1 Department of Microbiology and Immunology, Thomas Jefferson University, 1020
Locust Street, Philadelphia, PA 19107, USA
2 Department of Anatomy and Organismal Biology and HHMI, University of Chicago,
MC1028, AMBN101, 5841 South Maryland Avenue, Chicago, IL 60637, USA
3 Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, UK
Author for correspondence (e-mail:
jaynes{at}mail.jci.tju.edu)
Accepted 4 August 2003
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SUMMARY |
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Key words: Axon guidance, Homeodomain, Transcriptional repressor, Evx, Hb9, Grunge, Atrophin, Groucho, Eve, Nervous system
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Introduction |
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Later during Drosophila development, eve is expressed in
the nervous system, in the mesoderm in cells which develop into dorsal muscles
and pericardial cells, and in the anal plate ring
(Frasch et al., 1987).
Regulatory elements sufficient to drive each of these aspects of the pattern
were localized, downstream of the coding region
(Fujioka et al., 1999
;
Sackerson et al., 1999
). In
the nervous system, Eve is expressed in some ganglion mother cells (GMCs) and
in their daughter neurons (Frasch et al.,
1987
; Patel et al.,
1989
): the aCC and pCC neurons (derived from GMC 1-1a), the RP2
and RP2-sibling neurons (from GMC 4-2a; eve expression in RP2-sibling
is subsequently turned off), and the U/CQ neurons (which are generated by
several GMCs in the neuroblast 7-1 lineage)
(Bossing et al., 1996
;
Broadus et al., 1995
). The
other eve-expressing neurons, EL neurons, are derived from neuroblast
3-3 (Schmidt et al., 1997
);
however, the GMCs that produce them are eve negative
(Skeath and Doe, 1998
). The
aCC, RP2 and U/CQ neurons are motoneurons, and their axons innervate the
dorsal muscle field (Landgraf et al.,
1997
; Schmid et al.,
1999
; Sink and Whitington,
1991
), whereas the pCC and EL cells are interneurons. Expression
of eve in the nervous system is well conserved. For example, in the
grasshopper Schistocerca americana and in Crustaceans, Eve orthologs
are expressed in identified neurons that are homologous to those expressing
eve in Drosophila
(Duman-Scheel and Patel, 1999
;
Patel et al., 1992
;
Patel et al., 1994
). Studies
of Eve function in the Drosophila nervous system using the
temperature-sensitive allele eveID19 (also known as
eve1) showed that reduced Eve function causes alterations
of the RP2 and aCC axonal pathways (Doe et
al., 1988
), and that the axons of the Eve-positive motoneurons no
longer reach the dorsal muscle field
(Landgraf et al., 1999
).
Overexpression of Eve in the nervous system caused a redirection to the dorsal
muscle field of axons that normally innervate ventral or lateral muscles,
indicating that Eve function is both necessary and sufficient (at least in
some contexts) to direct motoneurons to innervate dorsal muscles
(Landgraf et al., 1999
). In
the mouse, expression of the eve homologue evx1 is
restricted in the developing spinal cord to V0 interneurons and is not
expressed in adjacent V1 interneurons. When evx1 function was
removed, the majority of V0 interneurons failed to extend commissural axons
and became similar to V1 neurons, suggesting that Evx1 is a determinant of V0
neuronal identity (Moran-Rivard et al.,
2001
). Consistent with the action of Eve and its homologues as
repressors that use conserved co-repressors, it has been suggested that the
pattern of neurogenesis in the mouse neural tube is regulated in part by the
spatially controlled repression of transcriptional repressors, through a
Groucho/TLE-dependent mechanism (Muhr et
al., 2001
), while in humans, a mutation (expansion of a
polyglutamine tract) in Atrophin is associated with the neurodegenerative
disease DRPLA (Koide et al.,
1994
; Nagafuchi et al.,
1994
).
Recent studies showed that several HD proteins are involved in the
regulation of neuronal identity (Thor and
Thomas, 2002). In Drosophila, the identities of ventrally
projecting motoneurons appear to be specified by Islet, Lim3 and
Drosophila Hb9 (Exex FlyBase), while Eve regulates the
identity of dorsally projecting motoneurons
(Broihier and Skeath, 2002
;
Landgraf et al., 1999
;
Odden et al., 2002
;
Thor et al., 1999
;
Thor and Thomas, 1997
). The
expression patterns of Drosophila Hb9 and Eve do not overlap in the
wild-type CNS (Broihier and Skeath,
2002
; Odden et al.,
2002
), and ectopic expression of Eve represses Drosophila
Hb9 expression, indicating that Drosophila Hb9 might be a direct
target of Eve (Broihier and Skeath,
2002
). Expression of Islet and Eve is also non-overlapping in the
wild-type CNS, and ectopic expression of Eve represses islet
expression in most motoneurons, although neither the absence of Islet nor its
ectopic expression was found to change the eve expression pattern
(Landgraf et al., 1999
;
Thor and Thomas, 1997
). Lim3
and Eve are co-expressed in the EL neurons, but not in other Eve-positive
neurons (Broihier and Skeath,
2002
).
