E(spl) region transcript m4
Cell-cell signaling through the Notch receptor is a principal
mechanism underlying cell fate specification in a variety
of developmental processes in metazoans, such as
neurogenesis. An investigation is described
of seven members of a novel gene family in Drosophila with
important connections to Notch signaling. These genes
all encode small proteins containing predicted basic
amphipathic alpha-helical domains in their amino-terminal
regions, as described originally for Bearded; accordingly,
they are referred to as Bearded family genes. Five members
of the Bearded family are located in a newly discovered
gene complex, the Bearded Complex; two others reside in
the previously identified Enhancer of split Complex. All
members of this family contain, in their proximal upstream
regions, at least one high-affinity binding site for the Notch-activated
transcription factor Suppressor of Hairless,
suggesting that all are directly regulated by the Notch
pathway. Consistent with this, it has been shown that Bearded family
genes are expressed in a variety of territories in imaginal
tissue that correspond to sites of active Notch signaling. Overexpression of any family member
antagonizes the activity of the Notch pathway in multiple
cell fate decisions during adult sensory organ development.
These results suggest that Bearded family genes encode a
novel class of effectors or modulators of Notch signaling (Lai, 2000).
Overexpression of Brd causes adult phenotypes closely resembling those conferred by loss-of-function mutations in N pathway genes. These phenotypes include both bristle multiplication and bristle loss; the former is due to the specification of supernumerary SOPs, while the latter is caused by inappropriate allocation of cell fates within the bristle lineage. Likewise, overexpression of other Brd family genes similarly interferes with cell fate specification events controlled by N pathway activity.
By comparison with the results with Brd, m4, and malpha, overexpression of Bob and Tom each cause much
stronger mutant phenotypes. With one copy of
UAS-Bob, a strong tufting or lethal phenotype is observed,
with bristle tufting extending to most macrochaetes and
microchaetes, as well as occasional bristle loss. UAS-Tom causes the most severe effects of all Brd
family members when expressed under the control of sca-GAL4,
with most lines giving high percentages of lethality at
late pupal/pharate adult stages. The relatively infrequent
escapers typically exhibit strong tufting of nearly all
macrochaetes and microchaetes and frequently display some
degree of bristle loss, especially on the legs (Lai, 2000).
If the gain-of-function effects reported here are indicative
of the normal direction of Brd family protein function, and if all
members of the Brd gene family are indeed targets of
transcriptional activation by this pathway, as has been
hypothesized, then Brd family proteins are excellent candidates
to mediate a negative feedback mechanism in N signaling.
However, a full understanding of Brd family
protein function must ultimately incorporate loss-of-function
genetic data, which, owing to apparent functional overlap
among these genes, is not available. Thus, it is
entirely possible that overexpression of Brd family proteins,
rather than reinforcing or exaggerating their wild-type activity,
instead causes a 'dominant negative' effect; in this case, these
proteins may normally function as positive effectors of N
signaling (Lai, 2000).
An important issue concerning the function of Brd family
proteins is whether they exert their effects on N signaling on
the sending or receiving side of the process, or both. Preliminary evidence suggests that
overexpression of Brd family genes is able to exert a cell non-autonomous
effect on lateral inhibition in proneural clusters, consistent with the possibility
that these proteins can antagonize the ability of a cell to send
an inhibitory signal. This is of considerable interest, since
relatively little is known about the detailed structure and
function of the N pathway upstream of the N receptor (Lai, 2000).
Because of the high degree of structural similarity
between malpha and m4, the expression patterns of both genes
during embryonic development and in imaginal discs were
compared. Embryonic expression patterns
are nearly indistinguishable, and appear very similar to
those of E(spl) bHLH genes, particularly for m5, m7 and m8. Characteristic for this group of genes is
the early mesectodermal expression, which appears shortly
before the onset of gastrulation. Later on, transcripts of malpha
and m4 are detected in the neuro-ectoderm as well as in the
mesoderm in a highly dynamic pattern in many stages of
embryogenesis. In imaginal discs, the expression domains
of malpha and m4 are similar to those described for different
E(spl) bHLH genes. Transcripts of m4 accumulate primarily within
presumptive proneural clusters of eye-antennal, wing and
leg discs, a pattern remarkably similar to that of m8 or m7
expression. However, malpha transcripts are detected in a pattern
matching very closely that of mbeta expression. In the eye
disc, malpha is expressed not only within but also posterior to
the morphogenetic furrow. In the wing pouch, staining of
presumptive intervein regions and wing margin is apparent.
