1 Department of Molecular and Cellular Biology, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
2 Program in Developmental Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030, USA
3 Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
* Author for correspondence (e-mail: kchoi{at}bcm.tmc.edu)
Accepted 26 August 2003
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SUMMARY |
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Key words: Bar homeodomain protein, atonal, Proneural, Retinal neurogenesis, Drosophila eye
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Introduction |
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An important question is how proneural gene expression is regulated to
either promote or inhibit neurogenesis. The Drosophila eye is an
excellent genetic model with which to address this question as the waves of
repetitive neurogenesis can be easily identified at the cellular level and
analyzed using powerful genetic tools. The adult compound eyes are composed of
800 unit eyes, or ommatidia, each of which contains eight photoreceptor
(R1-R8) neurons and 12 accessory cells
(Wolff and Ready, 1993
).
Retinal differentiation begins as the morphogenetic furrow moves anteriorly
from the posterior margin of the eye imaginal disc during larval development
(Wolff and Ready, 1993
).
Posterior to the furrow, R8 founder cells are specified first, and then they
recruit other photoreceptors in the sequential order of R2/5, R3/4 and finally
R1/6/7 (Wolff and Ready,
1993
). Therefore, the selection of the founder R8 cell in each
ommatidium is a critical step in retinal neurogenesis.
Neurogenesis of R8 founder cells from the undifferentiated retinal
epithelium of the eye disc requires the function of the proneural gene
ato (Jarman et al.,
1994). The dynamic pattern of ato expression can be
divided into four steps (Frankfort and
Mardon, 2002
) (see Fig.
1C). During stage 1, Ato is expressed as a stripe pattern across
the disc in the most anterior region of the furrow. Next, just posterior to
the stripe, Ato is expressed in columns of repetitive groups of about 20 cells
called intermediate groups (stage 2). At stage 3, Ato is expressed in subsets
of two or three cells called R8 equivalence groups. In the final step (stage
4), Ato expression becomes restricted to a single R8 founder cell. The
sequential restriction of Ato-expressing cells from the initial stripe of
cells (stage 1) to the founder R8 neuron (stage 4) is strictly regulated by
the interaction of positive and negative signaling
(Dokucu et al., 1996
;
Baker and Yu, 1998
;
Frankfort and Mardon, 2002
).
At stage 1, Hedgehog (Hh) and N signaling act positively on the expression of
Ato. During stage 2, Scabrous (Sca) are important for the formation of
regularly spaced intermediate groups. During stages 3 and 4, N-mediated
lateral inhibition, Rough (Ro) and Senseless (Sens) are involved in the
selection of single R8 founder cells.
|
One of the candidate genes involved in ato repression is
Bar, which encodes two functionally redundant homeodomain proteins,
BarH1 and BarH2. Bar proteins are expressed in undifferentiated retinal
precursor cells behind the furrow in the developing eye disc
(Higashijima et al., 1992),
showing a complementary expression pattern to Ato (see
Fig. 1D,E). They are also
expressed in two specific photoreceptors, R1 and R6, and other accessory cells
(see Fig. 1F). Studies on
Bar function in the eye have mostly been focused on its roles in the
differentiation of R1/R6 and pigment cells
(Higashijima et al., 1992
;
Hayashi et al., 1998
), but
little is known about its role in the population of undifferentiated cells.
Interestingly, there have been hints that Bar may play a role in
photoreceptor patterning. For example, ubiquitous Bar overexpression by
heat-shock (hs)-Bar or a duplication of BarH1 gene
in Bar1 mutant leads to downregulation of Hh,
Decapentaplegic (Dpp) and/or Ato expression in the eye disc, resulting in the
furrow-stop phenotype and reduced eyes
(Heberlein et al., 1993
;
Epps et al., 1997
;
Hayashi and Saigo, 2001
).
However, it has not been determined whether the Bar gene is directly
related to ato repression.
To understand the precise function of Bar in retinal neurogenesis,
we analyzed the regulatory relationships between Bar and
ato. We show that Bar is required to repress Ato expression behind
the furrow. Ectopic induction of Ato in the absence of Bar is
sufficient to form ectopic photoreceptor clusters. Furthermore,
ato-repression is mediated at the transcriptional level through the
regulatory elements of ato. This function of Bar on
ato-repression is independent of Ci, a mediator of Hh signaling
(Ingham, 1998) known to
activate ato expression. Therefore, Bar expression is crucial for the
control of ato and neurogenesis in the eye. The Bar class
homeobox genes are evolutionarily conserved
(Jones et al., 1997
;
Saito et al., 1998
;
Bulfone et al., 2000
;
Patterson et al., 2000
).