In this study, we address aspects of Eve protein function and conservation, and investigate in detail the requirements for eve function in the nervous system, by creating eve neuron-specific mutants. We accomplish this by rescuing eve-null mutants with transgenes containing the entire eve locus deleted for individual neuronal regulatory elements. This results in a complete loss of detectable Eve expression in the corresponding neurons, without affecting other aspects of the expression pattern. Combining these with reporter transgenes that specifically mark RP2 and a/pCC, we analyze the mutant phenotype. The lack of Eve causes severe alterations in axonal morphology, among other defects. We find that our constructed mutants have a more severe mutant phenotype than that caused by eveID19, and so apparently have a more complete loss of eve function. Furthermore, in situations of partially penetrant rescue, we observe a strong correlation between a mutant axonal morphology and derepression of Drosophila Hb9. We test which domains of Eve are required for rescue, and find that the Eve HD alone can partially rescue some aspects of the mutant phenotype. However, for full rescue, both of the Eve repressor domains are required, and this requirement can be fully supplied by a heterologous repressor domain from Engrailed. Homologues of Eve from species as diverse as the mouse are also able to rescue eve function in the developing nervous system.
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Materials and methods |
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A Gal4-Eve fusion protein construct has been described previously
(Fujioka et al., 2002). This
fusion protein coding region was placed downstream of either two tandem
repeats of the RP2+a/pCC element (the eve region from +7.9 to +8.6
kb), two tandem repeats of the U/CQ element (the region from +3.5 to +4.3 kb),
or the EL element (the region from +1.9 kb, MluI, to +3.0 kb). Two
reporter UAS constructs were made in order to test the ability of Gal4-Eve to
actively repress transcription in vivo. For testing the activity in RP2+a/pCC
neurons, the mesodermal element (the region from +5.7, SphI, to +6.7
kb, SmaI) and the RP2+a/pCC element (+7.9 to +8.6 kb) were cloned
upstream of the eve 5' region from 275 to +99 bp,
followed by the EGFP coding sequence (Clontech) and the eve 3'
untranslated region from +1306 to +1521 bp, followed by the EL element from
+1.9 to +3.5 kb. The UAS sequence, amplified by PCR using as a template the
pUAST plasmid (Brand and Perrimon,
1993
), was inserted between the mesodermal element and
eve 5' promoter. To test the repression activity in U/CQ
neurons, the fragment from +1.9 to +4.5 kb (BamHI) was cloned
upstream of the eve 5' region from 275 to +166 bp,
followed by the lacZ-coding region and eve 3'
sequences from +1306 to +1521 bp. The UAS sequence was inserted between the
regulatory fragment and the eve 5' region.
The constructs for expressing modified versions of Eve, as well as the Eve
orthologs Tc-eve and Sa-eve, from a complete rescue transgene, have been
described previously (Fujioka et al.,
2002). For expressing mouse Evx1, the mesodermal element from +5.7
to +6.7 kb and the RP2+a/pCC element from +7.9 to +8.6 kb were cloned upstream
of the eve 5' region from 275 to +99 bp followed by the
ATG of a Flag tag fused to the Evx1-coding sequence and the eve
3' untranslated region from +1306 to +1521 bp. This transgene expresses
Evx1 in the mesoderm and in the RP2, aCC and pCC neurons.
Drosophila strains
Transgenic lines were established as described previously
(Fujioka et al., 2000;
Rubin and Spradling, 1982
).
When either
RP2A,
RP2B or
RP2C
was placed in an eve-null mutant background, expression of Eve at the
blastoderm stage appeared to be normal. When either the
RP2A
or
RP2B transgene insertion was homozygous, engrailed
expression was regularly spaced as in the wild type, indicating that the
segmentation function of eve was fully rescued (data not shown).
