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First published online 22 March 2006
doi: 10.1242/dev.02338
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1 Department of Molecular and Human Genetics, 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 Division of Neuroscience, Department of Molecular and Cellular Biology, Baylor
College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Author for correspondence (e-mail:
hbellen{at}bcm.tmc.edu)
Accepted 23 February 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neurogenesis, Proneural genes, PNS, Sensory organs, bHLH proteins
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Most
organs perform a mechanosensory function, including the SOs on the thorax
(macro- and microchaetae), and those with stout and slender bristles at the
anterior wing margin (AWM). Others serve a chemosensory function, like the
organs with recurved bristles at the AWM
(Hartenstein and Posakony,
1989). Each mechanosensory organ is composed of a shaft and a
socket cell visible from the outside, and a neuron and a sheath cell
underneath the cuticle. The development of the adult SOs can be roughly
divided into the following steps
(Hartenstein and Posakony,
1989; Jan and Jan,
1993). The potential to become a sensory precursor is first
refined from a `proneural cluster' (Cubas
et al., 1991; Skeath and
Carroll, 1991) - a group of cells expressing low levels of
proneural proteins - to a primary SO precursor (pI), which expresses high
levels of proneural proteins. This process of pI selection is mediated via
Notch-mediated lateral inhibition
(Heitzler and Simpson, 1991;
Lai, 2004;
Schweisguth, 2004;
Simpson, 1990), and the
resulting accumulation of proneural proteins specifies pI cells as neural
precursors. Each pI then undergoes an asymmetric division to generate a pIIa
and a pIIb. The pIIa will further divide to generate the shaft and socket
cells. The pIIb will give rise to a glial cell, which migrates away and
undergoes apoptosis in some lineages (Gho
et al., 1999), and the pIIIb, which will divide asymmetrically to
generate neuron and sheath cells
(Posakony, 1994). Finally,
these cells differentiate to form the adult SO.
The neural commitment of pIs results from the function of evolutionarily
conserved proneural genes. All known proneural genes encode bHLH-type
transcription factors, which are expressed before and during pI specification,
and are necessary and sufficient to generate SOs in the ectoderm
(Cubas et al., 1991;
Ghysen and Dambly-Chaudiere,
1989; Romani et al.,
1989; Skeath and Carroll,
1991). Tissue-specific bHLH proneural proteins like Achaete (Ac)
and Scute (Sc) (Villares and Cabrera,
1987) form functional heterodimers with the ubiquitously expressed
bHLH protein Daughterless (Da) (Caudy et
al., 1988), and bind E-box sequences in target enhancers to
activate transcription (Murre et al.,
1989). As the expression of Ac and Sc usually stops before pIs
undergo asymmetric divisions (Modolell,
1997), it is thought that, by activating the expression of a host
of `neural-specific genes' in pI cells, proneural genes coordinate the genetic
program that governs the entire SO development
(Reeves and Posakony, 2005).
One of these targets is senseless (sens), which encodes a
zinc finger transcription factor
(Jafar-Nejad et al., 2003;
Nolo et al., 2000). Loss and
gain of sens function result in loss and gain of SOs in flies
(Nolo et al., 2000). Sens
functions as a binary switch during the selection of the AWM chemosensory pIs
(Jafar-Nejad et al., 2003).
Specifically, in proneural clusters that will give rise to chemosensory pIs,
low levels of Sens repress ac and sc expression in epidermal
cells, but high levels activate proneural gene expression in presumptive pIs
and thereby contribute to the selection and specification of sensory
precursors. However, the molecular mechanism of the loss of mechanosensory
bristle subtypes in sens clones is not known.
