Drosophila has four types of sensory elements: external sense organs, multiple dendritic neurons, chordotonal

neurons, and photoreceptors. The latter two do not require the proneural genes of the achaete-scute complex
(AS-C) in order to develop and function properly, but they do need atonal, a neurogenic gene that behaves in
many ways like the genes of AS-C. atonal gets its name from the disruptive effects the gene's mutation has on
chordotonal neuron differentiation. Mutants are completely difficient in this lateral sense organ (Jarman, 1995).
Early in embryonic development, atonal is expressed in all chordotonal organ progenitor cells, but its later
expression is restricted to only a particular set of precursors, through a process of lateral inhibition. Notch does
not seem to be required for this process in chordotonal organs, and the mechanism is not well understood.
Mutation in atonal also disrupts eye development (Jarman, 1995).
Atonal protein is produced in all cells just anterior to the morphogenic furrow. Expression is downstream of
hedgehog, an important gene involved in regulating the progression of the furrow. As the furrow progresses,
most cells that express atonal in front of the furrow lose that capacity. atonal expression behind the furrow is
confined to the R8 progenitors, whose fate atonal determines. atonal is produced within the context of the
furrow, which is only rudimentary in atonal mutants. atonal is a neurogenic gene, functioning in the place of
achaete-scute complex bHLH genes, both in the eye and in chordotonal organs.
The function of Atonal is best illustrated by its role in chordotonal organ development. A scolopidium, the basic
unit of chordotonal organs, consists of four cells: a neuron with a single dendrite, the scolopale cell, cap cell and
ligament cell. The scolopale cell (a glial cell) forms a sheath around the dendrite, while the cap cell and ligament
cell mediate the attachment of the chordotonal organ to the body cell. Expression of atonal is restricted to a
subset of atonal-requiring chordotonal precursors, called founder precursors. In atonal mutants, all chordotonal
organs are absent except for one scolopidium of Ich5, the abdominal pentascolopidial organ (Ich5 consists of
five scolopidia organized in a linear array). This one scolopidium formed in atonal mutants is atonal
independent. The atonal independent precursor corresponds to the earliest chordotonal precursor (precursor C1)
and corresponds to the P cell which gives rise to the anterior-most scolopidium of Ich5 (zur Lage, 1997).
EGF receptor signaling is required in neural recruitment during formation of Drosophila chordotonal sense
organ clusters. A total of five neural precursors express atonal in abdominal segments, and this number is too
few to explain the formation of the eight scolopidia in each abdominal segment. Of the five precursors, C-1, C2
and C3 contribute to Ich5, C4 migrates slightly anterodorsally and gives rise to v'ch1, the dorsal most
scolopidium, which is solitary. C5 contributes to vchAB, a more ventral pair of scolopidia. The remaining
precursors require Egf-R signaling for their selection. Signaling by the founder precursors is initiated by atonal
activating (directly or indirectly) rhomboid expression in the founder cells. It should be noted that in some
developmental processes, rhomboid appears to function in the signal-receiving cells, such as in the patterning of
ovarian follicle cells. It is not believed that this is the case in chordotonal-precursor formation, because rho is
expressed in precursors that do not require rhomboid function (C1-C5 are formed even in rhomboid mutants).
Signaling by these founder precursors, presumably through the EGF receptor ligand Spitz, then provokes a
response in the surrounding ectodermal cells, as shown by the activation of expression of the Egf-R target genes
pointed and argos. The signal and response then leads to recruitment of some of the ectodermal cells to the
chordotonal precursor cell fate. Egf-R hyperactivation by misexpression of rhomboid results in excessive
chordotonal precursor recruitment. Argos functions in a feedback mechanism to prevent the excess recruitment
of additional ectodermal cells. The increase in the number of scolopidia caused by Egf-R hyperactivation is
confined to an enlargement of existing cluster sizes: no new chordotonal clusters are formed. A two step
mechanism is postulated for the formation of clusters of chordotonal precursors. In the first step, precursors C1C5 are selected as founder precursors by the conventional route of proneural gene expression and lateral
inhibition. In a distinct second phase, these precursors then signal to adjacent ectodermal cells via the Egf-r
pathway, inducing some of them to become chordotonal precursors (secondary or recruited precursors). This
two-step process is strongly reminiscent of the way atonal acts in neurogenesis in compound eyes. Here, atonal
expression is initially refined by lateral inhibition, until atonal is expressed in only the founding R8 precursor,