Recent studies suggest that mammalian Bar class genes may function as
regulators for the expression of bHLH proneural genes during neurogenesis
(Saito et al., 1998
). Hence,
our study may contribute to understand the mechanism of Bar homolog
function in vertebrate neurogenesis.
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Materials and methods |
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Generation of loss-of-function mosaic clones and misexpression
studies
Bar loss-of-function clones were generated using
Df(1)B263-20 with the FLP/FRT system
(Xu and Rubin, 1993). First
instar larvae from the cross between yw Df(1)B263-20
FRT18A/FM7 females and w Ubi-GFPS65T FRT18A; hs-FLP3
males were treated for 1 hour at 37°C and incubated at room temperature
until dissection. For misexpression of Bar, progeny from the cross between
dpp-Gal4/TM6B males and UAS-BarH1M13 (or
UAS-BarH2F11) females were cultured at 25°C until
dissection at the third instar larval stage. lz-Gal4 was used to
express Bar (or CiFL) in the basal cells.
Immunocytochemistry
Third instar eye imaginal discs were dissected in phosphate-buffered saline
(PBS) on ice, fixed in 2% paraformaldehyde-lysine-periodate fixative and
stained as described (Carroll and Whyte,
1989). The following primary antibodies were used in this study:
mouse anti-Arm (1:200; Developmental Studies Hybridoma Bank [DSHB]), mouse
anti-Boss (1:2000; DSHB), mouse anti-Sca (1:200; DSHB), mouse anti-ß-gal
(1:250; Promega), mouse anti-dpERK (1:250; Sigma), mouse anti-Elav (1:10;
DSHB), rabbit anti-Ato (1:5000) (Jarman et
al., 1995
), rabbit anti-BarH1 (1:20)
(Higashijima et al., 1992
),
rabbit anti-GFP (1:2000; Molecular Probes), rat anti-CiFL (1:10)
(Motzny and Holmgren, 1995
),
guinea pig anti-Ato (1:1000) (Hassan et
al., 2000
), guinea pig anti-Dlg (1:1000; provided by P. Bryant)
and guinea pig anti-Sens (1:1000) (Nolo et
al., 2000
). Secondary antibodies were anti-mouse-CY3,
anti-mouse-fluorescein isothocyanate (FITC), anti-rabbit-CY3, anti-rabbit-FITC
and anti-guinea pig-CY5 (Jackson Immunochemicals). Fluorescent images were
scanned using Zeiss LSM laser-scanning confocal microscope and processed with
Adobe Photoshop.
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Results |
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Bar is necessary and sufficient to repress Ato expression
The complementary pattern of Bar and Ato expression raises the possibility
that Bar may be involved in inhibition of Ato expression in the basal cells
behind the furrow. To test this possibility, we generated loss-of-function
clones of Bar using a Bar deficiency,
Df(1)B263-20, with the FLP/FRT system
(Xu and Rubin, 1993).
Df(1)B263-20 uncovers BarH1 and BarH2,
along with forked (f), Fimbrin (Fim), a
broken 297 retrotransposon, and an uncharacterized transcript X2
(Higashijima et al., 1992
;
Sato et al., 1999
). It has
been shown that defects in wing and leg discs associated with
Df(1)B263-20 can be fully rescued by overexpression of
either BarH1 or BarH2 (Sato et al.,
1999
; Kojima et al.,
2000
). To test further whether Df(1)B263-20
can be used as a Bar mutant in the eye disc, we generated
Df(1)B263-20 loss-of-function clones with a background of
overexpression of BarH1 (or BarH2) gene by lozenge
(lz)-Gal4, which drives Gal4 expression in the basal cells
and in the R1, R6 and R7 photoreceptor cells behind the furrow
(Flores et al., 1998
). The
expression of either BarH1 or BarH2 under the control of lz-Gal4 in
the basal cells strongly rescued the eye phenotypes found in the
Df(1)B263-20 loss-of-function clones (data not shown).
Therefore, Df(1)B263-20 was used as a null mutant of two
Bar genes in the eye for the following studies.
We then examined the expression of Ato in the mutant cells of Bar loss-of-function clones (Fig. 2A-C). The expression level of Ato was strongly elevated in the basal cell layer and the photoreceptor cell layer within Bar loss-of-function clones behind the furrow (Fig. 2C; data not shown). In the basal layer, all undifferentiated cells showed Ato upregulation (Fig. 2C, white arrow). Additionally, many individually singled-out Ato+ cells showed even stronger Ato expression in the basal and apical layers than other basal cells (Fig. 2C, yellow arrow).
|
Ectopic Ato+ cells are sufficient to recruit other
photoreceptors
As shown in Fig. 2C, the
elevated expression of Ato within Bar mutant cells could eventually
resolve into single Ato+ cells, as individual R8 cells are selected
from equivalence groups of Ato+ cells near the furrow.