Ac and Sc are the proneural proteins for the majority of the adult external
SOs (Garcia-Bellido and Santamaria,
1978; Rodriguez et al.,
1990). However, it has been reported that the mechanosensory
organs of the AWM and the non-innervated bristles of the posterior wing margin
(PWM) are not affected by the loss of Ac and Sc function
(Garcia-Bellido and Santamaria,
1978; Jack et al.,
1991). Here, we report that sens and da provide
the pIs of the AWM mechanosensory organs and PWM bristles with neural
identity. In addition, we identify a novel role for Ac and Sc in the survival
of the mechanosensory pI progeny that is independent of pI selection and
specification. Gain-of-function experiments in the thorax indicate that Da and
Sens are able to generate ectopic SOs in mitotic clones of the ac-sc
complex (ASC). However, unlike in the wing margin (WM), sens
function is not required for the selection or specification of the pIs of the
thoracic SOs in wild-type animals. Altogether, our data indicate that Sens and
the bHLH proteins Ac and Sc serve clearly different functions during the
development of mechanosensory organs of the thorax and the WM.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), (8)
UAS-sensC12, (9) UAS-sensC5, (10)
UAS-sensC6, (11) y w; src (sens rescue
construct) (Nolo et al.,
2000), (12) UAS-Wg::GFP, (13) UAS-CD8::GFP hs-FLP;
tub-GAL80 FRT40A; tub-GAL4/TM6B (gift from S. Cohen, EMBL),
(14)mwh1 sensE2 FRT80B/TM6B, (15)
da3 ck13 FRT40A/CyO, (16) Df(1)260-1,
y- sn3 FRT19A/FM7, (17) Df(1)sc-B57,
sn3 FRT19A/FM7, (18) hs-FLP tub-GAL80 FRT19A; act-GAL4
UAS-CD8::GFP/CyO, (19) hs-FLP tub-GAL4 UAS-GFPnls;
tub-GAL80 M(3)67C FRT80B/TM6B, (20) y w Ubx-FLP; ubi-GFP FRT80B,
(21) y w hs-FLP; y+ ck13 FRT40A/CyO (this
study), (22) UAS-P35 (Huh et al.,
2004), (23) wgCX4 FRT40A/CyO (gift from G.
Mardon, Baylor College of Medicine), (24) UAS-da45, (25)
UAS-da52 (Cadigan et
al., 2002), (26) UAS-TCFDN
(van de Wetering et al.,
1997), (27) Eq-GAL4 /TM6B
(Pi et al., 2001), (28)
sca109-68-GAL4 (Frise
et al., 1996), (29) C96-GAL4
(Gustafson and Boulianne,
1996), (30) dpp-GAL4
(Staehling-Hampton et al.,
1994), and (31) A101-lacZ/TM3, Sb1
(Huang et al., 1991).
da3 (Cronmiller and
Cummings, 1993), sensE2
(Nolo et al., 2000),
WgCX4 (Bejsovec and
Wieschaus, 1993) and dsh3
(Perrimon and Mahowald, 1987)
are null alleles. All crosses were set at 25°C. To generate mitotic clones
either Ubx-FLP was used, which generates rather large clones in the
thorax, or hs-FLP, in which case first instar larvae of the desired
genotype were heat shocked for 1 hour at 37°C. See Table S1 in the
supplementary material for the full genotypes of flies used in clonal
analysis.
Immunohistochemistry and imaging
Dissections and antibody staining were performed using standard protocols.
The following antibodies were used: rabbit 
-Amos (1:2000)
(Goulding et al., 2000),
rabbit -Atonal (1:5000) (Jarman et
al., 1994), mouse -ßGal (1:1000; Promega), mouse
-Da (1:50) (Cronmiller and
Cummings, 1993), mouse anti-Elav (1:200; DSHB)
(Robinow and White, 1991),
rabbit -HRP (1:1000; Jackson Laboratories)
(Jan and Jan, 1982), mouse
-P35 (1:1000) (Huh et al.,
2004), rabbit -Prospero (1:1000)
(Justice et al., 2003), rabbit
-Sc (1:200) (Skeath and Carroll,
1991), guinea pig -Sens (1:1000)
(Nolo et al., 2000), Rat
-Su(H) (1:2000) (Gho et al.,
1996), mouse -Wg (1:10; DSHB)
(Brook and Cohen, 1996), Cy3-
and Cy5-conjugated secondary antibodies (1:500; Jackson Laboratories). Images
were captured using a LSM510 confocal microscope, processed using Amira 3.1
and Adobe PhotoShop 7.0, and assembled using Adobe Illustrator 10.0.
Cell culture, transcription assays and GST pull-down experiments
The S2 cell transfections, luciferase assays and the GST pull down were
performed as described previously
(Jafar-Nejad et al.,
2003).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Fig. 1A
shows part of the AWM of a wild-type male Drosophila, with a dorsal
row of spaced chemosensory bristles (arrow) and a medial uninterrupted row of
stout mechanosensory bristles (arrowhead). As reported previously, the AWM of
sc10-1 males lacks chemosensory bristles but still
contains mechanosensory bristles
(Garcia-Bellido and Santamaria,
1978; Jack et al.,
1991) (Fig. 1B).
