scute
The highly complex pattern of proneural clusters in imaginal discs is constructed piecemeal, by the action on achaete and scute of
site-specific, enhancer-like elements distributed along most of the AS-C (approximately 90 kb).
Fragments of AS-C DNA containing these enhancers drive reporter genes in only one or a
few proneural clusters. This expression is independent of the ac and sc endogenous genes, indicating that the enhancers respond to local combinations of factors (prepattern). The cross-activation between ac and sc has been thought to explain the almost identical patterns of ac and sc expression (Martinez, 1991), but cross-activation does not occur detectably between the endogenous ac and sc genes in most proneural clusters. Coexpression is accomplished by activation
of both ac and sc by the same set of position-specific enhancers (Gomez-Skarmeta, 1995).
The genes araucan and caupolican code for two divergent homeodomain proteins that regulate transcription from the position-specific enhancers of ac-sc. Expression in the wing imaginal disc starts during the second larval instar at the presumptive notum region and is increased in two large areas of the presumptive lateral heminotum. From the mid-third instar, expression occurs at the presumptive distal tegula, the dorsal radium, proximal vein L1, veins L3 and L5, the allula, and the pleura. This distribution suggests that araucan and caupolican in fact establish the prepattern for proneural clusters in the wing, and interact with the position-specific enhancers to regulate ac-sc in the precise pattern displayed by these proneural clusters. Expression of ac-sc at the presumptive vein L3 depends on enhancer sequences located 0.2-0.6 kb upstream of the scute transcriptional start. This enhancer also drives expression at the twin sensilla of the wing margin (TSM) proneural cluster, located on the proximal vein L1, a site of ara/caup expression. These sequences contain binding sites for IROC proteins. Two contiguous short stretches of DNA are revealed in a DNase1 protection assay. One of these contains the TAAT motif found in the consensus binding sites of many homeoproteins. A mutagenized enhancer fails to be expressed in both vein L3 and TSM territories (Gomez-Skarmeta, 1996).
scute's regulation, with the exception of its of in activation Sex-lethal, is similar to that of achaete. Zygotic scute expression begins earlier than the expression of achaete.
Complex patterns of ac and sc expression are
constructed by separable cis-controlling elements present within a large (ca. 90-kb) region. The yellow gene located 10 kb from
ac has completely different expression patterns and is activated by different enhancers. Therefore, these genes may serve as
a good model system for the analysis of proper enhancer-promoter recognition. Autologous recognition between genes and their respective promoters may depend on the existence of an
interdomain boundary between AS-C and the yellow locus, or it may be determined by the specificity of the proteins assembled on a
certain enhancer and promoter. An inversion is described that puts the yellow gene between the ac and sc genes and almost all of their cis-regulatory elements. This
inversion shows only weak interference with the expression of the ac and sc genes. When the suppressor of Hairy wing-binding region of the su(Hw) insulator is deleted or inactivated by the
su(Hw) mutation, the sc phenotype of the flies is practically indistinguishable from that of the wild type. The presence of the yellow gene between the AS-C
enhancers and the promoters of the ac and sc genes does not interfere with ac and sc expression in most areas. This work shows, however, that it is not required that the su(Hw) insulator separate promoters from enhancers to allow inhibition of transcription by
the su(Hw) protein. The presence of the su(Hw) insulator, located more than 20 kb away from the inversion, facilitates strong
suppression of achaete and scute gene expression, although is does not separate the promoters from the AS-C enhancers (Golovnin, 1999).
The mechanism of direct interaction between AS-C enhancers and su(Hw) insulator is not yet clear. One possibility is that the pairing between the P elements
located at the breakpoints of the inversion facilitates such interaction. However, deletions of the P elements on both sides of the inversion fail to influence the repression mediated by the su(Hw) insulator. Another possibility is that the inversion brings the su(Hw)-binding region into a
close contact with the AS-C cis-regulatory elements due to changes in chromatin folding, which then leads to new long-range contacts between certain chromatin
regions. As a result, the su(Hw)-mod(mdg4) complex formed on the su(Hw) insulator becomes capable of interacting directly with enhancer-bound transcription
activators or with proteins responsible for enhancer-promoter interactions. The fact that the inactivation of AS-C control elements by the su(Hw)-binding region in
the inversion is only partial may be explained by reversible interactions between the insulator and enhancers similar to the normal dynamic interactions observed
between enhancers and promoters (Golovnin, 1999).
The best characterized chromatin insulator in Drosophila is the Suppressor of Hairy wing binding region contained within the gypsy retrotransposon. Although cellular functions have been suggested, no role has been found yet for the multitude of endogenous Suppressor of Hairy wing binding sites. Two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex are shown to form a functional insulator. Genetic analysis shows that at least two proteins, Suppressor of Hairy wing and Modifier of MDG4, required for the activity of this insulator, are involved in the transcriptional regulation of Achaete-scute (Golovnin, 2003).