which then recruits R1-R7 in a mechanism that does not require the activation of atonal in these cells (zur Lage,
1997).
The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each
neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and
neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike
sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that
for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from
the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by
a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR
acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral
inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus
maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes
from previously recruited SOPs (zur Lage, 1999).
The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis
of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region
of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a
sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is
persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC,
cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes
visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the
stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by
upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein
and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only
apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore
referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the
stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not
have a counterpart in external sense organ precursor formation, although there is some evidence for multiple
steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato
remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since
AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately
6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs,
although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between
Ato and Ase or A101 (zur Lage, 1999).
The process of chordotonal SOP formation described above is at odds in several respects with the well-known
paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers
expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of
AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells
from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or
not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of
committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway
are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that
they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these
cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout
the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory
signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N
signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some
mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage,
1999).

The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the
clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in
development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal
precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation
in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming
the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing
a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal
SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line).
This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In
contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on
SOPs in general (zur Lage, 1999).
To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that
determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an
effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho
and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases
chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or
by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current
investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed
reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced
extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment
upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato
expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP
commitment (zur Lage, 1999).
Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time
that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so
far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the
spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key
components of the pathway were characterized. Localized expression of rho appears to play a central role in
spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that
are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very
restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to
the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody
that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg
imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs.
Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dpERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above
the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they
funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling
from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this
process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells.
That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural
competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling
are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells,
which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals
through the Spitz ligand (zur Lage, 1999).
Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also
relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C
PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and
resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid

shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene
expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the
femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating
lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but
instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing
general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of
Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves.
The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of
dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct
and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999).

The neurogenetic role of lethal of scute resembles that of the other three proneural
gene members in the achaete-scute complex (achaete, scute and asense). This
overview will examine instead the role of lethal of scute in the specification of muscle
progenitors.
Muscle development takes place in two phases. First, the pattern of muscle
development is laid down by allocation of founder cells, specific cells in the
mesoderm, each one serving as a founder for a unique muscle. Second, founder cells
recruit neighboring myoblasts to form the syncylial precursors of mature muscle by
fusion. Initially l'sc expression is widespread in cell clusters, but becomes allocated to
muscle founder cells through the action of the Notch pathway.
The expression of lethal of scute in mesoderm is transient, occuring in twist
expressing cells. From late stage 9 until stage 12, there are at least 19 clusters of l'sc
expressing cells in each hemisegment. In each cluster, one cell accumulates higher
levels of l'sc than the other cells in the cluster. It is this single cell, allocated from a
cluster of cells, that moves to a position close to the ectoderm and eventually becomes
a muscle founder cell.
Genes coding for three transcription factors, (nautilus, S59 and msh1) are each
expressed in small groups of cells destined to differentiate into muscle cells. Another
transcription factor, MEF2, is required for myosin expression and the fusion of
myoblasts. None of these are selector genes initiating muscle fate. Genes with a
decisive role in myogenesis, similar to members of the MYO-D family in vertebrates,
have not been found in Drosophila.
In neurogenic mutants, the domains of mesodermal S59 expression are expanded. This
suggests the Notch pathway is involved in restricting the expression of l'sc and S59 to
single cells, the same way it functions in neuroblast differentiation. l'sc is not the only
factor involved in founder cell specification. In some instances no l'sc is found in the
founder cell, and l'sc mutation does not completely upset muscle specification.
Founder cell specification is easily comparable to neuroblast specification. The role of
l'sc in muscle specification is analagous to the role of achaete-scute genes in neural
growth. Specification of muscle founder cells is one of the many different processes
involving the Notch pathway, a significant proportion of which involve proneural
genes (Carmena, 1994).