Interestingly, similar ectopic Ato expression has been observed in
loss-of-function clones of Drosophila homologs of the transcriptional
coactivator complex subunits, blind spot (bli;
TRAP240) and kohtalo (kto; TRAP230)
(Treisman, 2001). However,
singled-out Ato+ cells within TRAP loss-of-function clones
could not further differentiate into mature R8 cells, thus resulting in
failure to form photoreceptor clusters
(Treisman, 2001
).
To examine whether ectopic singled-out Ato+ cells within
Bar loss-of-function clones differentiate into mature R8
photoreceptor cells, we checked the process of R8 cell differentiation using
early or late R8 cell-specific markers
(Fig. 3). Sca is an early R8
cell marker normally expressed in the intermediate groups and the R8 cells
within a few columns posterior to the furrow
(Fig. 3C, bracket)
(Mlodzik et al., 1990;
Lee et al., 1996
). Sca was
ectopically expressed within Bar loss-of-function clones behind the
furrow (Fig. 3A-C). We also
checked the expression of Sens, which is expressed in the R8 equivalence
groups and all singled-out R8 photoreceptor cells within and behind the furrow
(Nolo et al., 2000
;
Frankfort et al., 2001
). The
number of cells expressing Sens was increased within Bar
loss-of-function clones (Fig.
3D-F). Finally, we checked the expression of bride-of-sevenless
(Boss), a late differentiation marker for R8 cells
(Fig. 3G-I). The number of
Boss+ cells was increased within Bar loss-of-function
clones (Fig. 3I). Taken
together, increased numbers of Sca+, Sens+ and
Boss+ cells within Bar loss-of-function clones suggest
that ectopic singled-out Ato+ cells are able to differentiate into
mature R8 photoreceptor cells.
|
Bar is independent of lateral inhibition
The fact that Bar mutant cells with the elevated Ato expression
could eventually resolve into single Ato+ R8 cells
(Fig. 2C) suggests that
N-mediated lateral inhibition may function within Bar
loss-of-function clones behind the furrow. In addition to lateral inhibition,
Sca and Epidermal Growth Factor Receptor (Egfr) seem to be essential for
interommatidial spacing between the intermediate groups of Ato+
cells in the furrow (Baker et al.,
1990; Baker and Yu,
1997
; Chen and Chien,
1999
; Baonza et al.,
2001
; Frankfort and Mardon,
2002
). The expression of Sens in the R8 equivalence groups within
Bar loss-of-function clones showed regularly spaced staining
(Fig. 4A-C), suggesting that
loss of Bar does not affect the initial spacing of intermediate
groups. Thus, Sca, Egfr and N signaling pathways involved in ommatidial
spacing may function normally near the furrow within Bar
loss-of-function clones.
|
Bar is required for transcriptional repression of ato
As Bar is a DNA-binding homeodomain transcription factor, ectopic induction
of Ato in Bar loss-of-function clones suggests that Bar is required
to repress ato expression at the transcription level. However, it is
equally possible that Bar may be involved in destabilizing Ato protein rather
than repressing ato transcription. To test whether Bar is necessary
for transcriptional repression of ato, we used ato-lacZ
reporters in which lacZ is under the control of two regulatory
regions of ato, 3'F:5.8 or
5'F:9.3 (Sun et
al., 1998). The 5.8 kb of ato 3' sequence
(3'F:5.8) specifies the initial stripe of ato
expression and is Ato-independent (stage 1;
Fig. 1C,
Fig. 5A). By contrast, the 9.3
kb of ato 5' sequence (5'F:9.3) is
responsible for ato expression in the equivalence groups and the R8
founder cells, but is auto-regulated by Ato itself (stages 2-4;
Fig. 1C,
Fig. 5E,I).
|
Next, we examined the ß-gal activity of 5'F:9.3
ato-lacZ within Bar mutant clones to test whether Bar may also
repress ato transcription through 5'-regulatory region of
ato (Fig. 5F-H). In
the wild-type eye disc, antibody staining of ß-gal in the
5':9.3 ato-lacZ eye disc shows precise co-localization
with Sens in the R8 equivalence groups and the singled-out R8 cells behind the
furrow (Fig. 5E). The
expression level of 5'F:9.3 ato-lacZ was dramatically
elevated within Bar loss-of-function clones
(Fig. 5H), suggesting that Bar
might also repress ato transcription through 5'-regulatory
elements of ato. However, as 5'F:9.3 is
dependent on Ato for driving its expression
(Sun et al., 1998), it is
possible that increased ß-gal expression of 5'F:9.3
ato-lacZ within Bar mutant cells might be caused by increased
endogenous expression of Ato protein, which can activate
5'F:9.3 enhancer, rather than by direct
transcriptional derepression through 5'F:9.3.