However, at the extended germ-band stage, eve expression in the
mesoderm was often weak or missing in these lines. By contrast, mesodermal
expression was normal in the
RP2C-rescued lines (data not
shown). However, in the
RP2C lines, the odd-numbered
parasegments were narrower than normal, indicating a lower activity at the
blastoderm stage. Because of this, only one out of five lines with this
construct gave rescue to adulthood. When one copy of the
RP2C
transgene was combined (in trans) with one copy of the
RP2A
transgene, mesodermal expression was normal, segmentation was normal and these
heterozygotes were efficiently rescued to adulthood. In these rescued embryos,
eve expression was never observed in RP2 and a/pCC neurons, whereas
eve expression was normal in EL and U/CQ neurons in all lines (see
Results). Unlike some of the RP2-element deletions, deletion of the EL or U/CQ
element did not cause a reduction of either mesodermal expression or
segmentation function, and both were able to be maintained as stocks. Rescue
transgenic lines were crossed into a Df(2R)eve mutant background
unless otherwise indicated.
In order to mark clearly the axons of RP2, aCC and pCC neurons, we used the
RP2 element to express Gal4 (Brand and
Perrimon, 1993), and this in turn to drive
UAS-
lacZ. In our first such attempt using a single copy
of the RP2+a/pCC element (from +7.9 to +9.2 kb) upstream of GAL4
(GAL4-RRC and RRK), only two out of 40 lines were able to
reliably activate UAS reporter gene expression
(Baines et al., 1999
), and even
in these cases, activity was rather weak. We tested whether this low activity
might be due to a lack of efficient translation in the nervous system by
changing the translational initiation signal (which was derived from the
GAL4 gene of yeast) to that found in the eve gene (see above
for details). With this modification, 11 out of 11 lines were able to drive
strong UAS reporter expression (data not shown), albeit with some
neuromere-toneuromere variability. We then tested two tandem repeats of the
region from +7.9 to +8.6 kb upstream of GAL4 (RN2-GAL4), and
found that this drove expression that was more consistently strong in all
neuromeres (see Results). RN2-Gal4 on the third chromosome was
recombined with either UAS-
lacZ
(Callahan et al., 1995
) or
UASCD8GFP (Lee and Luo,
1999
), and used in this study.
Genetic crosses and analysis of embryos
To analyze GFP expression in the RP2 mutant (described in Results), each of
the following four lines was self-crossed: (1)
Df(2R)eve,RP2A/SM6a;RN2-GAL4,UAS-CD8GFP; (2)
Df(2R)eve,
RP2C/SM6a;RN2-GAL4,UAS-CD8GFP; (3)
eveR13,
RP2C/SM6a; RN2-GAL4,UAS-CD8GFP; or
(4) Df(2R)eve,
RP2A/eveR13,
RP2C;RN2-GAL4,UAS-CD8GFP/TM3. In the case of the fourth
line, the analyzed population of GFP-expressing progeny contained the
following three second chromosome genotypes:
Df(2R)eve,
RP2A homozygotes,
eveR13,
RP2C homozygotes, and
Df(2R)eve,
RP2A/eveR13,
RP2C.
The subpopulation of these embryos homozygous for the
RN2-GAL4,UAS-CD8GFP third chromosome (which gave a stronger GFP
signal than RN2-GAL4,UAS-CD8GFP/TM3) was used for the
analysis.
For all experiments using a CyO balancer chromosome (below), the progeny not carrying this chromosome (negative for wg-lacZ staining) were analyzed. For the rescue experiments with modified Eve proteins, embryos from the following crosses were analyzed. A line carrying
Df(2R)eve,RP2A/CyO,P[wg-lacZ];RN2-GAL4,UAS-
lacZ
was crossed with lines of each of the following second and third chromosome
genotypes:
More than 2 lines of each were analyzed, and the lines showing better
rescue for each of the constructs were used for further analysis (however,
each of the lines examined showed the same overall trends, with only small
variations among them). For two-copy rescue by EveR and Eve
C,
the following lines were used: Df(2R)eve, P[Eve
R] (or
P[Eve
C])/CyO,P[wg-lacZ];RN2-GAL4,UAS-
lacZ
was crossed with Df(2R)eve,P[Eve
R] (or
P[Eve
C])/CyO,P[wglacZ];
RP2B.
In all cases, the combination of rescue transgenes provided complete rescue of
eve segmentation function (data not shown).
For analysis of the temperature-sensitive allele eveID19,
eveID19/CyO,P[wg-lacZ];RN2-GAL4,UAS-lacZ was
self-crossed for two copies of eveID19, or for one copy,
was crossed with
Df(2R)eve,
RP2A/CyO,P[wg-lacZ];RN2-GAL4,UAS-
lacZ.