To explain how the long-range activation potential of eukaryotic enhancers are restricted to the relevant target promoter, it has been proposed that eukaryotic chromatin is organized into functionally independent domains that prevent illegitimate enhancer-promoter communication. Recent publications suggest a model in which distant chromosomal binding sites of Su(Hw) are brought together by Mod(mdg4) into a small number of insulator bodies located at the nuclear periphery. In this way Su(Hw) marks the base of topologically independent looped chromatin domains. However, despite the presence of many endogenous Su(Hw) binding sites in polytene chromosomes, no specific function has been attributed to any site in a particular gene (Golovnin, 2003).
Using in vivo and in vitro assays, it has been shown that there exists a functional Su(Hw) insulator between a P-element inserted yellow gene and AS-C. At least four Su(Hw) binding sites have been shown to be required for effective enhancer blocking. It has been shown that a 125 bp fragment including only two Su(Hw) binding sites can partially block the strong yellow enhancer, while a larger 454 bp fragment including the same Su(Hw) sites completely blocks yellow enhancers. Thus, additional proteins binding to neighboring sequences are required for strong insulator action of the element between yellow and AS-C. The sequencing of the Drosophila genome shows the absence of large clusters of endogenous Su(Hw) binding sites, such as are found in the gypsy retrotransposon. It seems possible that in endogenous insulators, Su(Hw) cooperates with additional DNA-binding proteins to produce insulator activity. This assumption may also explain the absence of lethal phenotypes in the su(Hw)- background since other proteins would partly compensate for the loss of Su(Hw) function (Golovnin, 2003).
The results further confirm the initial observation of the interaction between two gypsy insulators. The two Su(Hw) binding sites in the 125 bp fragment and the gypsy insulator mutually neutralize each other's enhancer-blocking activity. Thus, the difference in the number of Su(Hw) binding sites between interacting insulators is not critical for the effective neutralization of the enhancer blocking activity (Golovnin, 2003).
Increasing the number of Su(Hw) binding sites increases insulator strength, and three copies of the 125 bp insulator block better than a single copy. How can this be reconciled with the observation that two Su(Hw) insulators neutralize one another? It is supposed that the neutralization requires the pairing between two insulators. Interaction between neighboring insulators would pre-empt their interaction with larger assemblies of Su(Hw) binding sites that have been proposed to associate together at the nuclear periphery through the Mod(mdg4) protein. Thus, for neutralization, it is supposed that the Su(Hw) binding sites must adopt a paired configuration, therefore requiring a sufficient distance between them for DNA to form a loop. In contrast, putting more Su(Hw) binding sites very close together merely ensures that enough Su(Hw) protein will be bound at any one time to produce insulator action (Golovnin, 2003).
The role of the Su(Hw) and Mod(mdg4) proteins in the expression of ASC genes becomes obvious when the normal architecture of the ASC regulatory region is altered by chromosome rearrangements. Many previously described inversions with breakpoints in the AS-C regulatory region and centric heterochromatin have weak mutant phenotypes, suggesting the presence of sequences that effectively impede the spread of heterochromatic silencing. The appearance of strong variegating repression of the ac and sc genes when the inversions are combined with loss of su(Hw) or mod(mdg4) function suggests that the Su(Hw) and Mod(mdg4) proteins are involved in the stability of the ac and sc expression (Golovnin, 2003).
In the In(1)y3p mutation, a heterochromatic breakpoint in the upstream regulatory region does not effect yellow expression suggesting that the yellow promoter is relatively resistant to heterochromatin proximity at this breakpoint. At the same time, ac and sc expression is strongly affected by su(Hw) or mod(mdg4) mutations, supporting the idea that Su(Hw) binding sites between yellow and ac block heterochromatin spreading (Golovnin, 2003).
The In(1)sc8 and In(1)scv2 inversions separate the ac and sc genes. The requirement of the Su(Hw) and Mod(mdg4) proteins for normal sc expression suggests the existence of additional Su(Hw) binding sites in the AS-C regulatory region. The strong genetic interaction between sc2 and mutations in mod(mdg4) or su(Hw) also supports the presence of additional Su(Hw) binding sites in ASC. The expression of ASC genes is regulated by a large number of enhancer-like elements. It seems reasonable that these ASC enhancers should be separated by boundary elements as was found for the 3' cis-regulatory region of Abdominal B (Abd-B), which is subdivided into a series of iab domains. Boundary elements like MCP, Fab-7 and Fab-8 separate the iab domains and protect each against positive and negative chromatin modifications induced by neighboring iab domains. The genetic results might be explained by the assumption that the Su(Hw)-Mod(mdg4) protein complex participates in formation of boundary elements between certain AS-C enhancers. The absence of noticeable changes in the wild-type AS-C gene expression on the su(Hw) or mod(mdg4) mutant background might be the consequence of the functional redundancy of the Su(Hw)-Mod(mdg4) protein complex. No clusters of potential endogenous Su(Hw) binding sites are found inside the AS-C sequence. Thus, it seems possible that Su(Hw)-Mod(mdg4) cooperates with other non-identified proteins in formation of the functional boundaries in the regulatory region of AS-C. The identification and characterization of new Su(Hw) binding sites may help in understanding the role of Su(Hw)/Mod(mdg4) in transcriptional regulation of AS-C genes and provide new insights into the mechanisms of the insulator action (Golovnin, 2003).