GENE STRUCTURE
cDNA clone length - 1067
Bases in 5' UTR - 46
Exons - one
Bases in 3' UTR - 268
PROTEIN STRUCTURE
Amino Acids - 257
Structural Domains
Lethal of scute has a basic helix-loop-helix domain, and an C-terminal acidic domain
(Cabrera, 1988 and Martin-Bermudo, 1993). A PEST domain is located just Cterminal of the bHLH domain. Deletion of the N terminal amino acids up to the basic
domain, or deletion of the C terminal domain including the PEST domain, still leaves
a functional protein (Hinz, 1994).
Evolutionary Homologies
The lin-32 gene of C. elegans codes for an Achaete-Scute homolog, sufficient for
specification of neuroblast fate (Zhao, 1995). Chicken Achaete-Scute homolog
(CASH-1) is one element in a multiple parallel pathway involving notochord or floor
plate-derived signals for the specification and development of chick sympathetic
neurons (Groves, 1995). A Xenopus Achaete-Scute homolog, XASH-3, when
dimerized with the promiscuous binding partner XE12, specifically activates the
expression of neural genes in naive ectoderm (Ferreiro, 1994). Xenopus AchaeteScute homologs XASH-1a and XASH-1b appear in defined regions of the developing
central nervous system. The pattern of expression of the Xenopus genes is modified
by the cyclops mutant (Allende, 1994).
The study of achaete-scute (ac/sc) genes is a paradigm to understand the evolution
and development of the arthropod nervous system. The ac/sc genes have been
identified in the coleopteran insect species Tribolium castaneum. Two Tribolium ac/sc
genes have been identified -- 1) a proneural achaete-scute homolog (Tc-ASH) and 2)
asense (Tc-ase), a neural precursor gene that reside in a gene complex. These genes
reside 55 kb apart from each other and thus define the Tribolium ac/sc complex.
Focusing on the embryonic central nervous system it is found that Tc ASH is
expressed in all neural precursors and the proneural clusters from which they
segregate. Through RNAi and misexpression studies it has been shown that Tc-ASH is
necessary for neural precursor formation in Tribolium and sufficient for neural
precursor formation in Drosophila. Comparison of the function of the Drosophila and
Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes
have maintained their ancestral function in neural precursor formation and have
acquired a new role in the fate specification of individual neural precursors. These
studies, however, do not support a role for Tc-ASH in specifying the individual fate of
neural precursors, suggesting that the ability of ac and sc to separately regulate this

process may represent a recent evolutionary specialization within the Diptera.

Furthermore, it is found that Tc-ase is expressed in all neural precursors, suggesting
an important and conserved role for asense genes in insect nervous system
development. This analysis of the Tribolium ac/sc genes indicates significant
plasticity in gene number, expression and function, and implicates these modifications
in the evolution of arthropod neural development (Wheeler, 2003).
The work presented in this paper together with studies on ac/sc gene function in
Drosophila provide strong evidence that serial duplications of proneural ac/sc genes
in the dipteran lineage led to the diversification of proneural ac/sc gene function in
Drosophila. In Drosophila, ac and sc carry out functions distinct from l'sc in
specifying the individual fate of the MP2 precursor. Tc-ASH can function in
Drosophila as a proneural gene but like Drosophila l'sc fails to specify efficiently the
MP2 fate in the CNS. Together these results suggest the ability of ac and sc to specify
MP2 fate in Drosophila arose after the divergence of Drosophila and Tribolium. These
data provide an example whereby a subset of duplicated genes has evolved a new
genetic function while the entire set of duplicate genes has retained the ancestral
function (Wheeler, 2003).
In addition to functional changes, the generation of multiple proneural ac/sc genes in
the insects was paralleled by modifications to the expression profiles of these genes.
In Anopheles (a basal dipteran), and Tribolium a single proneural ac/sc gene is
expressed in all CNS proneural clusters. In more derived Diptera the presence of
multiple ac/sc genes allows for more complex proneural ac/sc gene expression
patterns. For example, Ceratitis contains two proneural ac/sc genes, l'sc and sc; l'sc is
expressed in all CNS proneural clusters while sc is expressed in a subset of these
clusters. In Drosophila, ac and sc are expressed in the identical pattern of proneural
clusters and their expression is largely complementary to that of l'sc. The sum of
proneural ac/sc expression in each species then marks all CNS proneural clusters
despite differences in the expression pattern of individual proneural ac/sc genes. Thus,
in Drosophila, the complete expression pattern of proneural ac/sc genes is divided
between the largely complementary expression profiles of ac and sc relative to l'sc.
The division of labor between proneural ac/sc genes in Drosophila has resulted in
mutually exclusive expression patterns for ac and sc relative to l'sc in proneural
clusters like MP2. This spatial separation of proneural gene expression probably
facilitated the potential for ac and sc to acquire developmental functions distinct from
l'sc (Wheeler, 2003).
Together this work and that of others on arthropod ac/sc genes highlights the utility of
studying ac/sc genes in elucidating the genetic basis of the development and evolution
of arthropod nervous system pattern. These studies illustrate the dynamic nature of
ac/sc gene number, expression and function over a relatively short evolutionary time.
Based on this, future work on ac/sc genes in additional arthropod species should
continue to provide insight into the molecular basis of the evolution of arthropod
nervous system development (Wheeler, 2003).
REGULATION
Promoter Structure