In each case, embryos were collected at 25°C for 1 hour, and were allowed
to develop further at 18.5°C for 4 hours. Segmentation was rescued well
under these conditions, as determined from analyzing cuticle preparations
(Nüsslein-Volhard and Wieschaus,
1980
). The embryos were then incubated at 18.5°C (permissive
temperature) or 30°C (restrictive temperature), until they developed to
the appropriate stages.
Antibody staining was performed as described previously using biotinylated
secondary antibodies and SA-HRP (Patel,
1994). The staining was visualized using the HRP-DAB reaction with
or without nickel. For immunofluorescent staining, FITC-conjugated anti-mouse
(1:1000) and Texas Red-conjugated anti-rabbit (1:500, Jackson ImmunoResearch)
secondary antibodies were used. The following primary antibodies were used:
polyclonal anti-Eve at 1:10,000, a gift from M. Frasch
(Frasch et al., 1987
);
anti-Eve monoclonal 2B8 (Patel et al.,
1994
) at 1:20; polyclonal anti-ß-gal at 1:200 (ICN);
anti-Fas2 monoclonal 1D4 (Vactor et al.,
1993
) at 1:10; rabbit anti-Drosophila Hb9, a gift from J.
B. Skeath (Broihier and Skeath,
2002
) at 1:500; anti-Futsch monoclonal 22C10
(Fujita et al., 1982
) at 1:5;
and rat anti-Islet, a gift from J. B. Skeath
(Broihier and Skeath, 2002
) at
1:200. For the
lacZ marker, the following modifications to the
published protocol were used: secondary antibody was incubated overnight at
4°C (instead of 1-2 hours at room temperature), and the incubation time
for SA-HRP was prolonged to 2 hours (from 1 hour) at room temperature.
Lucifer Yellow injections
Late stage 16 (14 hours 15 minutes±15 minutes) wild-type and RP2
mutant embryos were dissected in Sorensen phosphate buffer as described
previously (Landgraf et al.,
1997), with the modifications that collagenase treatment was
omitted, and dissected embryos were fixed in 3.7% formaldehyde for 20 minutes.
Neurons (RP2, aCC, and pCC) were identified by a combination of GFP expression
(using RN2-GAL4;UAS-CD8GFP) and their position in the nerve cord.
Cells were filled with Lucifer Yellow, and preparations were processed, as
described previously (Zlatic et al.,
2003
).
Immunocytochemistry for single-cell labeling
We used the following primary antibodies: anti-Lucifer Yellow (at 1:1000,
Molecular Probes), anti-CD8 (at 1:50, Caltag Laboratories) and Cy5-conjugated
goat anti-Horseradish Peroxidase (at 1:200, Jackson ImmunoResearch); and
secondary antibody Alexa488-conjugated goat anti-rabbit (at 1:500, Molecular
Probes). The images shown are maximum projections of confocal
z-series acquired with a Leica SP confocal microscope and processed
with Adobe Photoshop software.
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Results |
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These data indicate that each of the neuronal regulatory elements is not only sufficient, but is also necessary for eve expression in the corresponding set of neurons. Somewhat surprisingly, each of these neuronal specific mutants survived to adulthood, and neither mutant adults nor larvae showed any obvious behavioral abnormalities.
eve expression is independent of eve function in
the nervous system
Although eve expression was eliminated in specific subsets of
neurons in these transgenic lines, we did not know whether the neurons
themselves were eliminated, or whether a change in neuronal cell fate had
occurred that might alter the activity of the eve neuronal enhancer
elements. To test whether eve regulatory element activity is affected
by the loss of eve function, we crossed into these lines additional
transgenes in which the regulatory elements directly drive lacZ
expression (Fujioka et al.,
1999). In each case, ß-gal expression was able to clearly
mark the eve mutant cells, showing that eve function is not
required to maintain the activity of the eve neuronal enhancers, and
that these neurons still exist without eve function
(Fig. 2E,H,K).
As we were able to mark the mutant cells in our RP2 mutants, we could
analyze the resulting morphological changes in detail. (Note that these
mutants lack eve expression in RP2, aCC, and pCC neurons.) In order
to mark axons more clearly, we used a Gal4 driver transgene with a
multimerized enhancer region and a modified translation initiation site (see
Materials and methods). We used this Gal4 driver (RN2-GAL4) in
combination with either UAS-lacZ or UAS-CD8GFP
to examine the mutant phenotype.
Loss of eve function causes aberrant positioning of cell
bodies and abnormal axonal morphology
In combination with the modified Gal4 driver,
UAS-lacZ generated strong marker expression that was
consistent from neuromere to neuromere, and clearly marked individual RP2 and
a/pCC axons (Fig. 3A). When
this reporter was placed in the RP2 mutant background, ß-gal staining
revealed several abnormalities (Fig.