In the leg disc as well as in the notal part of the wing disc, a
more general expression is observed with highest concentration
in areas encompassing proneural clusters. The expression patterns suggest that
both genes are under the same regulatory control as are the
different E(spl) bHLH genes and thus, might have a role in
Notch mediated cell differentiation as well.
Surprisingly, m2 transcripts also accumulate in a pattern
reminiscent of the transcript distribution of E(spl) bHLH
genes although there are no structural similarities with
either the bHLH or the m4/malpha genes. At
first, m2 transcripts are detected in a very dynamic pattern in
the neuro-ectoderm of stage 9 embryos. At stage
10/11 the transcripts accumulate at high levels in the presumptive
mesoderm, however, they disappear quickly
with the onset of germ band retraction. Imaginal
disc expression is rather weak (Wurmbach, 1999).
In eye disc, m2 transcripts
are observed close to as well as posterior to the morphogenetic
furrow. In the wing disc, areas of proneural clusters
stain weakly, as does the dorso-ventral boundary and vein/intervein regions. These patterns are typical of
E(spl) bHLH gene expression. Therefore,
m2 appears to be regulated in a similar manner like
E(spl) bHLH and m4/malpha genes and could also serve as a
Notch target gene (Wurmbach, 1999).
Postembryonic expression patterns of Brd family genes were examined by in situ hybridization. In wing imaginal discs of third-instar larvae, Brd and E(spl)m4 transcripts accumulate specifically in the full complement of sensory organ proneural clusters. Similarly, the complex pattern of E(spl)malpha expression in the wing disc includes proneural clusters, although malpha transcript accumulation in the clusters consistently appears broader and more diffuse than that of Brd or m4. In addition, malpha transcripts appear in a narrow stripe along the dorsoventral boundary of the wing pouch, as well as along wing vein borders. In contrast, neither Bob nor Tom exhibit any patterned expression in the wing disc, although Tom may be generally expressed at a very low level in this tissue. In the eye imaginal disc, four of the five Brd family genes in this study are expressed in the vicinity of the morphogenetic furrow, the exception being Bob, which is not detectably expressed in either the eye or antenna discs. Transcripts from the different Brd family genes accumulate with distinct spatial profiles relative to the morphogenetic furrow. Brd is expressed in two closely spaced stripes, one just anterior to, and one within and posterior to, the dpp furrow stripe. Transcripts from m4, by contrast, appear in a strong band that is largely just anterior to the zone of dpp-lacZ expression. malpha shows expression in a pattern that overlaps, and extends posterior to, the marker stripe. Finally, Tom expression somewhat resembles that of Brd, in that its transcripts accumulate in two stripes lying anterior and posterior to the dpp-lacZ stripe (Lai, 2000).