The proneural genes achaete (ac) and scute<
(sc) are necessary for the formation of the external sensory organs
(SOs) of Drosophila. ac and sc are expressed in
proneural clusters and impart their cells with neural potential. For this
potential to be realized, and the SO precursor cell (SOP) to arise within a
cluster, sufficient proneural protein must accumulate in the cluster. charlatan (chn) encodes a zinc
finger transcription factor that facilitates this accumulation by forming a
stimulatory loop with ac/sc. Loss of function of
chn decreases the accumulation of Sc in proneural clusters and
partially removes notum macrochaetae, while overexpression of chn
enhances ac/sc expression and the formation of extra SOs. Moreover,
chn is activated by ac/sc in proneural clusters. Chn
apparently stimulates ac/sc by physically interacting with the
proneural cluster-specific enhancers and increasing enhancer efficiency, thus
acting as a stimulator of ac/sc expression in proneural clusters.
chn is also required for the proper development of the embryonic
peripheral nervous system; its absence leads to loss of neurons and causes
aberrant development of chordotonal organs (Escudero, 2005).
The strong genetic interaction between the LOF conditions for chn
and ac/sc, together with the presumed activation of chn by
ac/sc, led
to an examination of whether chn might in turn stimulate ac/sc
expression. Initially examined was whether the overexpression of chn
affects Sc accumulation in third instar wing discs. In these discs,
ac and sc are coexpressed in a stereotyped pattern of
well-resolved proneural clusters from which SOPs emerge.
With the MS248-Gal4 driver, UAS-chn promotes strong and
generalized expression of sc in most of the domain of expression of
the driver, namely, the medial and part of the lateral prospective notum. Many SOPs arose from
this enlarged region of Sc accumulation, as detected by the Sens marker,
consistent with the additional macrochaetae that develop on the notum of
these flies. With the MS1096-Gal4 driver, which is expressed most strongly in the dorsal
part of the wing anlage, there is also ectopic expression of sc
and emergence of extra SOPs in the wing territory. Interestingly,
expression of UAS-chn disrupts the characteristic double row
expression of sc and sens at the wing margin, suggesting
interference with its formation. This is also consistent with the presence of small, crumpled
adult wings that carry many bristles and other types of sensilla. Overexpression of UAS-chn with the ubiquitous wing
disc driver C765-Gal4 activates sc but fails to stimulate
atonal, a proneural gene which is not a member of the ASC and is
normally expressed in a few cells at the presumptive tegula and ventral radius. Conversely,
overexpression of atonal does not stimulate chn in the wing
disc (Escudero, 2005).
The expression of ac/sc in proneural clusters is controlled by a
series of separable enhancer elements in the ASC. Each enhancer is responsible
for expression in one or in a few proneural clusters. Thus, whether the ectopic activation of
sc could be mediated by the overexpression of UAS-chn acting
upon these enhancers was examined. UAS-chn strongly stimulates the activity of a
construct in which the lacZ gene is under the control of the ASC
L3/TSM enhancer [construct 2.3-lacZ; MS1096-Gal4 driver], which directs expression at
the wing vein L3 and the twin sensilla of the wing margin proneural clusters.
Similar observations were made with the dorsocentral (DC) enhancer [construct
AS1.4DC=DC-lacZ; C765-Gal4 ubiquitous driver], which promotes
expression in the central part of the notum. It also activates expression
directed by the ANP enhancer. Since the DC-lacZ construct bears
the heterologous hsp70 promoter, these data indicate that the
sc endogenous promoter is dispensable for the stimulation by
UAS-chn. The Ac and Sc proneural proteins are also not essential for
the increased activity of the enhancers; DC-lacZ expression is
strongly increased by Chn in an In(1)sc10.1 background. By contrast, the
sc SOP-dedicated enhancer (SRV-lacZ construct), which is
responsible for the strong accumulation of Sc in SOPs, is only clearly activated by UAS-chn
(C734-Gal4 driver) in the presence of ac/sc, and this
stimulation occurred in individual cells. This observation
suggests that the upregulation of this enhancer results from the formation of
ectopic SOPs by the UAS-chn-induced overexpression of sc,
rather than from a direct effect of Chn on the enhancer. Still, the
possibility remains that Chn and Sc cooperate in the activation of this enhancer (Escudero, 2005).