To test these two possibilities, we ectopically expressed BarH1 (or BarH2)
in the antennal disc using the dpp-Gal4 driver
(Fig. 5I-L). The expression of
ato in the antennal disc is regulated by 2.1 kb enhancer regions of
7.2 kb upstream to the ato-coding region and is Ato independent
(Sun et al., 1998). The
5'F:9.3 and 5'F:7.2 ato-lacZ
lines include the antenna-specific enhancer regions and thus express
ß-gal even in the ato1 mutant discs
(Sun et al., 1998
). The
ato1 encodes a non-functional Ato protein because of a
mutation in the DNA-binding domain (Jarman
et al., 1994
). Misexpression of BarH1 or BarH2 showed the strong
repression of ß-gal expression of 5'F:9.3 or
5'F:7.2 ato-lacZ in the ventral sector of the antennal
disc both in the wild-type and the ato1 mutant backgrounds
(Fig. 5J-L, arrows; data not
shown). These observations suggest that Bar represses ato
transcription directly through the 5'-regulatory region of ato,
probably as well as by autoregulation. Consistent with this interpretation,
some Bar mutant cells were lacZ positive but Sens negative
(Fig. 5M-P, arrows), indicating
that increase in number of lacZ+ cells within Bar
mutant clones might be due to the direct transcriptional derepression by the
loss of Bar. Taken together, these data suggest that Bar represses
ato expression at the transcription level through 3'- and
5'-regulatory regions of ato.
Bar repression of ato is CiFL-independent
After furrow is initiated, Hh is secreted anteriorly from differentiating
photoreceptors to activate the expression of target genes within the furrow
(Dominguez and Hafen, 1997;
Strutt and Mlodzik, 1997
;
Borod and Heberlein, 1998
;
Dominguez, 1999
;
Greenwood and Struhl, 1999
).
In the absence of Hh signal, Ci exists as an inactive cleaved form, whereas in
the presence of Hh, the full-length active form of Ci (CiFL) is
produced (Chen et al., 1999
;
Methot and Basler, 1999
;
Price and Kalderon, 1999
).
CiFL is strongly expressed anterior to the furrow but expressed at
a low level posterior to the furrow (Fig.
6A,B) (Strutt and Mlodzik,
1997
). Ato is one of the targets of CiFL in the stripe
of the furrow (Fig. 6A,C)
(Dominguez, 1999
), showing an
overlapping expression with CiFL
(Fig. 6A,D).
|
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Discussion |
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Bar represses ato transcription through the regulatory
regions of ato
Ato expression is highly elevated in the absence of Bar behind the furrow
(Fig. 2C), suggesting that Bar
is necessary for downregulation of Ato expression. Furthermore, we showed that
Bar repressed ato expression at the transcriptional level through
both 3'- and 5'-regulatory regions of ato
(Fig. 5). The 9.3 kb of
ato 5' sequence (5'F:9.3) has been
shown to be responsible for ato expression in the equivalence groups
and the R8 founder cells in Ato-dependent manner (stages 2-4;
Fig. 1C,
Fig. 5E)
(Sun et al., 1998). Sca and
Egfr-mediated MAP kinase signaling may inhibit this enhancer function of
5'-regulatory element of ato within interommatidial regions to
establish regularly spaced intermediate groups
(Chen and Chien, 1999
). By
contrast, the 5.8 kb of ato 3' enhancer
(3'F:5.8) is only activated anterior to the furrow to
drive the initial stripe of ato expression (stage 1;
Fig. 1C,
Fig. 5A)
(Sun et al., 1998
). How this
enhancer activity is inhibited posterior to the stage 1 Ato domain of the eye
disc is unknown. Our results now indicate that the initial stripe (stage 1) of
ato expression driven by 3'-regulatory element is strongly
inhibited by Bar behind the furrow.
Ectopically elevated Ato expression within Bar loss-of-function clones was sufficient to induce the formation of mature ectopic photoreceptor clusters (Fig. 3). This suggests that Bar mutations specifically eliminate the repression of initial ato expression with little effects on subsequent steps of photoreceptor recruitment. This is consistent with the observations that Sca, Egfr signaling and N-mediated lateral inhibitions function properly within Bar loss-of-function clones (Fig. 4). Therefore, the major role of Bar during retinal neurogenesis appears to be the inhibition of initial stripe ato expression through 3'-regulatory elements of ato behind the furrow.