However, quantification of the number of stout bristles indicates a small yet
statistically significant reduction in sc10-1 compared
with wild-type males (Fig. 1E),
suggesting a minor role for Ac and Sc in AWM mechanosensory bristle formation.
As sens has been shown to be expressed in pIs and to genetically
interact with proneural genes during the development of the SO precursors
(Jafar-Nejad et al., 2003;
Nolo et al., 2000;
Quan et al., 2004), we tested
whether modifying the sens dosage alters bristle number in the AWM.
We find that removal of one copy of sens in
sc10-1 males results in a very severe decrease in the
number of stout bristles (Fig.
1C,E). Conversely, adding an extra genomic copy of wild-type
sens restores the number of stout bristles of the
sc10-1 flies to near wild-type numbers
(Fig. 1D,E). Neither removing
nor adding a copy of sens shows a bristle phenotype in an otherwise
wild-type background (data not shown). Therefore, our observations indicate an
important role for sens during stout bristle formation, and an
accessory role for ac and sc. This is different from
thoracic SOs, which are completely lost in the absence of ac and
sc function.
|
;
Jafar-Nejad et al., 2003;
Nolo et al., 2000). We first
established that Sens marks AWM mechanosensory organ pIs by double-labeling
A101-lacZ pupae for Sens and ß-Gal. A101-lacZ is an
enhancer trap inserted in the neuralized locus that drives
lacZ expression in pIs and their progeny
(Blair, 1993;
Huang et al., 1991). At 2
hours after puparium formation (2 hours APF), high levels of Sens colocalize
with ß-Gal in chemosensory organ pIs, and lower levels of Sens are
detected in two broad bands that extend along the whole WM (see Fig. S1 in the
supplementary material). Around 8-10 hours APF, when the mechanosensory pIs
are being specified (Hartenstein and
Posakony, 1989), Sens is strongly expressed in these cells, as
evidenced by its colocalization with ß-Gal
(Fig. 1F-F''). At this
time point, the chemosensory pIs have already undergone their asymmetric
divisions and formed sensory clusters, whose internal cells are marked by Sens
at this stage. Staining of pupal wings at 12-14 hours APF with an anti-Sens
antibody does not show a significant difference in the number of AWM
mechanosensory precursors among wild-type, sc10-1 and
sc10-1; sens+/- pupae
(Fig. 1G-I). Note that one row
of Sens positive cells that are lost in the mutant wings are the chemosensory
clusters, which depend on ac and sc. These observations
indicate that a defect in pI formation does not account for the
sc10-1 stout bristle loss, and that another proneural gene
is required for the specification of the stout SOs.
We next examined whether the progeny of mechanosensory pIs are properly
formed during pupal development. We stained 24-hour APF pupal wings with
antibodies raised against Elav (Robinow
and White, 1991) and Su(H)
(Gho et al., 1996), which mark
neurons and socket cells, respectively. In wild-type pupae, neurons and socket
cells of both mechano- and chemosensory organs stain strongly at this stage
(Fig. 2A). In contrast to the
modest AWM bristle loss in the sc10-1 adults, the number
of neurons is severely reduced in sc10-1 pupae
(Fig. 2B). In addition, removal
of one copy of sens virtually eliminates the neurons that persist in
sc10-1 AWM, although a few socket cells are still present
in the sc10-1; sens+/- pupae
(Fig. 2C,E). This indicates
that ac, sc and sens are required for proper pI progeny
development at the AWM. Taken together, the data indicate that ac and
sc are not required for the selection, specification and division of
the mechanosensory pIs at the AWM. However, they contribute to the normal
development of the pI progeny.
|
)
have been shown to prevent apoptosis in several contexts
(Chandrasekaran and Beckendorf,
2003; Grimes et al.,
1996; Jafar-Nejad and Bellen,
2004; Nolo et al.,
2000; Wallis et al.,
2003; Yucel et al.,
2003), we examined whether blocking apoptosis rescues the
sc10-1 mechanosensory bristle loss. We find that
overexpression of the baculovirus anti-apoptotic protein P35
(Hay et al., 1994) in the wing
margin restores sc10-1 stout bristles to wild-type numbers
(Fig. 2F,H; data not shown).
Moreover, the neuronal loss is also efficiently rescued, as evidenced by the
staining patterns of Elav and the neuronal membrane marker HRP
(Jan and Jan, 1982)
(Fig. 2G-I'). These
observations indicate that ac and sc, as well as
sens, promote the survival of mechanosensory pIs or their progeny in
the AWM.
