the anterior edge of the MF is essential for their regular
spacing. We find that uniform expression of Sca within the MF
(roE-sca) or anterior to its normal expression domain (hG-sca)
causes defects that are very similar to the loss-of-function sca
phenotype. In all three genotypes the spacing of ommatidial
preclusters is irregular, and too many of them emerge from the
MF. This primary defect does not appear to disrupt the
inductive events that govern subsequent ommatidial assembly
and R-cell determination.
Sca and spacing in the furrow
With regard to precluster spacing, the loss-of-function and
gain-of-function (ectopic uniform expression) phenotypes are
very similar. The only clear phenotypic difference between eye
discs that are sca− and those with ectopic Sca expression is the
presence of directly neighboring R8 cells in sca−, which is not
observed in the other genotypes. Interestingly, no (sca−), intermediate uniform (2×roE-sca in sca−/+ or sca−) or high uniform
expression (4×roE-sca in wt or sca−) all cause the same
precluster spacing defects. This suggests that the relative quantitative difference of sca expression between neighboring
groups of cells is an essential factor for the correct establishment of the spacing pattern, whereas the absolute Sca protein
levels in the MF are less important. For example, with two
copies of roE-sca in wild type, there remains a sufficient difference between cells expressing endogenous sca and their
neighbors expressing the transgene only, to allow most of the
normal patterning process to take place. The removal of one
endogenous copy of sca reduces this difference and leads to a
more uniform Sca distribution with stronger patterning defects.
In this situation, 2×roE-sca behaves similarly to a weak
recessive sca allele.
A striking result of the ectopic expression is the observed
down regulation of the endogenous sca gene. In both cases
analyzed (roE-sca and hG-sca), indiscriminate high expression
sca and the morphogenetic furrow 1967
within or anterior to the furrow leads to repression of endogenous sca expression in the furrow, suggesting that sca is
involved in a feedback loop affecting its own transcription.
Indeed, in protein-expressing sca mutants, the distribution of
Sca is not as restricted as in wild type (Baker et al., 1990). Thus
it appears that the wild-type sca expression pattern depends on
sca function. Furthermore, its patterned expression established
by the negative feedback mechanism may also be essential for
regionalization in the furrow, giving rise to a regular latticelike array of proneural regions. Accordingly, the sca-expressing groups of cells in each column are exactly out of register
with the groups in the next posterior column and thus are as
far away as possible from other cells expressing sca. Negative
feedback might also be utilized in the subsequent restriction of
sca expression to the R8 precursor within each group.
Fig. 6. Eye phenotype in hG-sca flies. All combinations tested with
independent UAS-sca insertions show a very similar phenotype.
(A,B) Adult eye phenotype: SEM picture and eye section,
respectively (magnification 800×). Note oversized and fused
ommatidia. (C,D) Disc phenotype as analyzed with anti-Elav and
anti-Boss antibodies, respectively. The MF is on the left edge of the
panels and posterior is to the right. Compare with wild-type and roEsca expression patterns in Figs 3 and 4. It is worth noting that there
are some minor differences between the roE-sca and hG-sca induced
phenotypes. The initial spacing defects in the MF are similar, but
there are slightly fewer fusions and oversized ommatidia in adult hGsca eyes. Nevertheless, these eyes appear rougher exteriorly than
roE-sca eyes.
Two-tiered regulation of sca
The observation that the second phase of sca expression in R8
precursors is not affected by ectopic Sca suggests a two-tiered
regulation of sca transcription. First, during the early phase,
sca expression and the establishment of regionalization within
the MF depend on interactions between sca-expressing and
non-expressing groups of cells. Ectopic expression in the MF
interferes with these processes and leads to the observed
spacing defects. During the following R8-specific phase,
however, sca appears to be regulated by a different mechanism
so that it is always expressed transiently in any given R8
precursor established. This aspect of sca regulation is not
affected by ectopic Sca. Interestingly, the two phases of sca
expression also seem to be separated in eye discs mutant for
Ellipse, a dominant Egfr allele (Baker et al., 1990; Baker and
Rubin, 1992), where Sca can only be detected in established
R8 precursors.
In parallel to the biphasic regulation of expression there
might also be two separate functions for sca in R8 cell spacing.
The earlier function would be responsible for the regular
precluster spacing in the furrow, whereas the later function
would lead to the establishment of a single R8 precursor in
each precluster. The view that these roles are separable is
supported by the observation that ectopic Sca is able to mimic
the early furrow defects, but has no apparent negative effect
on the later function. In fact, in sca−; roE-sca discs, ectopic
Sca is mostly sufficient to rescue the R8 duplication feature of
the sca− phenotype (Fig. 4). However, it is not clear whether
there is any direct relationship between the two phases of sca
regulation and the two apparently distinct functions. It is conceivable that sca might accomplish both precluster spacing and
suppression of duplicated R8 during its expression in groups
of cells in the MF.