3B). RP2 neurons were positioned further posterior than normal,
lying almost adjacent to aCC, instead of the normal position intermediate
between segmentally reiterated groups of a/pCC neurons (arrows,
Fig. 3C,D). The positions of
mutant RP2s were also often abnormal laterally (out of line with the a/pCCs)
and dorsoventrally (data not shown). By contrast, the positions of a/pCC
neurons were more normal, lying just dorsal to the positions of the most
medially located U/CQ neurons (Fig.
3D), as in the wild type; however, their positions relative to
each other were abnormal (see below).
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In the wild type, aCC neurons extend their axons posteroperipherally
through the aISN and have predominantly anteriorly projecting dendrites
(Fig. 3E, Fig. 4E)
(Landgraf et al., 1997;
Schmid et al., 1999
;
Sink and Whitington, 1991
). In
the mutant, virtually all aCC neurons showed clear abnormalities
(Table 2;
Fig. 3F,H,J,
Fig. 4F,G). Many aCCs extended
short axons anteriorly or posteriorly that usually seemed to attach to RP2
axons. Some aCC axons were also observed to cross the midline, mostly at the
posterior commissure (Fig. 4F).
Unlike the RP2s, one-third of aCCs had no axon
(Table 2), and by stage 16,
some of these cells appeared to be fragmenting, possibly as part of a cell
death process (data not shown). We were able to label only two mutant aCC
neurons by intracellular injection. Both had very abnormal morphologies: axons
did not exit the CNS, and only one was bipolar
(Fig. 4F,G).
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In order to test whether our cell-specific mutants have residual
eve function, we compared our observed phenotypes with those of the
previously studied temperature-sensitive allele eveID19
(Doe et al., 1988;
Landgraf et al., 1999
).
Compared with our RP2 mutant, eveID19 (at the restrictive
temperature) exhibited more RP2 and aCC neurons extending axons toward the
muscle field in almost every segment, and fewer pCC axons crossing the midline
(Fig. 3Q). We also crossed
these mutants to generate transheterozygotes of eveID19
and
RP2A. Their phenotype was intermediate between that of our
RP2 mutants and that of eveID19 homozygotes
(Fig. 3R). These data show
that, even though eveID19 exhibits cuticle (segmentation)
defects very similar to those of eve nulls, it has residual function
in the nervous system, while our mutant has less (if any) such residual
function.
Even in wild-type embryos, we were not able to use the lacZ
marker to follow the axons to the dorsal muscle field. However, we were able
to do so using UAS-CD8GFP, and we were also able to clearly visualize
neuromuscular junctions (Fig.
5A-D,G,H). When this marker was placed in the RP2 mutant
background, although axons were strongly marked in the CNS
(Fig. 5B), only a few were
observed in the muscle field. About a third of embryos examined showed none
(17 out of 50), while most of the remainder showed one or two axons extending
outside the CNS. However, there were a few embryos (three out of 50) that had
short axons outside the CNS in almost all segments, although these were never
seen to reach the dorsal muscle field. In total, axons were observed in the
muscle field in 19% of hemisegments (n=366), consistent with the data
obtained using
lacZ (21-23%, see above) and single-cell labeling
(six out of 23). For those axons observed in the muscle field, the GFP
intensity was weak relative to that in eve+ embryos. This
is also consistent with our results described above using the
lacZ marker, which showed almost no aCC axons exiting the CNS,
so that we would expect only one axon to be present at each position (from
RP2) rather than the Wild-type combination of two in each hemisegment (from
RP2 and aCC). When embryos were stained with anti-Fas2, sites of contact to
DA2 (also known as muscle 2), a normal target muscle of both RP2 and U/CQ
(Landgraf et al., 1997
;
Schmid et al., 1999
;
Sink and Whitington, 1991
) and
DO2 (a.k.a. muscle 10), a normal target muscle of U/CQ
(Landgraf et al., 1997
;
Schmid et al., 1999
) were
visible in the mutants, although they were possibly less extensive than in the
wild type (arrowheads in Fig.
5I-L), perhaps reflecting the lack of RP2 axons. However, contacts
with DA1 muscles (also known as muscle 1), normal targets of aCC
(Landgraf et al., 1997
;
Schmid et al., 1999
;
Sink and Whitington, 1991
)
were just barely visible in the mutant (arrows in
Fig. 5K,L; compared with the
wild type in Fig. 5I,J). The
apparent residual innervation beyond the DA2/DO2 neuromuscular junction may be
due to U/CQ neurons targeting the DO1 muscle
(Schmid et al., 1999
).