While carrying out these experiments, we observed the appearance of several
neurons in the PWM of the sc10-1 pupae upon P35
overexpression (Fig.
2I,I'). This is different from the phenotype in wild-type
wings, where all SOs and nerves reside in the anterior compartment
(Palka et al., 1983)
(Fig. 2J,J'). The adult
non-innervated bristles at the PWM do not normally contain a sheath cell, a
neuron or a socket cell, although presumptive socket cells are found in early
pupae along the PWM (Hartenstein and
Posakony, 1989) (data not shown). As shown in
Fig. 2K,K', inhibition of
apoptosis in the wing margin of wild-type pupae results in the generation of a
large number of neurons at the PWM. These neurons are able to send out axons,
which grow along the PWM towards the distal end, where they merge with the
marginal nerve that runs along the AWM towards the thorax
(Fig. 2K,K')
(Palka et al., 1983). These
data indicate that PWM bristles do have the potential to generate neurons and
send out axons, but that they are normally non-innervated because the neurons
or their precursors undergo apoptosis
(Blair, 1992;
Lawrence, 1966). It should be
noted that as these non-innervated bristles are not lost in
sc10-1 male flies (data not shown), the proneural gene for
the PWM bristles is unknown.
sens and da are required for the specification of the AWM mechanosensory and PWM bristle precursors
Our data indicate that rather than playing the proneural role for the AWM
mechanosensory organs, Ac and Sc provide a differentiation/survival signal
during the development of these bristles. One of the candidates for the AWM
proneural gene is asense, another bHLH gene in the ASC that
has previously been shown to be required for AWM mechanosensory bristle
development (Brand et al.,
1993; Dominguez and Campuzano,
1993). To explore if, in the absence of Ac and Sc function,
asense can assume a proneural role for the stout bristles, we
generated marked mitotic clones of deficiencies that remove the whole
ASC (ac, sc, lethal of scute and asense) and
examined the AWM bristles. As shown in Fig.
3A, lack of the ASC is compatible with bristle formation,
although many of the mutant bristles show abnormal morphology. Although this
observation is in agreement with previous data on the role of asense
in SO differentiation, it precludes asense from substituting for the
proneural role of ac and sc in the AWM.
The other known tissue-specific bHLH proneural genes, atonal and
amos, are not normally expressed in the wing margin
(Goulding et al., 2000;
Jarman et al., 1993). However,
it has been shown that bHLH proteins can repress the expression of one another
in the vertebrate spinal neural tube
(Gowan et al., 2001),
suggesting that amos and/or atonal might be ectopically
expressed upon removal of the ASC and serve as the proneural gene(s)
for the AWM mechanosensory organs. We therefore stained mitotic clones of the
ASC in the AWM with anti-Atonal and anti-Amos antibodies
(Jarman et al., 1994;
zur Lage et al., 2003).
Although Sens is strongly expressed in mutant mechanosensory pIs that lack the
ASC, we could not detect Atonal or Amos staining in these cells
(Fig. 3B,B'; data not
shown). We therefore conclude that another gene or set of genes is required to
provide the AWM mechanosensory organs with neuronal identity.
|
; Nolo et al.,
2000), we examined whether sens is required for WM
bristle formation. As shown in Fig.
3C, sens clones in the AWM are devoid of mechanosensory
bristles. In addition, the non-innervated bristles of the PWM are lost in the
absence of sens function (Fig.
3D), in agreement with our observation that Sens is expressed in
wild-type PWM bristle precursors (see Fig. S2 in the supplementary material).
Together, the data suggest a proneural role for sens in the
specification of the precursors of mechanosensory organs and non-innervated
bristles of the WM. To rule out the possibility that loss of WM bristles in
the absence of sens function is due to apoptosis, we used the MARCM
(Lee et al., 2000) system to
overexpress P35 in sens clones, and found that P35 is unable to
rescue the neurons and support cells in sens clones (see Fig. S3 in
the supplementary material). This shows that loss of Sens cannot be overcome
by suppressing cell death in WM mechanosensory lineages and provides further
evidence for a proneural role for sens. Because none of the known tissue-specific bHLH proneural proteins seems to be involved in the specification of these precursors, we investigated whether Da is required for this process. We generated clones of a null da allele and observed that both AWM and PWM bristles are lost in da clones (Fig. 3E,F). These observations provide strong evidence that Da and Sens cooperate to specify the precursors of the WM SOs.