The patterns of transcript accumulation from these genes were examined during pupal wing development, to assess the possible expression of these transcripts in sensory organ lineages and in the vicinity of the developing wing veins. The members of the Brd-C are not expressed at detectable levels in the pupal wing at 8 hours after puparium formation (APF), although Brd and Tom transcripts are present in the large clusters of proximal campaniform sensilla at this time. By contrast, m4 and malpha in the E(spl)-C display both proximal campaniform expression and specific wing margin expression at 8 hours APF. m4 transcripts accumulate in a set of anterior wing margin cells at this stage. Based on their spacing, they are likely to represent cells in the lineage of the chemosensory organs that appear in dorsal and ventral rows on the margin. Since transcripts from E(spl)mgamma accumulate in these organs, it appears that at least one Brd family member and at least one bHLH gene in the E(spl)-C share this aspect of their expression. It has been found that malpha is expressed at this time (8 hours APF) in a broad domain of wing margin cells that includes cells of the posterior as well as the anterior margin, and also in an incomplete wing vein boundary pattern. This latter observation prompted an examination of the accumulation of malpha transcripts in later pupal wing discs. At 24 hours APF, malpha is indeed expressed in a largely complete pattern consisting of thin rows of cells at all vein/intervein boundaries. This is highly reminiscent of the pattern of transcript accumulation from both Notch and the bHLH gene E(spl)mbeta at approximately the same time. In addition, malpha transcripts remain present throughout the wing margin (both posterior and anterior) at this stage. malpha expression in the pupal wing is highly dynamic, however: by 30 hours APF, its transcripts have nearly gone from the margin, are excluded from vein/intervein borders, and appear instead in the veins themselves and in non-vein wing blade tissue. Taken together, these observations strongly suggest that at least one Brd family member may have a role in wing vein development. In summary, in developing imaginal tissue, Brd family members are expressed specifically in multiple territories in which Notch signaling-dependent cell fate decisions take place (Lai, 2000).
Overexpression UAS-m4 and UAS-malpha causes downregulation of E(spl) bHLH expression accompanied by cell autonomous overcommitment of sensory organ precursors and tufting of bristles. Experiments by Nagel (2000), suggest that the overexpression phenotype of E(spl) m4 and E(spl) malpha obrained by Apidianakis (1999) is likely to be due to a dominant negative effect and does not reflect the biological function of these two genes. The results of and interpretation of Apidianakis (1999) are presented here for a complete record.
Negative regulation of the Notch pathway by overexpression of E(spl) m4 and malpha is specific to the process of sensory organ precursor singularization and does not impinge on other instances of Notch signaling. Identical effects were produced by both
UAS-m4 and UAS-malpha transgenes, with quantitative variations among lines attributed to position effects; therefore they are collectively referred to as m4/alpha. Macrochaetae, microchaetae and campaniform sensilla were similarly affected by m4/alpha overexpression. For example,
expression of UAS-m4 ahead of the antero-posterior boundary of the wing disk (by ptc-GAL4) produces excess scutellar bristles and anterior cross-vein (ACV) campaniform
sensilla (the sensory organs that arise from this
region of the disc). Characteristic of the m4/alpha overexpression phenotype is the multiplication of sensory organs at
their normal locations; the extra bristles are frequently
tufted together. Other than sensillum multiplication,
no effects are observed in the wing, where Notch signaling is known to affect wing margin integrity and vein thickness. No wing pattern aberrations are observed
when single copy UAS transgenes are driven with GAL4
lines that express predominantly in the wing pouch, such as
32B and ombmd653. This has been confirmed by examining
vg(boundary)-lacZ and wg-lacZ in m4/alpha overexpression
backgrounds; the patterns of these wing-margin specific
markers were identical to wild-type (Apidianakis, 1999).
Overexpression of proneural genes of the bHLH type, like
those of the Achaete-Scute complex (see Achaete), also gives rise to extra
sensory organs. Yet, the phenotype
observed for m4/alpha is quite distinct from that of overexpression of proneural genes. The latter produce ectopic bristles
at new locations, such as the wing blade, something never
observed with m4/alpha. Also, the ectopic bristles produced by
UAS-lethal of scute are spaced, suggesting that they arise from individually spaced SOPs. To compare the SOP pattern produced
by UAS-m4/alpha with that of UAS-l'sc, each one was expressed with
the ap-GAL4 driver in the background of the SOP specific enhancer trap neurA101. Although l'sc yields a pattern of
discrete SOPs randomly dispersed throughout the dorsal
half of the wing pouch (the apterous expression domain), m4/alpha
gives clustered SOPs at the normal locations where single
SOPs would have arisen in the wild-type, as
expected from the adult phenotype. Therefore, it appears
that m4 and malpha do not have proneural function; rather,
they rely upon proneural gene expression to promote the
SOP fate. This was confirmed by combining ectopically
expressed m4 (ap-GAL4;UAS-m4) with a deficiency for
the proneural genes ac and sc, Df(1)sc10-1. Flies hemizygous
for sc10-1 have bald nota, as no SOPs are specified in the
absence of proneural gene activity. Expression of m4 in this
background does not restore any chaetae,
suggesting that m4 is unable to induce sensory organs in
the absence of proneural proteins (Apidianakis, 1999).