Thirty nucleotides upstream from the start of transcription is an unconventional TATA

box (TATTTAAA). Seven CANNTG motifs (E-boxes), putitive binding sites for
bHLH transcription factors, are located in this upstream region 200 to 1000 bp above
the start site. These local upstream elements could function in l'sc autoregulation or
activation by Achaete or Scute (Martin-Bermudo, 1993).
Transcriptional Regulation
Snail represses l'sc transcription in the presumptive embryonic mesoderm (Kosman,
1991). Elements regulating l'sc are scattered throughout 75 kb between achaete and
asense. These elements activate l'sc in specific proneural clusters and as a
consequence, also in their corresponding neuroblasts (Martin-Bermudo, 1993).
Short gastrulation prevents Decapentaplegic from suppressing neurogenesis laterally
in the blastoderm embryo. It is possible to exacerbate defects in sog mutants by
increasing the level of DPP. The earliest neuroectodermal marker affected in sog
mutants with a double dose of dpp is rhomboid, which is normally expressed in lateral
stripes 8-10 cells wide in wild-type embryos but rapidly narrows to stripes 4-6 cells
across in sog mutants with elevated DPP. Similarly l'sc expression is reduced in sog
mutants with elevated DPP. A striking feature of the affects of DPP on neural
suppression and dorsalization is that neuronal suppression is induced by a lower
threshold of DPP activity than is dorsalization. Much less DPP is required to suppress
expression of neuroectodermal genes than is required to activate dorsal markers. For
example, brief submaximal heat induction of heat shock dpp in a wild type sog
background leads to nearly maximal suppression of lethal of scute, scratch and snail
expression during germ band extension, but there is no detectable ectopic expression
of zerknllt in the neuroectoderm (Biehs, 1996).
The segmented portion of the Drosophila embryonic central nervous system develops
from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem
cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after
gastrulation in two sequential waves of neuroblast segregation and are arranged in
three dorsoventral columns and four anteroposterior rows per hemisegment. Each
neuroblast acquires a unique identity, based on gene expression and the unique and
nearly invariant cell lineage that this expression produces. Little is known as to the
control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr)
has been shown to promote the formation, patterning and individual fate specification
of early forming neuroblasts along the DV axis. Molecular markers identify particular
neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and
show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form;
(2) medial column neuroblasts often acquire identities inappropriate for their position,
while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the
regions from which the medial and intermediate neuroblasts will later delaminate. The
concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from
Egfr mutant embryos, even though loss of either gene alone yields minor CNS
phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast
formation, patterning and specification along the DV axis within the developing
Drosophila embryonic CNS (Skeath, 1998).