Wingless (Wg) signaling is necessary and sufficient to induce sens expression in the wing
In all cases reported so far, sens is a downstream target of Ac
and Sc in bristle precursors (Frankfort et
al., 2004; Jafar-Nejad et al.,
2003; Nolo et al.,
2000). However, expression of sens at the AWM and PWM
(Fig. 3G) is independent of
ac, sc and da function
(Jafar-Nejad et al., 2003). It
has been suggested that Wg signaling at the WM is responsible for
sens expression (Parker et al.,
2002). To test this, we stained wing imaginal discs harboring
clones of a null wg allele for Wg and Sens, and observed that Sens
expression in the WM is lost in wg clones
(Fig. 3H,H'). Also,
ectopic expression of Wg-GFP along the anteroposterior boundary of the wing
imaginal disc results in a broad ectopic domain of sens expression
(Fig. 3I,I'). Finally, we
find that Sens expression in the WM is lost in mitotic clones of the essential
Wg signaling component dishevelled
(Klingensmith et al., 1994),
and also upon misexpression of a dominant-negative form of the Wg transducer
Tcf (van de Wetering et al.,
1997) (data not shown). Therefore, in agreement with a previous
report (Parker et al., 2002),
these observations place sens downstream of Wg in the WM, unlike in
other tissues where sens is activated by proneural proteins.
Sens and Da synergize in vivo and in transcription assays, and physically interact
It has previously been shown that co-expression of Sens with Ac or Sc
results in a synergistic increase in the number of ectopic bristles generated
in transgenic flies (Frankfort et al.,
2004; Jafar-Nejad et al.,
2003; Nolo et al.,
2000). As our data indicate that Sens and Da, but not other
proneural proteins, are required to specify AWM mechanosensory and PWM
non-innervated bristle precursors, we wondered whether a synergistic
relationship also exists between Da and Sens. Overexpression of da
using the sca109-68-GAL4 driver generates 17.1±0.5
extra SOs along the third wing vein (Fig.
4A,D; n=29). A weak UAS-sens transgene produces
7.0±0.5 extra SOs, preferentially of the dome-shaped, campaniform
sensilla type, along the third wing vein
(Fig. 4B,D; n=25).
Co-expression of da and sens generates 63.5±2.1 SOs
composed of both bristles and campaniform sensilla in the same region
(Fig. 4C,D; n=17). We
conclude that sens and da synergize to promote pI
formation.
Parallel to their in vivo synergy, Sens and proneural proteins have been
shown to synergize in S2 cell transcription assays
(Jafar-Nejad et al., 2003). To
examine if Sens can transcriptionally synergize with Da, we performed
transcription assays in Drosophila S2 cells using an
ac-luciferase construct that contains multiple E-boxes and a
Sens-binding site as a reporter. As shown in
Fig. 4E, while Sens alone does
not affect luciferase expression, it significantly increases the expression
induced by Da, providing further evidence for synergy between the two
proteins. Moreover, GST pull-down experiments indicate that Da and Sens
physically interact (Fig. 4F).
Altogether, these data support a model in which sens and da
cooperate to specify bristle precursor cells in the WM via the transcriptional
activation of key target genes.
|
|
),
sens expression in the WM is controlled by Wg. Accordingly, we
hypothesized that in precursors of thoracic bristles, the primary task of
proneural proteins with respect to providing neural identity to ectodermal
cells might be to upregulate Sens, which will then specify the pI fate
together with Da. If this hypothesis is correct, one would predict that, if
expressed at sufficiently high levels, sens and da should be
able to substitute for the function of ac and sc. Indeed,
overexpression of da in the thorax using Eq-GAL4 generates
numerous bristles in sc10-1 males
(Fig. 5A,B). Similarly, driving
a strong UAS-sens transgene with Eq-GAL4 can induce multiple
bristles in a sc10-1 background, in agreement with a
previous report (Pi et al.,
2004) (Fig. 5C). Of
note, Sens and Da synergize to promote bristle formation even in the absence
of ac and sc function: although overexpression of Sens with
Eq-GAL4 using a moderate transgene (UAS-sensC6)
only generates a few extra SOs in sc10-1 males
(Fig. 5D), the number of SOs
generated in a sc10-1 background by the co-expression of
Da and Sens is much larger than that induced by UAS-da or
UAS-sensC6 alone (Fig.