Overexpression of m4/alpha blocks lateral inhibition.
The SOP pattern is a result of an interplay between
proneural proteins, which promote neural fate, and Notch
signaling, which inhibits it. The fact that supernumerary
chaetael/SOPs arise in close apposition to each other suggests that the contact-dependent Notch
signaling that normally counters SOP fate may be compromised. To locally block lateral inhibition for comparison
purposes, a well characterized negative
regulator of Notch signaling, Hairless (H) was overexpressed. H is known to negatively modulate Notch signaling by interfering with the
activity of the transcriptional activator Su(H), by which at
least part of the Notch signal is transduced to the nucleus (Apidianakis, 1999).
Generally, the effect of H overexpression is similar to that of m4/alpha. At the ACV
(anterior cross-vein campaniform sensillum), L3 (third
longitudinal vein campaniform sensillum) and wing margin
clusters, the extent of SOP overcommitment is comparable
to that caused by m4/alpha, whereas at the dorsal radius H gives
much higher numbers of supernumerary SOPs. The effects
of H differ from those of m4/alpha in two further respects.
(1) H abolishes some wing margin sensilla, presumably
by interfering with the Su(H)-dependent inductive Notch
signaling that sets up the dorsoventral boundary, which subsequently induces margin SOPs. m4/alpha does not affect the process of dorsoventral wing
patterning, consistent with the presence of a full complement of margin SOPs. (2) In the
adult phenotype, whereas m4/alpha produces
solely bristle tufting, H overexpression variably
produces naked patches or double-shaft socketless bristles, consistent
with its proposed role in the SOP lineage cell fate decisions.
Ectopic expression of m4/alpha gives neither of these phenotypes, suggesting that it affects only
SOP singularization but not the SOP lineage. In order to
test this hypothesis, pupal nota were stained with antibodies
directed against Elav, a neuron specific marker, and Pros,
specific to the sheath cell. There is a one-to-one correspondence between Elav positive and Pros positive cells. Therefore, overexpression of m4/alpha does not upset
the Notch/Numb mediated asymmetric divisions in the SOP
lineage. The only step in sensory organ
development that m4/alpha seem to affect is that of lateral inhibition, which restricts the number of SOPs produced per
proneural cluster (Apidianakis, 1999).
As it is likely that m4/alpha affect a pathway of cell-cell communication, it is important to determine whether they
do so by interfering with signal emission or with signal
reception. In the former case, their effect would be non
cell-autonomous. To test this, UAS-m4 was overexpressed in
clones of cells. Patches overexpressing m4 give
the expected phenotype of microchaeta tufting marked
with f, a marker carried by the Ubx transgene, whereas adjacent non-expressing f1 bristles are
always single. Moreover, f marked bristles at clone boundaries are usually multiplied, that is, they are not
'rescued' by their proximity to wild-type tissue. It cannot
be concluded that this autonomy holds down to the single cell
level: the epidermal cell phenotypes could not be scored. Still, it is tentatively concluded that the effect of m4 is
achieved through blocking of signal reception (Apidianakis, 1999).
How do m4/alpha act to negatively modulate lateral inhibition? This new family of proteins contains no known structural motifs that would point toward a possible function. To study
the level of functioning of the Notch pathway a
number of molecular markers have been used. One indicator of Notch
signaling is the expression of the E(spl) bHLH genes, a
subset of which are recognized by the monoclonal antibody
323. Wild type proneural clusters are
positive for mAb323 immunoreactivity with the exception
of single cells, which represent the committed SOPs. Unlike
severe Notch loss of function, which abolishes mAb323
immunoreactivity, overexpression
of m4/alpha causes a milder overall loss of staining with a
subset of cells within the proneural cluster displaying undetectable levels of mAB323. The negative cells are always
in contact, surrounded by E(spl) positive cells. Most likely,
these cells correspond to the clustered neurA101 positive
SOPs, since loss of E(spl)bHLH expression favors the SOP fate (Apidianakis, 1999).