UAS-chn upregulates the activity of these enhancers, but it does
not lead to a generalized expression of lacZ in all the domains of
UAS-chn expression. These data indicate that despite the elevated
activation, the enhancers are still dependent on the prepattern factors that
define their spatial domains of activity. This fact was verified by the
observation that the overstimulation of DC-lacZ is strongly
dependent on its prepattern activator, the transcription factor Pnr. Moreover,
2.3-lacZ, which is active only in the wing pouch, is not stimulated
by the overexpression of UAS-chn in the prospective notum
(MS248-Gal4); this indicates that the sc promoter present
in this construct was not responsive to UAS-chn (Escudero, 2005).
Next examined were mosaic wing discs to determine whether removal of chn
function affects expression of sc or enhancer-lacZ
constructs in proneural clusters. Homozygous chnECJ1 cells
generally display reduced expression of sc or
ß-galactosidase under the control of proneural enhancers, when compared
with neighboring heterozygous chnECJ1/+ cells. Note
however, that the expression is not completely abolished. Similar decreased
expression of sc is observed by misexpressing
UAS-chniS in cell clones. These effects appear
to be cell-autonomous. While SOPs can still emerge from homozygous
chnECJ1 cells with reduced levels of Sc,
SOPs were often missing, in agreement with the partial suppression of macrochaetae
observed within the chnECJ1 clones. When both homozygous
and heterozygous cells were near a position where an SOP emerged, a
heterozygous cell appeared to be preferentially selected. These findings
clearly indicate that chn+ is required for proneural
proteins to accumulate in proneural clusters at levels sufficient to ensure
SOP selection. Moreover, the observation that expression of
enhancer-lacZ constructs is reduced in chn-
cells and increased in chn overexpressing cells indicates that the
effect of chn+ is not due to an enhanced perdurance of
the Sc protein, but to the increased transcription of the sc gene (Escudero, 2005).
To analyze whether Chn is a direct regulator of the ASC enhancers, the ability of the Chn protein to bind to the DC enhancer was assayed in vitro. A
fragment of the Chn protein containing the five zinc fingers was produced in
and purified from E. coli. The enhancer DNA was divided into six
partially overlapping fragments of approximately 300 bp each, and each of them
was assayed in gel retardation experiments. Only the fragment
that comprised the proximal-most region of the enhancer (fragment DC6) shows
binding of the Chn polypeptide. Interestingly, the DC6 fragment is included within the
PB0.5DC sequence, the smallest subfragment of AS1.4DC, which still retains
enhancer activity. For unclear reasons, the PB0.5DC enhancer drives
expression only in the PDC SOP. Still, misexpression of UAS-chn expands this
expression to many cells of the posterior notum. This suggests that
the binding of Chn protein to the DC6 region of the DC enhancer may prompt its
response to Chn in vivo (Escudero, 2005).
Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. A new gene has been identified involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos (French for 'hairy back'), that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null alleles of poils au dos are associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete–scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, pad has been shown to interact synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively (Gibert, 2005).
Expression of ac-sc in proneural clusters is regulated by independently-acting cis-regulatory enhancers. The enhancer responsible for activation of ac-sc in the cluster giving rise to the DC bristles has been characterized in detail. The activity of this enhancer in a reporter construct was examined using lacZ expression. The activity of this enhancer is modified in pad1. The domain of expression of lacZ appears wider. At the same time, the anterior limit of the cluster is retracted in a posterior direction. It is possible that this is in part due to the slight distortion of the overall shape of the notum seen in pad1 mutants. Interestingly, the ectopic bristles do not arise within the misshapen proneural cluster. They are therefore formed independently of the activity of the DC enhancer used for activation. In fact, the aDC, as well as the ectopic DC precursors, are both clearly situated outside the DC cluster. Another characterized enhancer of ac-sc, the L3-TSM enhancer involved in the formation of the sensilla on the anterior wing margin, anterior cross vein and third vein was examined and no significant modification was observed. These results suggest that poils au dos does not act through the cis-regulatory sequences controlling expression in the proneural clusters (Gibert, 2005).