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Eve can act as a direct repressor in RP2 and a/pCC
Whether Eve acts as a transcriptional repressor of direct endogenous target
genes in these neurons could not be addressed extensively, as the target genes
in this tissue are only beginning to be identified. Therefore, we tested
whether a chimeric protein of the Gal4 DNA-binding domain fused with the Eve
repressor domains (Fujioka et al.,
2002) could repress an activated UAS-containing target gene in
these cells. Expression of such a transgene in RP2 and a/pCC, as well as in EL
neurons to provide an internal control
(Fig. 7A), was monitored in the
presence or absence of Gal4-EveRC, expressed from a transgene specifically in
RP2 and a/pCC (but not ELs). We found that the reporter GFP expression was
clearly reduced in both RP2 and a/pCC neurons in the presence of Gal4-EveRC,
indicating that Eve can, indeed, act as a direct transcriptional repressor in
these cells (Fig. 7B,C). Using
a similar strategy, we found that Eve can also act as a direct repressor in
U/CQ neurons (Fig. 7D,E).
However, we were not able to detect repressor activity clearly in EL neurons
using a similar assay (data not shown).
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The monoclonal antibody 22C10 (Fujita
et al., 1982) recognizes a subset of neurons, including RP2 (green
arrows, Fig. 8E-H) and aCC
(yellow arrow, Fig. 8E,F), but
not pCC. This antibody was recently shown to recognize the futsch
gene product, which is homologous to vertebrate MAP1B, a
microtubule-associated protein (Hummel et
al., 2000
). We used this antibody to examine expression of the
antigen in our mutants. We found that although it was still detectable in the
RP2 mutant, its expression was weaker than in the wild type, especially in aCC
neurons (23 out of 24 neurons showed decreased intensity; yellow arrow,
Fig. 8G,H), indicating that it
is a downstream target of Eve regulation. We also examined expression of the
transcription factor Islet, which is known to be involved in axonal guidance
(Thor and Thomas, 1997
).
However, we did not observe clear derepression in the RP2 mutant (data not
shown).
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Discussion |
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The mutants that we constructed lack all detectable Eve expression either in the combination of RP2 and a/pCC neurons, or in the U/CQ neurons, or in the EL neurons (Fig. 2), and here we analyzed in detail the defects in the first of these. In this case, despite significant defects in axonal architecture (discussed below), individuals were able to survive to fertile adulthood. In a preliminary analysis, we did not observe any behavioral abnormalities in either larvae or adult flies (M.F. and J.B.J., unpublished). Eve is normally also expressed in the parental GMCs of RP2, aCC, pCC and the U/CQ neurons, and this expression is also eliminated in our mutants. The removal of Eve from these neuronal lineages did not cause a loss of neurons (Fig. 2), showing that eve function in the GMCs is not required for their normal cell division to occur.
In order to identify the mutant neurons and to analyze the axonal
phenotypes, the eve neuronal elements were used to drive marker gene
expression. Expression in RP2+a/pCC was enhanced using the Gal4-UAS system
(Brand and Perrimon, 1993). In
order to be effective, we found that Gal4 activity needed to be increased by
replacing the yeast translational initiation signal in the Gal4 driver
transgene with that from eve, and by multimerizing the enhancer (see
Materials and methods). With these modifications, in combination with a
UAS-
lacZ transgene
(Callahan et al., 1995
), the
axons of RP2, aCC and pCC were clearly marked (Figs
3,
6 and
8). Combining the same Gal4
driver with UAS-CD8GFP (Lee and
Luo, 1999
) allowed us to mark axons with membrane-localized GFP.
This allowed us to examine how far mutant axons grew towards the muscle field,
and to visualize connections to specific target muscles
(Fig. 5).
The regulation of eve neuronal elements
The locations of individual neuronal regulatory elements sufficient for
eve expression in subsets of neurons were identified previously
(Fujioka et al., 1999).
Because deleting each of these regulatory elements in the context of the
rescue transgene eliminates eve expression in the corresponding
neurons, the elements are also necessary in the context of the entire gene
(Fig. 2), as was found for the
eve mesodermal enhancer (Han et
al., 2002
).
In the absence of eve function, all neuronal enhancer elements remain active. This suggests that the cells do not completely change their identities in the absence of Eve, but continue to express the combination of factors that normally initiate and maintain eve expression, and which presumably act in concert with Eve to specify the phenotype.
Requirements for Eve in RP2 motoneurons
Using a combination of the constructed cell-specific mutant and the marker
transgenes described above, we analyzed the morphology of RP2 mutant neurons.