In a screen to identify mutations that disrupt embryonic CNS development, one P

element mutation, l(2)03033, was identified that causes a loss of essentially all Evepositive RP2/RP2 sib neurons. This P element maps to cytological position 57F1-2 in
the right arm of the second chromosome and is known to be inserted within the Egfr
locus. To verify that lesions in Egfr result in a nearly complete loss of RP2
motoneurons, three additional Egfr mutants were obtained, including the Egfr null
allele, flb 1K35Egfr allele (Skeath, 1998).
The first phase of CNS development, as gastrulation commences, involves the
activation of the Ac-S proneural genes in a precise pattern of proneural clusters. To
investigate whether Egfr regulates As-C expression in the neuroectoderm, the
expression patterns of the achaete (ac) and lethal of scute (lsc) genes were followed
in Egfr mutant embryos. Loss of Egfr causes specific defects to the DV registration of
ac and lsc gene expression in the neuroectoderm; however, no defects to the AP
registration for either ac or lsc gene expression were found. In wild-type embryos
during stages 8/9, ac is expressed in the medial and lateral, but not intermediate,
clusters of rows 3 and 7; lsc is expressed in the medial and lateral, but not
intermediate, clusters of row 7 and in the medial, intermediate and lateral clusters of
rows 1 and 5. A single neuroblast subsequently forms from each proneural cluster. In
Egfr mutant embryos, ac gene expression expands into the intermediate column in
rows 3 and 7 and lsc expression expands into the intermediate column in row 7; lsc is
expressed normally in rows 1 and 5. The lateral limits of ac and lsc gene expression
in the neuroectoderm are unaltered in Egfr mutant embryos. The changes to the DV
registration of ac and lsc gene expression in Egfr mutant embryos suggest that
neuroectodermal cells in the intermediate column change their fate. Both ac and lsc
are normally expressed in the medial and lateral columns in the affected rows, thus the
phenotype is consistent with intermediate cells acquiring either a lateral or a medial
fate. msh-1, which is expressed exclusively in the lateral column, expands into the
intermediate column in Egfr mutant embryos. In this context, it appears that ac and
lsc expression expand from the lateral column into the intermediate column in the
absence of Egfr (Skeath, 1998).
The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm,
neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each
tissue is subsequently subdivided into multiple cell types during gastrulation. This
study investigates the formation of the mesectoderm within the ventral-most region of
the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in
concert with Notch signaling to specify the mesectoderm through the activation of the
regulatory gene sim within single lines of cells that straddle the presumptive
mesoderm. This model was confirmed by misexpressing a constitutively activated
form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2
enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic
ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the
ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor,
which is selectively expressed in the ventral mesoderm. Additional studies suggest
that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene
appears to induce Notch signaling in 'nave' embryos that contain low uniform levels
of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch
target genes and stimulation of Notch signaling -- help define precise lines of sim
expression within the neurogenic ectoderm. It is proposed that Snail functions as a

gradient repressor to restrict Notch signaling. In precellular embryos, the initial snail
expression pattern is broad and extends into the future mesectoderm. During
cellularization, the pattern is refined and snail expression is lost in the mesectoderm
and restricted to the mesoderm. The early, broad snail pattern might create a broad
domain of potential Notch signaling by repressing components of the Notch pathway,
such as Delta and lethal of scute. After cellularization, Notch signaling is blocked in
the presumptive mesoderm by sustained, high levels of the Snail repressor. However,
Notch can be activated in the mesectoderm because of the loss of Notch inhibitors
repressed by transient expression of the Snail repressor. According to this model, the
dynamic snail expression pattern determines both the timing and limits of Notch
signaling (Cowden, 2002).
Targets of Activity
L'SC activates Enhancer of split and HLH-M5 of the Enhancer of split complex, and
possibly dorsal as well (Hinz, 1994).
Protein Interactions
Extramachrochaete forms inactivating heterodimers with L'SC as it does with AC and
SC (Cabrera, 1994). Daughterless is required as a dimerization partner for L'SC
function (Hinz, 1994).
Classical genetics indicates that the achaete-scute gene complex (AS-C) of
Drosophila promotes development of neural progenitor cells. To further analyze the
function of proneural genes, the effects of Gal4-mediated expression of lethal of
scute, a member of the AS-C, were studied during embryogenesis. Expression of
lethal of scute forces progenitor cells of larval internal sensory organs to take on
features of external sensory organs. Normally, these cells are committed to this fate
independent of AS-C activity. Surprisingly, overexpression of l'sc does not result in
supernumerary neural cells. Supernumerary neural cells can be induced ectopically
only if daughterless is overexpressed, either alone or together with lethal of scute:
cells of the amnioserosa and the hindgut then express neuronal markers. Cells of the
proctodeal anlage, which normally lack neural competence, acquire the ability to
develop as neuroblasts following transplantation into the neuroectoderm. Activated
Notch prevents the cells of the neuroectoderm from forming extra neural tissue when
they express an excess of proneural proteins. Under the present conditions, lateral
inhibition is thus dominant over the activity of proneural genes (Giebel, 1997).
DEVELOPMENTAL BIOLOGY
Embryonic
See the embryonic expression pattern of l(sc) at the Berkeley Drosophila Genome
Project Patterns of Gene Expression Site.
Like achaete and scute, l'sc is expressed in very specific subsets of cells in
neuroblasts of the ventral neurectoderm (Martin-Bermudo, 1993). Gene expression in
the ventral neuroectoderm is discussed at the achaete-scute complex site.