To determine which regions of the AS-C are required for the formation of the ectopic bristles in pad, the pad1 mutant was placed in various ac-sc mutant backgrounds. These included several deletions generated by excision of the P-element in the line NP-6066. In(1)ac3, an inversion separating sequences located 1 kb upstream of ac, including the DC enhancer, was used, as well as Df(1)91B (which deletes 45 kb from a position 10.3 kb upstream of sc that includes ac and the DC enhancer); Df(1)115 (which deletes 7.8 kb between the positions 14.5 and 6.7 kb upstream of the scute ATG), and In(1)sc4 (an inversion with a breakpoint 7-8 kb downstream of sc). None of these rearrangements prevent formation of the ectopic bristles present in pad1. In(1)sc4 causes a loss of all scutellar bristles, because the relevant enhancer, located 40 kb downstream of sc, is translocated elsewhere and is thus not able to drive the expression of ac-sc in the scutellum. However, occasional scutellar bristles form in In(1)sc4; pad1 flies at the position normally occupied by the anterior scutellar bristle. In contrast to the rearrangements cited above, no, or very few, ectopic bristles are formed in scbald; pad1 flies. This hypomorphic sc allele carries the remains of a P element located 10 kb upstream of sc and displays a high frequency of missing SC, aDC and orbital bristles. Together, these results indicate that the target sequences are probably located in a fragment that extends 6.7 kb upstream and 7-8 kb downstream of sc (Gibert, 2005).
In order to visualize the precursors of the ectopic bristles in pad1, an antibody against Senseless, a marker of neural precursors, was used. A transgene was used driving the expression of LacZ under the control of the achaete/scute Sensory Organ Precursor enhancer (SOP-lacZ). The minimal SOP enhancer of 500 bp drives expression of lacZ exclusively in the bristle precursors and contains binding sites for Ac-Sc/Da (E boxes), as well as sites for the binding of repressors. It was observed that the precursors of ectopic bristles appear between 0 and 2 h after puparium formation. This is about the same time as the formation of the precursors for the anterior DC (aDC) bristles in wild-type flies. The posterior DC (pDC) precursors appear much earlier, around 24 to 12 h before puparium formation. In situ hybridization with a probe to sc, indicated that sc is expressed ectopically in third instar wing discs. Expression of ac was examined using an anti-Achaete antibody and is also significantly up-regulated in pad1. In both cases, the proneural clusters that give rise to the wild-type bristle precursors are clearly visible at wild-type locations, but they appear to be enlarged. In addition, many more cells express high levels of ac-sc outside the proneural clusters. These are mainly located in the future anterior and central regions of the notum, consistent with the fact that ectopic macrochaetes are found here. Weak sc expression can be detected in these areas in wild-type discs but does not give rise to sense organs. Ectopic expression in pad1 is particularly visible in the region of the presutural, DC and PSA bristles where many ectopic bristles form (Gibert, 2005).
To better visualize the regions of ectopic expression, the reporter construct EE4 containing an artificial SOP enhancer composed of four E-boxes and the binding sites for the Ac and Sc proteins was used. The EE4 construct lacks the sequences required for repression and so it is very sensitive to the levels of Ac-Sc and can be used to measure the increased amounts of Ac-Sc in the pad mutant. It was observed that expression driven by this enhancer in pad1 is significantly different from that seen in the wild type. In the wild type, it is expressed exclusively in the cells of the proneural clusters where it is present at high levels. In pad1, expression in the PSA region expands medially and expression in the DC region expands anteriorly. Some of the ectopic precursors appear within this expanded anterior region (Gibert, 2005).
Changes in cis-regulatory sequences are proposed to underlie much of morphological evolution. Yet, little is known about how such modifications translate into phenotypic differences. To address this problem, focus was placed on the dorsocentral bristles of Drosophilidae. In Drosophila melanogaster, development of these bristles depends on a cis-regulatory element, the dorsocentral enhancer, to activate scute in a cluster of cells from which two bristles on the posterior scutum arise. A few species however, such as D. quadrilineata, bear anterior dorsocentral bristles as well as posterior ones, a derived feature. This correlates with an anterior expansion of the scute expression domain. This study shows that the D. quadrilineata enhancer has evolved, and is now active in more anterior regions. When used to rescue scute expression in transgenic D. melanogaster, the D. quadrilineata enhancer is able to induce anterior bristles. Importantly, these properties are not displayed by homologous enhancers from control species bearing only two posterior bristles. Evidence is provied that upstream regulation of the enhancer, by the GATA transcription factor Pannier, has been evolutionarily conserved. This work illustrates how, in the context of a conserved trans-regulatory landscape, evolutionary tinkering of pre-existing enhancers can modify gene expression patterns and contribute to morphological diversification (Marcellini, 2006).
Unfortunately, to date, the mechanism responsible for restricting the activity of the DC enhancer in the anterior direction has not been discovered. However, the direct input of Pnr and U-shaped (Ush), essential for the correct activity of the Dm-DCE along the dorso-lateral axis, has been extensively analyzed. In order to shed light on the ancestry and the functional conservation of the regulation by Pnr and Ush, the sequences of orthologous DCEs, as well as their relative activities in various mutant backgrounds was tested (Marcellini, 2006).