The mutant RP2s exhibit a variety of defects in axonal morphology (Figs
3 and
4, summarized in
Table 1), and in addition they
are defective in their ability to migrate to their normal position within the
CNS (Fig. 2E,
Fig. 3). Although the abnormal
position of mutant RP2s might affect their ability to extend axons normally,
it does not seem to be a primary determinant of whether they extend to the
muscle field, because the position defect is often rescued by the Eve protein
without its repressor domains (EveNH, supplied by an additional rescue
transgene expressing this protein; Fig.
6C), and yet in this case, axons still fail to extend properly. In
addition, there are a few abnormally located RP2s in wild-type embryos, and
they still extend their axons normally (data not shown). The mutant RP2s seem
to retain some axonal guidance capability, as their axons often recognize
their normal point of exit from the CNS along the ISN, once they `happen' onto
it, although many of these fail to extend further (Figs
3,
4,
5,
Table 1). The RP2 mutant axons
were never observed to extend as far as their normal target muscles, and
indeed, many of them do not exit the CNS
(Fig. 5). Of those that do exit
the CNS, most appear to be unable to defasciculate from the ISN onto muscle
targets. It is unlikely that the failure of mutant RP2 axons to exit the CNS
by stage 16 is due solely to a delay in either axon outgrowth or recognition
of the nerve roots, because we find similar percentages of peripherally
projecting RP2 neurons in both stage 16 embryos and young first instar
larvae.
The inability of the mutant axons to reach the muscle field was partially
rescued by the Eve HD without its repressor domains
(Fig. 6B,C). However, in
addition to the HD, the two distinct repressor domains of Eve contribute
strongly to rescue of the mutant phenotype
(Fig. 6D,E). Interestingly,
complete rescue can also be provided by the Eve HD fused with a heterologous
repressor domain from Engrailed (Fig.
6G). Therefore, in addition to the functions provided by the
DNA-binding HD, a generic repressor function of sufficient strength is
required for normal function in these neurons. The fact that the HD alone can
provide a detectable degree of rescue, which contrasts with the lack of its
ability to rescue segmentation function
(Fujioka et al., 2002),
suggests that perhaps competition for binding sites with activators of
downstream target genes plays a more prominent role in Eve function in the
nervous system. Consistent with the requirement for its repressor domains, an
Eve-Gal4 fusion protein was able to actively repress a UAS-containing target
gene in RP2 neurons (Fig.
7B,C).
Requirements in aCC motoneurons
The requirement for Eve in axonal guidance is somewhat more stringent in
aCC than in RP2 neurons. Although a significant fraction of mutant RP2s
initially extend axons in the same direction as wild-type RP2s, essentially
none of mutant aCCs do so (Table
2). In addition, unlike for RP2s, the aCC phenotype was not
significantly rescued by either the HD alone or the HD with the N terminus
(which provides no detectable repression activity, but might stabilize the
protein). In aCC, as in RP2, the phenotype was partially rescued by including
either repressor domain, and the Engrailed repressor domain was able to
provide full activity (Fig. 6).
Furthermore, Eve repressor domains were able to actively repress a UAS target
gene in aCC neurons (Fig. 7).
These data indicate that the primary function of eve in aCC is to
actively repress target genes. The more stringent requirements in aCC versus
RP2 suggest that there may be different target genes in these two motoneurons,
although Drosophila Hb9 is a common target (discussed below).
Requirements in the interneuron pCC
In mutant pCC neurons, in contrast to the wild type, 40% of axons crossed
the ventral midline to the contralateral side
(Table 3). This phenotype was
rescued quite effectively by the HD alone, suggesting that the target gene(s)
involved may be passively repressed through a competition for activator
binding sites (although more complex possibilities cannot be ruled out).
Recent studies have shown that midline crossing is regulated by a complex
interplay of responses to attractive and repellent signals secreted by midline
cells (reviewed by Dickson,
2002). One possibility is that the midline crossing phenotype of
mutant pCCs might be caused by derepression of the DCC/Frazzled receptor
(Kolodziej et al., 1996
) for
the midline attractant Netrins (Harris et
al., 1996
; Mitchell et al.,
1996
).
In addition to the defects in axonal morphology, the cell body position of pCC relative to that of its sibling aCC is apparently randomized in the mutant (Fig. 3P). We do not know whether this is due to the lack of Eve in aCC, in pCC, or in both. As other neurons in the CNS are wild type, it is unlikely to be an effect involving surrounding neurons. This defect is rescued effectively only when the Eve HD is accompanied by at least one repressor domain. It is unclear whether the normally tight control of this characteristic has a role in the subsequent morphogenesis of the neurons.