As with achaete and scute, l'sc is expressed in every neurogenic region of the fly,
including the cephalic and gnathal regions. After stage 9, l'sc is expressed in the
mesectodermal, central and peripheral nervous system anlage, as well as the
stomatogastric nervous system and the optic lobes (Cabrera, 1990).
In head midline structures, in particular the optic lobe and stomatogastric nervous
system, there may be a late phase of EGFR signaling (as assayed by the expression of
aos and activated ERK) whose significance is not yet known. EGFR signaling could
be involved in modifying the inhibitory feed-back loop between neurogenic and
proneural genes that exists in other neurectoderm cells. In the head midline
neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus,
instead of a short burst of proneural gene expression in proneural clusters that is
resolved into expression in individual neuroblasts, proneural genes are expressed for a
long period of time; at the same time, the expression is never restricted to single
neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that
express lsc, the inhibitory loop between E(spl)-C and proneural genes must be
interrupted at some level. It is possible that Egfr signaling is causing the interruption
of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the
compound eye, it has been speculated that one of the consequences of Egfr activation
(which ultimately is required for all ommatidial cell types to differentiate) is to inhibit
N signaling, since constitutively active N inhibits ommatidial cell differentiation by
preventing response to differentiative signals. However, the same effect could be
achieved if Egfr signaling, similar to what is proposed here for the midline
neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this
would not prevent N signaling, it would cancel the effect of N signaling on
downregulating proneural genes and thereby keep cells in a state of competency to
respond to signals (Dumstrei, 1998).
The expression of the proneural gene lethal of scute is required for the development of
the majority of the procephalic neuroblasts. lethal of scute expression patterns
correspond to many of the identifiable 23 groups of neuroblasts in the developing
brain. l'sc expression in the procephalic neurectoderm is controlled in partially
overlapping domains of the neuroectoderm. Loss of function of a given head gap gene
results in the absence of l'sc expression in its domain, followed by the absence of
neuroblasts that would normally segregate from this domain (Younossi-Hartenstein,
1997).
Neuroblasts delaminate from the procephalic neurectoderm in a stereotyped
spatiotemporal pattern that is tightly correlated with the expression of l'sc. The pattern
of neuroblasts was reconstructed by using the marker asense; similar to its expression
in the ventral neuroblasts, asense labels all brain neuroblasts. seven-up, expressed in
specific subsets of neuroblasts making up approximately one-third of the total, is also
used as a marker. For most, if not all, of these clusters the number of neuroblasts and
the time of onset of svp expression are absolutely invariant (Younossi-Hartenstein,
1996).
Effects of Mutation or Deletion