5B,D,E). However, it is possible that overexpression of
da or sens in sc10-1 flies induces the
expression of Asense, which then substitutes for the function of Ac and Sc. To
address this issue, we overexpressed da or sens in MRACM
clones of two small deficiencies that lack the ASC, Df(1)260-1 and
Df(1)sc-B57 (see Materials and methods). As shown in
Fig. 5F, thoracic ASC
clones are devoid of bristles. However, overexpression of da
(Fig. 5G) or sens
(Fig. 5H) results in the
formation of bristles in these clones. Staining with anti-Sens antibody
(Nolo et al., 2000) indicates
that even in the absence of ASC function, overexpression of
da is sufficient to induce high levels of Sens in single cells, the
presumptive pIs (see Fig. S4 in the supplementary material). Together, these
data indicate that not only do Sens and Da endow the WM epidermal cells with
neural identity in the wild-type context, but their WM proneural function can
also be replicated in the thoracic bristle lineages in overexpression
studies.
Overexpression of da is able to induce pI formation in the absence of sens function
We next sought to determine whether sens and da require
the function of one another for pI specification in thorax. Analysis of MARCM
da clones that overexpress sens indicates that Sens cannot
generate bristles in the absence of da function
(Fig. 6A). Similarly, no
microchaetae are formed in MARCM sens clones in which da is
overexpressed, although the cuticle in these clones is abnormal
(Fig. 6B,C). These observations
suggest that sens and da require the function of one another
in order to generate extra bristles in the thorax. However, staining of the
clones with the anti-Elav antibody shows that the similarity between the adult
phenotypes of sens and da clones - namely, the loss of adult
bristles - is misleading: although no Elav+ cells are observed upon
overexpression of sens in da clones (data not shown), a
large number of neurons are formed in MARCM clones of sens in which
da is overexpressed (Fig.
6D-D''). These data indicate that although sens
requires da to induce pI formation in overexpression studies, high
levels of da can efficiently generate pIs in sens clones.
However, these pIs do not generate shaft or socket cells, only extra
neurons.
The above observations suggest that sens may be required for
proper cell fate determination of the pI progeny but not the pI itself. By
contrast, we have previously proposed that sens is involved in
specifying microchaetae pIs, based on the severe loss of thoracic bristles in
adult sens clones and the high level expression of sens in
the pIs of these bristles (Jafar-Nejad et
al., 2003). To clarify these discrepancies and further dissect the
function of sens in microchaetae development, we first stained pupae
harboring sens clones with an anti-Sc antibody at 12 hours APF, when
the single microchaetae pIs are being selected
(Hartenstein and Posakony,
1989). As shown in Fig.
7A-A'', single cells accumulate Sc in sens clones,
providing strong evidence that sens function is not required for
microchaetae pI selection. Although these pIs are able to divide, there is a
delay in their division compared with wild-type pIs (data not shown). The loss
of shaft and socket structures in adult sens clones on the thorax
(Nolo et al., 2000) indicates
that the sens- pIs develop highly aberrantly. Indeed, the
staining of sens clones for Elav and the sheath cell marker Prospero
(Justice et al., 2003) shows
that sens mutant sensory clusters contain multiple neurons and an
occasional sheath cell (Fig.
7B,B'). The mutant neurons are capable of sending out axons,
as indicated by rather thick HRP+ extensions that connect the
mutant neuronal clusters (Fig.
7C,C'). Also, staining sens clones with an
anti-Su(H) antibody shows that, unlike wild-type microchaetae clusters, which
contain one neuron and one socket cell, more than 98% of the mutant
microchaetae clusters lack a socket cell
(Fig. 7D-D'';
n=200). Together, these data indicate a gain of neurons in
sens mutant sensory clusters at the expense of the support cells,
which strongly suggests a pIIa-to-pIIb transformation and also a
sheath-to-neuron transformation later in the lineage
(Fig. 7E). Moreover, inhibition
of apoptosis via overexpression of P35 fails to restore shaft and socket cells
in MARCM sens clones, further supporting a fate change (data not
shown). In summary, these observations indicate that unlike the WM
mechanosensory bristles for which Sens plays a proneural role, in the
microchaetae lineage sens is not required for pI selection and
specification. However, it does regulate several key aspects of SO
development, including proper cell fate determination of the pI progeny.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The large body of work that followed this discovery led to
the realization that Ac and Sc are members of the bHLH proneural
(Ghysen and Dambly-Chaudiere,
1989; Romani et al.,
1989) protein family, which are involved in early steps of
neurogenesis in flies and vertebrates
(Bertrand et al., 2002;
Hassan and Bellen, 2000).