Sequence alignments reveal that the DCEs are greatly variable in size and have undergone considerable turnover. Only the extremities display significant levels of similarity between all species examined. The central region is poorly conserved. The elements from D. melanogaster (1.5 kb) and D. eugracilis (2 kb) are more similar to each other than to the others, in accordance with their closer phylogenetic relationship. The enhancers from D. virilis and D. quadrilineata share a relatively large size (4.1 and 3.3 kb, respectively) and a conserved stretch of about 300 nucleotides that is absent from the D. melanogaster and D. eugracilis sequences. Putative binding sites for Pnr are present in all species. Mutation of a specific Pnr binding site severely reduces activity of the Dm-DCE. This site is embedded within a stretch of 16 nucleotides perfectly conserved between the four species. Interestingly, two other neighbouring GATA sequences can be recognised as homologous between all species. Conservation overall, however, is low, and the number, spacing, and orientation of the remaining putative Pnr binding sites are extremely variable (Marcellini, 2006).
In D. melanogaster, pnr is expressed in a broad medial domain, but activates sc in discrete proneural clusters. Expression of sc mediated by the Dm-DCE is a direct consequence of Pnr binding. DCE function is restricted dorsally through the repressor activity of Ush, which forms heterodimers with Pnr and prevents activation of sc. It was found that the activity of the Dv-DCE and the Dq-DCE in D. melanogaster is restricted to a lateral cluster of cells completely included within the expression domain of pnr. This suggests that, despite significant sequence turnover, the divergent DCEs require Pnr and are efficiently repressed dorsally by Ush. Behaviour of the DCEs was examined in the context of various mutant alleles of pnr. pnrVX4, a strong loss of function allele, pnrV1, a hypomorphic allele and pnrD1, a gain of function allele with a missense mutation that disrupts the interaction of Pnr with Ush were used. Activity of the Dm-DCE was compared with that of the Dv-DCE and that of the Dq-DCE. It was observed that the enhancers react in a similar fashion to four different mutant backgrounds. The expression domains are reduced in loss of function genotypes and expanded in gain of function genotypes (Marcellini, 2006).
This study has presented evidence that the activity of Pnr is conserved and positively regulates the DC enhancers from distantly related Drosophilidae. When assayed in D. melanogaster, the Dv-DCE and Dq-DCE are active in groups of cells completely included within the expression domain of Dm-pnr. It is significant that an essential, high-affinity Pnr binding site in the Dm-DCE is conserved in the DCEs of the other species. Note that the three conserved Pnr binding sites are clustered in a region of the DCE that is required for activity and is sufficient in D. melanogaster to direct weak expression by itself. Expression of sc mediated by the Dm-DCE is restricted dorsally through the repressor activity of Ush that associates with Pnr to prevent activation. In gain-of-function pnr alleles that are insensitive to Ush, activity of the Dv-DCE and the Dq-DCE, like the Dm-DCE, expands dorsally. Most of the open reading frame of pnr was cloned from D. quadrilineata, and it was found that, as in D. virilis, the two zinc fingers are perfectly conserved, suggesting that Dq-Pnr and Dv-Pnr may also bind Ush within their respective species. Hence, it is most likely that Pnr and Ush are direct, evolutionarily conserved regulators of the DCE within Drosophilidae. Indeed the expression domain of pnr, as well as other upstream regulators, has been found to be conserved in other families of flies. Even Pnr from the mosquito Anopheles gambiae is able to regulate ac-sc in transgenic D. melanogaster, suggesting conservation of pnr function throughout the Diptera (Marcellini, 2006).
D. quadrilineata is phylogenetically distant from D. melanogaster and displays four instead of two DC bristles. The results demonstrate that this secondary gain is partly due to evolution of the cis-regulatory sequence that drives sc expression at the DC site. A Dq-DCE-sc minigene, present in transgenic mutant D. melanogaster devoid of the endogenous DC proneural cluster of ac-sc expression, is not only able to rescue posterior bristles, but also allows development of more anterior bristles. It thus mimics the DC phenotype of D. quadrilineata itself. Expression driven by the Dq-DCE in D. melanogaster extends anteriorly in a domain that is longer and thinner. Although the Dq-DCE was not tested in D. quadrilineata itself, it is active in D. melanogaster in a domain that is similar to the DC domain of sc expression in D. quadrilineata visualized by in situ hybridisation. This suggests that the Dq-DCE autonomously reproduces an expression pattern similar to the endogenous one in D. quadrilineata. Expression of sc mediated by the Dm-DCE is restricted laterally through lack of Pnr, dorsally through the repressor activity of Ush and posteriorly through the antagonistic activity of Islet, but it is not yet known what restricts expression in an anterior direction. The anterior expansion seen with the Dq-DCE indicates that this sequence may be at least partially insensitive to whatever factors limit anterior expression driven by the Dm-DCE. Alternatively it may contain new information not present in the other species (Marcellini, 2006).