Downregulation of the E(spl)bHLH protein levels by m4/alpha could be at the level of transcription or post-transcriptional. To test this, E(spl) derived reporter genes were used
in both the eye and in a tissue-culture assay. In the eye, m8-lacZ was used. This drives expression in a subset of
proneural clusters.
m4 was overexpressed using the omb-GAL4 driver, which
expresses in a broad central domain of the wing pouch, and
apmd544-GAL4, which expresses in the dorsal compartment
of the wing disc. In both cases a dramatic
reduction of m8-lacZ activity was observed within the m4 overexpression
domain. This occurs even in regions where no ectopic sensory organs are formed, such as the posterior wing
margin, suggesting that the loss of m8-lacZ staining is not simply a consequence of overcommitment of
SOPs. The effects observed with the 323 antibody are not
identical to those seen by X-gal staining, the latter displaying a spatially more uniform reduction in staining as a result
of m4 overexpression. The difference may be attributed to
the different sensitivity of the techniques used, or to the fact
that the two experiments assay the expression of different
genes [mAb323 does not recognize E(spl)m8]. Further
studies are needed to determine if m4/alpha affect the expression of different E(spl)bHLH genes differentially; the
conclusion at present is that overexpression of m4/alpha
decreases E(spl)bHLH protein levels, at least partly through
blocking transcription (Apidianakis, 1999).
There are a number of Notch pathway components that
m4/alpha could interact with to downregulate the expression of
E(spl)bHLH genes. In an exploratory experiment, m4 or malpha were over-expressed in genetic backgrounds heterozygous
for various Notch pathway mutations to detect possible
genetic interactions. Of the mutations tested,
five modify the phenotype: N264-40 and E(spl) b32.2
increase the number of supernumerary bristles, and N
Ax, Dp(1;2)N+ and H2
decrease it. The effects of N alleles agree with the
proposed negative regulation of Notch signaling by m4/alpha.
The further suppression of lateral inhibition by halving the
dose of E(spl) is also not surprising, given the data that
m4/alpha acts by downregulating the transcription of
E(spl)bHLH genes. The suppression of supernumerary bristles by a reduction in H can finally be accounted for by
increased transcriptional activation of the E(spl)bHLH
genes, since H normally blocks the activity of the Su(H)
transcriptional activator. Instances of
severe H LOF (H1/H2) or strong N GOF (NAxM1/Y) produce
nota that are essentially bald. In these backgrounds m4/alpha
overexpression appears unable to induce bristles or SOPs
(as revealed by anti-Asense staining) (Apidianakis, 1999).
Whereas the genes encoding m4 and malpha are located
within the E(spl) region at 96F, two other members, Brd and
a newly identifed EST transcript, reside in 71A. m4, malpha and
Brd, are normally expressed in proneural clusters from
which individual SOPs will arise, and m4/alpha (but not Brd)
are transcriptionally induced by Notch signaling. The expression pattern and Notch-dependence of the
fourth member is still unknown.
Loss of function of either Brd or m4 gives a wild-type
phenotype (Leviten, 1996 and Apidianakis, 1999), pointing to functional redundancy within this protein family.
GOF phenotypes of Brd and m4/alpha are also virtually identical: they produce supernumerary SOPs. The only known
difference to date is slight: m4/alpha seem unable to affect cell
fates in the sensory organ lineage, whereas strong Brd
gof
occasionally causes naked cuticle patches due to pIIa transformation to the pIIb fate.
Based on the similarity of both LOF and GOF phenotypes,
a model is suggested whereby all members of this
family have a similar mode of action, namely to antagonize
Notch. Their action (at least in the case
of m4 and malpha) can be accounted for by reduced expression
of E(spl)bHLH proteins, which are well known effectors of
Notch signaling and antagonists of the SOP fate. Despite functional
similarity, sequence similarity is not high between Brd and
the other members: most importantly, Brd lacks the well
conserved C-terminal domain present in all other members.