Later, two other bHLH genes, atonal and amos, were shown to
play the proneural role for almost all SOs that did not depend on Ac and Sc
function (Goulding et al.,
2000; Huang et al.,
2000; Jarman et al.,
1993; Jarman et al.,
1994), with the notable exception of the WM mechanosensory
bristles (Garcia-Bellido and Santamaria,
1978; Jack et al.,
1991). Here we show, based on multiple lines of evidence, that
Sens plays the proneural role for these bristles: sens expression in
the WM begins before the selection of mechanosensory pIs in a proneural
cluster (see Fig. S1 in the supplementary material), similar to other
proneural proteins (Cubas et al.,
1991; Skeath and Carroll,
1991); sens expression is upregulated in presumptive pIs
and is downregulated in ectodermal cells
(Fig. 1, see also Figs S1, S2
in the supplementary material), just like ac and sc
expression is refined to pIs in thoracic proneural clusters
(Cubas et al., 1991;
Skeath and Carroll, 1991);
loss and gain of sens function result in loss and gain of SOs in the
wing (Figs 3,
4); and Sens synergizes with
the Da protein in vivo and in transcription assays, and binds Da in a GST
pull-down assay (Fig. 4).
Unexpectedly, overexpression of the anti-apoptotic protein P35 in the WM
results in the generation of a large number of neurons along the PWM,
uncovering the neural identity of the PWM bristle precursors. Similar to the
AWM, the expression pattern and loss-of-function phenotype of sens in
the PWM indicate a proneural role for sens for the PWM bristles as
well. However, the neural potential of the PWM bristles is not realized in the
wild-type situation because of apoptosis of the pI progeny, providing an
example of the role of apoptotic machinery in diversifying the various sensory
lineages, as recently highlighted by Lai and Orgogozo
(Lai and Orgogozo, 2004). In
summary, Sens satisfies all the genetic and developmental criteria for being a
proneural protein for the WM bristles, and is the only zinc finger protein
shown to play a proneural role in SO development in flies.
As for other proneural proteins, the proneural function of Sens requires
the function of Da. Da serves as the binding partner for the bHLH proneural
proteins to bind E-box sequences (Huang et
al., 2000; Jarman et al.,
1993; Murre et al.,
1989) and is also able to bind DNA as homodimers
(Jafar-Nejad et al., 2003;
Murre et al., 1989). No
function has been assigned to Da homodimers in Drosophila, largely
because of the identification of tissue-specific bHLH proteins in most
contexts in which Da functions. In the WM mechanosensory precursors, however,
none of the known tissue-specific bHLH proneural proteins is expressed,
suggesting a proneural role for Da homodimers. One might argue that there is
probably an unknown dimerization partner for Da in these sensory precursors,
and we cannot exclude this possibility. However, two groups have independently
identified all Drosophila genes encoding bHLH proteins using database
searches of the complete Drosophila genome
(Moore et al., 2000;
Peyrefitte et al., 2001) and
none of the newly identified bHLH proteins are predicted to be a
transcriptional activator of the Ac-Sc or Atonal families
(Moore et al., 2000). Also,
none of these genes shows an embryonic expression pattern compatible with a
proneural function for the CNS (Moore et
al., 2000; Peyrefitte et al.,
2001). Because we find that da is required for
mechanosensory organ formation, and as it can efficiently generate bristles in
the absence of ASC, we propose that Da homodimers cooperate with Sens
to endow neural identity to AWM mechanosensory organs and PWM bristle
precursors. The physical interaction of these two proteins and the strong
transcriptional synergy between them strongly favors a role in activating key
target genes in SO development.
Our data also reveal that Ac and Sc promote the survival of the WM
mechanosensory neurons and support cells independently of pI selection. The
more severe loss of neurons compared with support cells associated with the
loss of Ac and Sc in sc10-1 suggests either that the
neurons (or their precursors) are more sensitive to the lack of ac
and sc function, or that the loss of support cells is secondary to
the neuronal death, as reported previously for another insect
(Garcia-Bellido and Santamaria,
1978). The observation that adding or removing one copy of
wild-type sens strongly modifies the sensory lineage apoptosis
observed in sc10-1 animals indicates that, in addition to
a proneural function, Sens also plays an anti-apoptotic role in these cells;
this is in agreement with many reports on the role of sens and its
homologues in mammals and C. elegans in preventing apoptosis
(Jafar-Nejad and Bellen,
2004). It is interesting to note that although Ac and Sc are not
detected in the PWM by antibody staining
(Cubas et al., 1991;
Skeath and Carroll, 1991), P35
overexpression rescues many more neurons in the PWM of wild-type flies than in
sc10-1 animals (Fig.