These observations demonstrate an altered response of the D. quadrilineata sequence to the upstream regulators of D. melanogaster. This response should reside in the sequence of the Dq-DCE itself that is sufficient to modify the phenotype of D. melanogaster when used to drive sc. Thus the exchange of a single, well-defined enhancer is sufficient, not only to reproduce an expression pattern, but also to partially transform a morphological trait of one species into that of another. It is proposed that a change in cis, within a pre-existing regulatory element of sc, contributed to the evolution of the bristle pattern observed in D. quadrilineata by altering the region where it is expressed (Marcellini, 2006).
The Dv-DCE, in D. melanogaster, drives expression in a larger cluster that expands predominantly in a dorsal direction. A Dv-DCE-sc minigene, however, allows the development of only two bristles positioned at the correct locations. The most likely explanation for the fact that the expanded expression driven by Dq-DCE-sc leads to additional bristles, whereas that of the Dv-DCE-sc does not, is probably linked to the different locations of the cells expressing sc. It seems that, in D. melanogaster, the region anterior to the two DC bristles is competent to produce bristles. This region is situated between the domains of expression of sr, a repressor of macrochaete development, and overlaps a band of expression of wingless (wg), a gene encoding a secreted factor that is required to maintain sc expression and to repress sr. It is possible to select for additional anterior DC bristles, but not for macrochaetes on either side of the DC row where sr is expressed but wg is not. Notably, anterior DC bristles were present in the ancestor common to D. melanogaster and D. virilis. The curved shape of the Dq-DCE–driven expression domain means that it avoids overlap with the domains of expression of sr and shows significant overlap with that of wg. Therefore only the Dq-DCE drives expression in an anterior location that is competent to produce bristles (Marcellini, 2006).
Nevertheless transgenic D. melanogaster expressing Dq-DCE-sc do not perfectly reproduce the bristle pattern of D. quadrilineata. The anterior-most DC bristle, the scapular bristle, is absent. This bristle is situated in the prescutum, anterior to the transverse suture. It may be that this difference is attributable to changes in factors that negatively or positively regulate the enhancer in trans. It is also possible that full enhancer activity requires sequences on either side of the fragment tested. Additionally, the modification of cis-regulatory elements lying elsewhere within the D. quadrilineata ac-sc complex could also have contributed to the emergence of the additional bristles. However, it is equally possible that other extraneous factors are responsible that cannot be controlled for in these experiments. For instance, it has been shown that differences in the timing of bristle precursor formation between species can influence the development of macrochaetes (Marcellini, 2006).
The two DC bristles resulting from the activity of Dv-DCE-sc are situated at exactly the correct positions despite the fact that the Dv-DCE drives expression in a cluster of cells that is larger and displaced dorsally when compared with that of D. melanogaster. Thus the fly can compensate for this degree of imprecision in sc expression at the DC site. The explanation for this probably lies in the manner in which the bristle precursors are selected from the proneural cluster. Notch-mediated lateral signalling allows the selection of only two cells destined to become precursors with the appropriate spacing. However, the choice of these cells is not random, but biased by external factors such as the repressors emc and sr, whose activity causes the precursors to arise at similar positions within the DC cluster of all individuals. Their site of origin is in fact located within the region of overlap of expression driven by the Dm-DCE and the Dv-DCE. Positioning of bristle precursors thus results from restricted expression of sc in the proneural clusters as well as other cues that constrain the choice of precursor cell. Together, these two inputs lead to a robust patterning mechanism that is resistant to mild perturbations such as the shifting of the proneural cluster observed for Dv-DCE activity (Marcellini, 2006).
The ability of poorly conserved enhancers to drive expression of reporter genes in homologous tissues when transferred between species of similar morphology has been widely documented in the literature. Where a detailed comparison of enhancer activity allowed a rigorous assessment of the degree of conservation, two different outcomes have been observed. On the one hand, transferring enhancers between related species of Drosophila (e.g., even-skipped), or of nematodes (e.g., lin-48) revealed a perfect conservation of activity, a phenomenon attributed to stabilizing selection. On the other hand, the regulatory regions exchanged between species of sea urchins (e.g., endo-16) or ascidians (e.g., Otx) did not perfectly recapitulate the endogenous expression pattern. The DCEs from D. eugracilis and D. virilis behave like the latter: they drive reporter gene expression in a cluster of cells that is not perfectly co-incident with that of the endogenous DC cluster. The slightly different expression patterns could be due to the divergent sequences, or could result from co-evolution between the enhancer and its regulatory environment. Indeed earlier experiments have hinted that co-evolution between Pnr and its target sequences may be occurring (Marcellini, 2006).