lethal of scute mutants may reach adulthood (Martin-Bermudo, 1993). This attests to
the many redundancies or biological fail safe mechanisms created by the duplication
of function in proneural genes.
klumpfuss shows genetic interactions with achaete, scute, lethal of scute and asense.
l'sc is able to activate klu expression, but apparently only in the wing disc. There
appears to be only a weak influence of the AS-C genes on klu expression, restricted to
the wing area of the wing disc. However, the overall expression pattern of klu is
largely independent of proneural genes. The assumption that SOPs enter apoptosis in
klu mutants is supported by the observation of abundant cell death in other developing
organs of klu mutants, like the legs. At certain bristle positions, such as that of the
anterior sternopleura, klu is required during early bristle development immediately
after proneural gene function, in order to allow a particular epidermal cell to develop
as a SOP. It is suggested that klu is required only for initiation of bristle development,
being downregulated once specification takes place (Klein, 1997).
During Drosophila embryogenesis, mesodermal cells are recruited to form a
stereotyped pattern of about 30 different larval muscles per hemisegment. The
formation of this pattern is initiated by the specification of a special class of
myoblasts, called founder cells, that are uniquely able to fuse with neighbouring
myoblasts. The COE transcription factor Collier plays a role in the formation of a
single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic
segments T2 and T3). Col expression is first observed in two promuscular clusters (in
segments A1-A7), corresponding to two progenitors and then their progeny founder
cells, but its transcription is maintained in only one of these four founder cells, the
founder of muscle DA3[A]. It is proposed that specification of the DA3[A] muscle
lineage requires both Col and at least one other transcription factor, supporting the
hypothesis of a combinatorial code of muscle-specific gene regulation controlling the
formation and diversification of individual somatic muscles (Crozatier, 1999).
Following establishment of the promuscular clusters, specification of the progenitors
is controlled by lateral inhibition, a cell-cell interaction process mediated by the
neurogenic genes Notch (N) and Delta (Dl)). In both N and Dl mutant embryos,
promuscular Col expression is initiated normally but fails to become restricted to a
single cell per cluster, similar to observations previously made for the expression of
lsc. As a consequence, a hyperplasic expression of Col is observed from stage 11.
Since it is expressed in promuscular clusters and segregating muscle progenitors, lsc
has been proposed to play a role in muscle progenitor selection similar to the role of
achaete and scute in neuroblast specification. However, in embryos lacking lsc
activity, selection of the Col-expressing progenitors occurs normally at stage 11 and
muscle DA3[A] forms as in wild type (Crozatier, 1999).
In the embryonic ventral neuroectoderm of Drosophila the proneural genes achaete,
scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts
delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm
before and during delamination of the first two populations of neuroblasts show that
cells in all regions of proneural gene activity change their form prior to delamination.
Furthermore, the form changes in the neuroectodermal cells of embryos lacking the
achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of

embryos overexpressing l'sc, suggest that these genes are responsible for most of the
morphological alterations observed (Stollewerk, 2000).
Almost all neuroectodermal cells are larger than the cells of the dorsal epidermal
anlage (DEA). In comparison with the cells of the DEA in early stage 8 embryos the
dorsoectodermal cells of mid-stage 8 embryos are clearly smaller. A comparison of
the neurogenic region in early and mid-stage 8 embryos shows that the medial and
intermediate regions of the ventral neuroectoderm (VNE) do not increase further in
size whereas the lateral region enlarges considerably during this time. Due to these
morphological changes the VNE can now be subdivided in relation to the cell sizes
into three longitudinal regions on both sides of the midline: medial, intermediate, and
lateral regions. In contrast to the cells of the medial and lateral regions, which now
have approximately the same average values, the intermediate cells are smaller. Only
20% of all cells in the intermediate region are larger than the average, whereas 63% of
the medial and 64% of the lateral cells exceed the average value. Most of the enlarged
cells have a cuboidal shape. In every hemisegment the apical surfaces of two to four
cells in the medial and lateral regions are very small (12-16 m2) but expand basally
to cover an area of 65-80 m2. One or two cells of this shape are also located in the
intermediate regions but are smaller basally (48-58 m2) than the medial and lateral
cells. The number and position of these cells suggest that they correspond to the
delaminating neuroblasts; this was confirmed by staining the embryos with antiHunchback antibody, an early marker for neuroblasts (Stollewerk, 2000).
During delamination of the SI neuroblasts the neuroectodermal cells gradually
decrease in size, with the exception of a few cells located close to the midline. The
cells that remain enlarged are either elongated perpendicularly to the midline or have
a rounded appearance. Basally, between neuroectoderm and mesoderm, large round
cells are located that lose contact with the apical surface at about 60% EL. On the
basis of their position and arrangement, as well as the analysis of embryos stained for
Hunchback, these cells can be identified as the SI neuroblasts. Before delamination of
the SII neuroblasts, cells in the intermediate region of the neuroectoderm increase in
size. Most of the SII neuroblasts delaminate from this region, whereas only a few
neuroblasts arise from the medial region, where enlarged cells can also be detected.
After delamination of the SII neuroblasts the enlarged cells shrink once again, as
revealed by double staining with anti-Hunchback antibody and phalloidin. Cells in all
regions of the VNE increase in size again prior to delamination of the SIII
neuroblasts. Thus, the VNE of wild-type embryos becomes morphologically
distinguishable from the DEA shortly before delamination of the SI neuroblasts. At
this point the cells of the DEA have already divided, and about two-thirds of all cells
in the medial and the lateral regions have become enlarged so that the DEA and the
VNE are clearly distinguishable due to differences in cell size. In addition, almost all
cells of the intermediate region increase in size prior to delamination of the SII
neuroblasts. These data are at odds with claims that only the neuroblasts enlarge prior
to delamination, both in grasshopper and Drosophila (Stollewerk, 2000).
Is there a correlation between the activity of the ASC genes and the observed
morphological changes? The results presented indicate that the ASC genes are not the
only ones responsible for the morphological changes that occur before delamination
of the SI neuroblasts. Although the number of enlarged cells corresponds closely to
the number of cells that express the ASC genes at this time point, the lack of the ASC