2). This indicates a requirement for Ac and Sc in these cells.
During the third instar larval period, low levels of Sens are expressed in
the proneural clusters along the AWM that will give rise to the pI cells of
the AWM chemosensory bristles. Using in vivo and in vitro assays, we have
previously shown that low levels of Sens repress, and high levels of Sens
activate, ac and sc expression in these proneural clusters,
and thereby that Sens is involved in pI selection
(Jafar-Nejad et al., 2003).
Given the similar low-level expression of Sens in thoracic microchaetae
proneural clusters and the severe loss of microchaetae in adult sens
clones, we had hypothesized that Sens also functions during proneural
upregulation and in the selection of the microchaetae pIs. We were therefore
surprised to find that microchaetae pI selection does not require Sens
function. A recent report by Pi and colleagues
(Pi et al., 2004) presented
data on the function of the adaptor protein Phyllopod and its relationship
with Sens in microchaetae development. On the one hand, Sens was shown to be
required for the function of Phyllopod in the pIs, as well as for timely
downregulation of phyllopod expression in epidermal cells. This
suggests a dual role for Sens in pIs and surrounding epidermal cells, in
agreement with the binary switch model
(Jafar-Nejad et al., 2003). On
the other hand, phyllopod expression could still be upregulated in
single cells in sens mutant clones, suggesting that pI selection is
not disrupted. We now present evidence that microchaetae pIs are indeed
selected in sens clones and that they divide to generate progeny.
However, the mutant pIs exhibit an abnormal division pattern, and we observe a
pIIa-to-pIIb transformation, as evidenced by a gain of neurons at the expense
of support cells. These data indicate that Sens regulates several aspects of
microchaetae precursor development after the pIs are selected.
In summary, the normal development of all adult bristles in flies relies on
the function of Ac and Sc, Da and Sens. Our data indicate that despite the
structural and functional similarities between various adult bristles,
sens functions at four distinct steps in different lineages
(Table 1). First, in the WM
mechanoreceptor and non-innervated lineages, very high levels of Wingless
induce the expression of Sens, which assumes a true proneural role and
specifies SO fate independently of the typical proneural proteins Ac and Sc.
Second, in the WM chemosensory lineages, for which ac and sc
are the proneural genes, Sens is required for pI selection, as it represses
proneural gene expression in ectodermal cells and activates proneural gene
expression in presumptive pIs (Jafar-Nejad
et al., 2003). Third, even though gain-of-function studies show
that Sens is able to induce pI formation in the thorax in the absence of Ac
and Sc function, it normally plays a later role in specification of the pIIa
versus the pIIb of microchaetae lineages. Fourth, Sens is required for the
survival of the pI progeny in the WM mechanosensory lineages. We also find
that ac and sc prevent apoptosis in this lineage
independently of pI specification. Finally, our data suggest that a typical Da
heterodimeric complex is not required during the formation of the WM
mechanosensory and non-innervated bristle pIs. Hence, the cooperation between
the same group of genes is adapted in different ways to ensure the proper
development of various SOs.
|
),
hematopoietic stem cell self-renewal rate
(Hock et al., 2004),
intestinal cell fate specification
(Shroyer et al., 2005) and
neutrophil differentiation (Hock et al.,
2003; Karsunky et al.,
2002). Moreover, Gfi1 has an oncogenic potential
(Moroy, 2005) and has been
implicated in several human diseases, such as hereditary neutropenia
(Person et al., 2003),
spinocerebellar ataxia type 1 (Tsuda et
al., 2005) and small cell lung carcinoma
(Kazanjian et al., 2004).
Therefore, given the structural and functional similarities between Gfi1 and
Sens (Jafar-Nejad and Bellen,
2004), further analysis of the various aspects of Sens function in
Drosophila SO development will continue to help unravel the
mechanisms of Gfi1 function in health and disease. |
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/9/1683/DC1
* Howard Hughes Medical Institute 
|
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|
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17). Arrows in insets indicate the extra campaniform
(dome-shaped) sensilla. (E) Sens strongly increases the level of
ac-luciferase transcription induced by Da. (F) In
vitro-translated Sens can be pulled-down using GST-Da, but not with GST alone.
Western blotting with an anti-Sens antibody was used for detection.