What might the role of this domain be? One possibility is
that it constitutes a regulatory domain whose function
becomes dispensable upon overexpression of the protein (Apidianakis, 1999).
The fact that m4/alpha are turned on by Notch signaling,
even though they act against the downstream implementation of the signal, might be counter-intuitive, yet is not
unprecedented. Other signaling pathways employ similar
mechanisms of negative autoregulation. For example, activation of the EGF receptor results in expression of argos,
which encodes an inhibitory ligand of Egfr. Another example is the Hh pathway, where
signaling upregulates expression of patched, coding for a transmembrane receptor of Hh, which inhibits signal transduction. A major difference, however, is that in
both examples, inhibition serves to spatially restrict the
effects of signaling, and, in the case of Argos, is not cell
autonomous. In contrast, m4/malpha cells act autonomously,
antagonizing the Notch signal within the same cell that
should be responding to it. In fact both signaling mediators
(E(spl)bHLH proteins) and antagonists (m4/alpha) are turned on
by Notch signaling, via the same mechanism, namely
Su(H)-dependent transcriptional activation. One possible
function of this apparently conficting co-expression is to
set a threshold for the level of Notch signaling needed to
divert a cell from the SOP fate. An alternative (and not
exclusive) possibility might be that these factors ensure
that the N effect is very transient. In this respect, it is
worth noting that all Notch-responsive genes within the
E(spl) locus, both the bHLH and m4/alpha, are short and intron-less, ensuring rapid accumulation of their products. Their
degradation is rapid, too: their transcripts contain special destabilizing signals, and the same
likely holds true for their protein products as well. These attributes make the
presence of these Notch responsive factors very transient
and dynamic, such that small differences in temporal accumulation might be hard to detect. For example, members of the m4/alpha family can be thought of as factors that are activated slightly
later than E(spl) bHLH to switch off a round of Notch
signaling, after the positive mediators [E(spl) bHLH
proteins] have accomplished their task. To address such a
function, detailed studies are needed, which will focus on
the precise temporal sequence of E(spl)bHLH versus m4/alpha
expression (Apidianakis, 1999).
What is more unexpected in these findings is the high specificity of m4/alpha for the process of SOP singularization and the
apparent indifference of other Notch mediated processes to
m4/alpha overexpression. Overexpression of Hairless or Numb,
two other well characterized Notch pathway inhibitors,
affects a much broader range of developmental processes,
e.g. cell fates in the SOP lineage, wing margin formation, and
wing pro-vein restriction. Hairless
may normally participate in all of these events. In contrast, Numb is specific for the asymmetric divisions in the
SOP lineage, but can mildly affect other processes when
ectopically expressed. Could the inability to detect phenotypes in other Notch-dependent processes
be simply due to low levels of transgene expression? This is believed not to be the case, since a large number of GAL4
drivers were utilized that otherwise give SOP phenotypes with high expressivity and penetrance. A possible
explanation for the refractoriness of other processes to m4/alpha expression is that these proteins are subject to post-translational regulation that masks their activity in most cell
types. One type of such regulation might be the association
with an essential co-factor, whose expression could be
restricted to the proneural cells. Yet, two-hybrid analysis fails to reveal such a co-factor among the various likely
candidates tested. The one lead regarding the molecular
mode of action of m4/alpha is the documented downregulation
of E(spl)bHLH gene expression. Since these are target genes of
Notch, such an effect could result from a block in any of the
steps involved in Notch signal transduction. The tissue
culture results suggest that m4/alpha can block E(spl)bHLH
genes even when activated Notch is exogenously provided,
suggesting that these factors act at a step after Notch activation. However, the effects observed in these experiments are
rather modest compared to the in vivo effects and thus it is uncertain that they reflect the same activity of the m4/alpha
molecules (Apidianakis, 1999).
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E(spl) region transcript m4:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 5 January 2001
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