Bar is a DNA-binding homeodomain transcription factor. Mammalian homolog
Barx2 was shown to bind directly to regulatory elements of several neural cell
adhesion molecules, which contains target sites including the core sequence
CCATTAGPyGA (Jones et al.,
1997). Interestingly, the 5'F9.3 and 3'F5.8 regulatory
regions of ato also have multiple potential Bar binding sties
containing the same core sequence (data not shown), suggesting that Bar may
directly bind to these target sites of ato regulatory elements and
repress ato transcription.
It is important to note that CiFL induced by Hh signaling can
activate ato expression
(Dominguez, 1999).
Furthermore, Bar and CiFL are expressed complementarily each other
(Fig. 6E-H). These observations
raise the possibility that ato repression by Bar may be mediated by
Bar repression of CiFL. However, our results indicate that Bar
function is independent of CiFL
(Fig. 6I-L), supporting that
the primary cause of ato repression behind the furrow may be a direct
function of Bar as a repressor rather than indirect effects of the removal of
the activator, CiFL. Furthermore, overexpression of CiFL
by the lz-Gal4 driver in the presence of Bar did not activate
ato expression (data not shown), indicating that Bar-mediated
ato repression is epistatic to an overexpression of CiFL
activator.
Based on our findings, we propose a model of Bar function in retinal neurogenesis as summarized in Fig. 7. Ato is expressed within the furrow and is required for the generation of R8 founder neurons. Bar homeodomain proteins are expressed in the basal cells behind the furrow and represses ato expression, showing a complementary expression pattern to Ato. This function of Bar on ato-repression occurs independent of CiFL, a transcriptional activator of ato. Rather, Bar may directly repress ato transcription by binding to 3'- and 5'-regulatory regions of ato through its potential binding sites.
|
It has been shown that loss of groucho (gro) results in
increased ato expression behind the furrow of the eye disc
(Chanut et al., 2000). Gro is
a member of E(spl) complex to repress the expression of proneural genes during
N-mediated lateral inhibition (Heitzler et
al., 1996
). It is interesting to note that Bar family homeodomain
proteins have a conserved
10 amino acid motif termed the FIL domain at
the N-terminal region of homeobox (Saito
et al., 1998
; Patterson et
al., 2000
). This domain shows sequence similarity to the core
region of the engrailed homology-1 (eh1) domain in Engrailed (En) repressor
(Smith and Jaynes, 1996
),
which can directly interact with Gro co-repressor through its eh1 motif
(Jiménez et al., 1997
).
Therefore, Bar may interact with Gro through its FIL domain for its repressor
function.
Conserved role of Bar family proteins
Bar class homeodomain proteins are evolutionarily highly conserved from
Drosophila to human. Vertebrate Bar homologs include
Xenopus XBH1 and XBH2
(Patterson et al., 2000),
mouse and human Barhl1 and Barhl2
(Bulfone et al., 2000
), rat
Mbh1 [same gene as Barhl2
(Saito et al., 1998
)], and
murine and human Barx1 and Barx2 genes
(Jones et al., 1997
). Although
in vivo function of Bar homologs has not been extensively analyzed,
some members of the Bar class homeobox genes may be involved in the
genesis and fate specification of neuronal cells. A mammalian homolog,
Mbh1, is expressed in a complementary pattern to Mash1, a
homolog of ASC, in the rat eye
(Saito et al., 1998
). Hence,
Mbh1 may be involved in inhibition of Mash1 expression, similar to
the ato repression by Drosophila Bar proteins.
In vertebrate eye development, a mammalian homolog of ato, Math5
(and/or Xath5), is crucial for the generation of retinal ganglion
cells, which are the first neurons to arise and therefore may be analogous to
the R8 founder cells in the Drosophila eye
(Kanekar et al., 1997;
Wang et al., 2001
). The
essential role of Math5 in the genesis of ganglion cells suggests that Math5
plays Ato-like proneural function in vertebrate eye development. It will be
interesting to see whether a specific Bar homolog(s) may be involved in the
repression of Math5 as the Drosophila Bar inhibits
ato expression. In addition, Bar class genes are attractive
candidates for many human genetic disorders, including Joubert syndrome and
Rieger syndrome (Hjalt and Murray,
1999
; Bulfone et al.,
2000
; Blair et al.,
2002
). The new function of Drosophila Bar in negative
regulation of neurogenesis may provide insights into the function of
Bar family genes in vertebrates and the molecular basis of human
diseases associated with altered Bar function.
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ACKNOWLEDGMENTS |
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