Null and hypomorphic neuronal phenotypes
Previous studies using the eve temperature-sensitive allele
eveID19 showed that eve is required in
eve-positive motoneurons for proper axonal morphology, including the
ability to reach the dorsal muscle field
(Doe et al., 1988;
Landgraf et al., 1999
). The
eveID19 allele contains a point mutation in the HD
(Frasch et al., 1988
), and
shows a near-null segmentation phenotype at the restrictive temperature
(Nüsslein-Volhard et al.,
1985
). However, our data indicate that eveID19
is not a true null in the nervous system
(Fig. 3). This might explain
the differences between the morphological phenotypes of mutant RP2, aCC and
pCC neurons that we observe in our mutant as compared with those seen in
eveID19 (Doe et al.,
1988
).
A significant variation in phenotype with a small change in the level of function is consistent with an interpretation wherein loss of Eve leads to the absence of a particular subset of neuronal properties. Interestingly, even in our mutant, which has no detectable Eve expression in these lineages, some RP2s as well as some pCCs show several of the characteristics of their wild-type counterparts. If we assume that our mutant represents the complete null phenotype, this indicates that, to a limited extent, Eve acts in parallel with other factors in the specification of these cell types, rather than being an overall determinant of the cell fate. This notion is also consistent with the fact that the eve regulatory elements, which are specific markers for these cell types, continue to be active in the absence of eve function.
Evolutionary conservation of function
A single copy of a wild-type transgene was sufficient to almost completely
rescue the mutant phenotypes in the nervous system, in contrast to the
requirement for two copies to rescue segmentation
(Fujioka et al., 1999). This
relative lack of eve dosage sensitivity in the nervous system might
be related to the apparently ancestral nature and greater conservation of the
nervous system function of eve.
We tested the extent to which Eve homologues from Tribolium, grasshopper, and mouse could rescue the mutant neuronal phenotypes. We found that a single copy of either of the insect orthologs could provide essentially complete function in Drosophila, while mouse Evx1 also showed quite strong rescuing capability. Coupled with the fact that active repression function is required for this degree of rescue, these results indicate that each of these homologues has retained both the ability to be targeted to and to repress the key direct target genes of Eve in the nervous system.
Target genes in the nervous system
It has previously been shown that Drosophila Hb9 and Eve are
expressed in a non-overlapping pattern in the wild-type CNS
(Broihier and Skeath, 2002;
Odden et al., 2002
), and that
ectopic Eve expression represses Hb9, indicating that Hb9 is
a target gene of Eve (Broihier and Skeath,
2002
). We found that Hb9 is derepressed in the RP2 mutant
in both RP2 and aCC (Fig. 8),
but not in pCC neurons (the RP2 mutant lacks Eve in all three cell types),
showing that there are significant differences in target gene regulation in
different neurons, even in those derived from the same GMC (in the case of aCC
and pCC).
When the Eve HD alone is used to rescue the RP2 mutant, Hb9 is repressed in
many of the RP2 neurons, and this seemingly stochastic repression correlates
with a more normal axonal morphology. However, effective repression,
particularly in aCC, requires active repression domains, with either of the
repressor domains of Eve alone providing partial activity (in the context of
the Eve HD). Although there is a strong correlation in situations of partial
rescue between the axonal phenotypes of individual neurons and derepression of
Hb9, this correlation is not 100%. This suggests that there may be other key
target genes that mediate Eve neuronal function in addition to Hb9. We also
found that the level of expression of the antigen (Futsch) of the monoclonal
antibody 22C10 (Hummel et al.,
2000) is reduced in RP2 and aCC in the absence of Eve
(Fig. 8). However, the gene
encoding this antigen is likely to be an indirect target of Eve, because its
expression is activated rather than repressed by Eve.
Either of the repressor domains of Eve is sufficient to give a similar
degree of partial rescue of each of the phenotypes we have studied in the
nervous system, including the repression of Hb9, showing that these
repressor domains provide a similar function. In fact, two copies of a
transgene expressing either EveC or Eve
R are able to rescue to a
similar degree as that of one copy of the wild-type transgene (data not
shown). Thus, we see that the recruitment of either of two apparently distinct
co-repressors, Groucho or Atrophin, produces the same net result. The two are
used in these neurons in an additive fashion to generate the appropriate level
of Eve repressor activity, with no apparent target gene specificity.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Integrative Biology, 3060 VLSB # 3140,
University of California, Berkeley, Berkeley, CA 94720-3140, USA
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