Molecular models for Sca action
An interesting molecular feature of Sca is that its fibrinogen
related part (Baker et al., 1990) also has homology to a globular
domain found in the newly discovered tenascin (TN) family of
vertebrate extracellular matrix (ECM) proteins (Fig. 7;
reviewed by Erickson, 1993). The homologous domain is
located at the C termini of these molecules and it has been
proposed that it participates in protein-protein interactions. The
vertebrate TN-proteins show a highly regulated expression that
is often restricted to developing neural tissues. Specifically,
chicken and rat TN-R (originally called restrictin and neural
1968 M. C. Ellis and others
Fig. 7. Sca homology to tenascin family ECM proteins in the fibrinogen related domain. Alignment of the sequences of chicken and rat TN-R
(TnR C; Nörenberg et al., 1992; TnR R; Fuss et al., 1993), chicken TN-C (TnC C; Chiquet et al., 1991), human adrenal medulla fibrinogen-like
protein or TN-X (TnX H; Morel et al., 1989), porcine ficolin (Fic P; Ichijo et al., 1993) and scabrous (Sca D) are shown. Amino acid numbers
within the respective peptides are given on right side. Within this domain of 171 amino acids sca has up to 42% (TN-X) identity to the proteins
listed. Identical amino acids are boxed in black and similar residues in grey. The following amino acids were considered similar: M, V, L, I; A,
G; F, Y, W; S, T; K, R; H, Q, N; D with E, N and E with D, Q. The homologous domain is always located at the C terminus. EGF-like repeats
and fibronectin type III repeats present in most of these proteins are not found in Sca. Databases were searched using the FASTA/TFASTA and
PROFILESEARCH/TPROFILESEARCH programs (EMBL GCG implementation). Sequences were aligned with PILEUP and displayed using
PRETTYBOX.
recognition molecule J1-160/180, respectively) and TN-C
(also called tenascin/cytotactin) have been implicated in the
modification of neuronal differentiation (Chiquet et al., 1991;
Nörenberg et al., 1992; Fuss et al., 1993; Ichijo et al., 1993).
This raises the intriguing possibility that Sca might interact
with or modify the ECM in a given neurogenic region. Alternatively, Sca could participate in the cell-cell interaction
process by affecting ligand presentation, ligand-receptor interaction or internalization. Some of the phenotypic features, such
as the aspect of R8 inhibition in neighboring cells, might still
suggest a diffusible inhibitor function.
Sca expression is one of the first molecular markers for neurogenic regions in imaginal discs, during eye as well as bristle
development. All features of the sca mutant and misexpression
phenotypes taken together with the molecular data fit well into
a model in which sca is involved in altering the cellular environment within clusters of neurogenic cells in the furrow. In
such a ‘positive modification’ model, sca would participate in
defining fields in which communication between cells of such
clusters is facilitated or modified in order to define a neural
precursor (possibly via interactions between neurogenic
genes). Moreover, such regions might also be temporarily
insulated from communication with adjacent cells that are not
part of a given sca-expressing region. The existence of these
conditioned fields should be short-lived and disappear quickly
after the relevant communications and decisions have been
achieved. Such a mechanism would permit the same set of
genes to be used again in different cell-cell interactions during
subsequent developmental decisions. Indeed, the neurogenic
gene Notch (as well as some other neurogenic genes) is not
only required for defining neural precursors, such as the R8 or
bristle organ precursors, but is being ‘reused’ at multiple stages
of cell fate decisions during the subsequent development of the
ommatidia (Cagan and Ready, 1989; Fortini et al., 1993) and
in decisions determining the differentiation of specific cell
types in the whole bristle organ (Hartenstein and Posakony,
1990). It is worthwhile to note that genetic interactions
between neurogenic genes and sca (Brand and Campos-Ortega,
1990; Rabinow and Birchler, 1990; Baker et al., 1990) are also
seen in roE-sca flies (our unpublished results).
Accordingly, in both sca mutant and misexpression
genotypes, all cells in the MF are exposed to similar amounts
of Sca, allowing more ‘random’ interactions that finally lead
to the observed spacing defects. The accessory function that
sca would serve in the model described above might also
explain the incomplete penetrance of the sca null phenotype.
Other genes, like the neurogenic loci, could still be active
(although with reduced efficiency) in the absence of sca
function. In restricting their possible inhibitory interactions to
discrete areas in the MF, Sca could be considered to play a
partially instructive role in the generation of the spacing pattern
in the Drosophila eye.
We are indebted to U. Heberlein for sharing unpublished information and providing the rough enhancer fragment prior to publication.
We thank A. Brand, Y. Hiromi, U. Gaul, M. Freeman and L. Zipursky
for providing fly strains, C. Thummel and A. Brand for their
expression vectors, and G. Rubin, L. Zipursky and K. Saigo for gifts
of antibodies. We are most grateful to Roger Wepf and Deryck Mills
for skilled and patient help with the SEM analysis. We thank David
Strutt, Martin Zeidler, Gerrit Begemann and Steve Cohen for valuable
comments and suggestions on the manuscript.
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(Accepted 11 April 1994)