does not result in all cells remaining the same size. Whereas in the medial region of
the VNE of Df (1)260-1 embryos (that is, those lacking the ASC) only about 50% of
the cells are smaller in size than in the wild type, and the lateral region is most
strongly affected in comparison to the medial and intermediate regions. Therefore the
enlargement of the neuroectodermal cells depends to a varying degree on the activity
of the ASC genes and is additionally influenced by other factors. However, a clear
correlation can be seen prior to delamination of the SII neuroblasts. At this time
almost all cells of the intermediate region increase in size, which coincides with the
expression of l'sc in this region. Furthermore, analysis of the VNE of embryos lacking
the ASC reveals that the intermediate cells do not become enlarged prior to
delamination of the SII neuroblasts, suggesting that the observed morphological
changes are due to the activity of the ASC genes at this point. In addition, the
shrinkage of the cells that had enlarged during delamination of the SI and SII
neuroblasts is correlated with the decrease in ASC gene expression in the VNE at
these time points (Stollewerk, 2000).
Analysis of wild-type and Delta mutant embryos also suggests that the ASC genes are
important for the maintenance of the morphology of the neuroectodermal cells.
Despite the fact that the total area of the intermediate region does not change
significantly between early and mid-stage 8, cell size changes can be detected in this
region shortly before delamination of the SI neuroblasts. While 20% of the
intermediate cells remain larger than the average, the cells that had an average cell
size in the VNE of early stage 8 embryos now split into groups of smaller cells. The
fact that the number of cells that remain larger than the average corresponds to the
number of cells that express the ASC genes in the intermediate region suggests that
the proneural genes are required to keep these cells enlarged. This view is confirmed
by analyses of the VNE of Delta mutant embryos. In Delta mutant embryos all cells
of a proneural cluster continue to express the proneural genes and become
neuroblasts. This altered gene expression causes all cells of a proneural cluster to
remain enlarged until proneural gene expression is turned off (Stollewerk, 2000).
A correlation between increase in cell size and ASC gene expression has also been
shown by the analysis of embryos labeled for ac protein and embryos overexpressing
l'sc. Area measurements reveal that 85% of all cells that express ac are enlarged in
these embryos. The fact that not all ac-expressing cells are larger than the average at
the time point analyzed may be due to the rapidity of the morphological changes
(enlargement and shrinkage) that occur immediately before and during delamination
of the neuroblasts. A clear influence of a proneural gene on the cell sizes in the VNE
can be seen in embryos overexpressing l'sc: 45% more cells become enlarged in the
intermediate region in comparison to the wild type. Only a minor increase in the
numbers of enlarged cells can be seen in the medial and lateral regions, because twothirds of these cells already express proneural genes. In addition, the high proneural
gene activity in the VNE of embryos overexpressing l'sc causes the future neuroblasts
to change their morphologies: they expand not only their basal but also their apical
surfaces. These data clearly show that the ASC genes have an influence on the
morphologies of the neuroectodermal cells (Stollewerk, 2000).