The role of the sensory macrochaetes in behaviour is not known. Many species of Acalyptrata have ancient stereotyped patterns in which the number and precise position of each bristle is invariant. The bristle patterns of the Drosophilidae are remarkably conserved, and the majority of the nearly 4,000 species have two DC bristles. The evolutionary stability of the many bristle patterns suggests a role for selective forces to maintain them. D. quadrilineata is unusual among Drosophilidae in having four or five DC bristles. The anterior-most DC bristles would allow additional positional sensory input, and it is possible that they confer a selective advantage. However, it is important to note that not all morphological change needs be driven by selection. Kimura proposed a neutral theory of molecular evolution in which mutations with null or negligible effect can become passively fixed in populations. Similarly, natural selection alone may not explain the infinite number of subtle morphological variations displayed by the many species of Drosophila described. Exploratory behaviour is an intrinsic property of biological systems, and one may therefore also speculate that evolution can proceed through a series of viable, seemingly useless, phenotypes (Marcellini, 2006).
Two distinct roles are described for Dorsal, Dif and Relish, the three NF-kappaB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-kappaB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. It is proposed that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis (Ayyar, 2007).
The results suggest a dual role for the NF-kappaB/Rel proteins of Drosophila in the formation of SOPs. First, they could be recruited directly to the sc promoter and regulate transcription. The SOP enhancer of sc, required for auto-regulation of sc in the SOPs, contains α boxes (ACTAGA), consensus sequences for NF-kappaB/Rel. Evidence has been obtained for a role of these sequences in both activation and repression of sc. Expression of Rel-VP16, a potent transcriptional activator form of Relish, is able to ectopically activate a reporter gene containing the intact sc SOP enhancer but not one in which the α3 box is mutated. So activation in this experimental situation requires the presence of an intact α3 site. The experiment does not rule out indirect effects, so further work is required to verify whether activation is direct. It is suggested the NF-kappaB/Rel proteins participate in activation and repression of transcription of sc, a hypothesis consistent with dl, Dif and Rel mutant phenotypes of additional as well as missing bristles. Second, unexpected role is described of Rel in mRNA turnover of sc, ase and sens, neuronal genes required to specify and/or maintain the neuronal fate of SOP cells. In Rel mutants, transcripts of sc, ase and sens accumulate due to increased transcript stability. Therefore in the wild type, Relish promotes rapid mRNA turnover, presumably indirectly through an unidentified transcriptional target. A similar phenotype is observed in Toll-8 mutants, which furthermore, interact genetically with Rel mutants. Transcripts for Rel are reduced in the Toll-8 mutant suggesting a role for Toll-8 in maintaining the levels of Rel transcript. This might be the reason for the genetic interaction (Ayyar, 2007).
A number of differences are apparent between mutants of the three NF-kappaB/Rel-encoding genes of Drosophila. Mutants triply homo- or hetero-zygous have a normal complement of bristles, while single homo- or hetero-zygous animals have either additional or missing bristles. This suggests possible opposing functions for these genes. Furthermore bristle phenotypes due to loss or gain of function differ in detail between the three mutants. Together these results point to the importance of the stoichiometric relationships between the three NF-kappaB/Rel proteins and raise the possibility that different Dorsal/Dif/Relish homo- or hetero-dimers may have distinct binding sites and therefore different targets. This merits further investigation (Ayyar, 2007).
If NF-kappaB/Rel proteins both activate and repress sc, then they are expected to activate in SOP cells and repress in cells of the proneural clusters not chosen to be SOPs. Two possible ways that this could occur are discussed. First, activation in the SOP may rely on high levels of proneural protein and low levels of NF-kappaB/Rel protein; conversely repression may require low levels of proneural and high levels of NF-kappaB/Rel protein. Notch-mediated lateral inhibition results in high levels of Sc in the SOP and lower levels in surrounding cells. Toll-8 expression is excluded from SOP cells suggesting, that, if Toll-8 affects NF-kappaB/Rel activity, there would be lower levels of NF-kappaB/Rel in SOPs. NF-kappaB has been shown to activate transcription even without stimulus if IkappaB levels are low enough to allow NF-kappaB-dependent gene expression in the basal state. Interestingly, it has been shown that low levels of Dorsal can act synergistically with bHLH proteins to activate target genes in the embryo. This depends on direct association of Dorsal and bHLH proteins and cooperative binding to closely linked binding sites for the two respective proteins. Furthermore cooperative binding for Sc and Dorsal has been demonstrated. In the sc SOP enhancer one of the alpha boxes is indeed close to an E box, so perhaps high levels of Sc and low levels of NF-kappaB/Rel combine to activate transcription in the SOP. Two observations are consistent with this hypothesis: Rel-VP16 was able to ectopically activate sc-SOPE-lacZ only at sites where ac and sc are expressed and, after over-expression of NF-kappaB/Rel proteins, bristles are generally missing on the lateral notum (where Toll-8 levels are high), whereas ectopic bristles are found on the medial notum (where Toll-8 levels are low) (Ayyar, 2007).
A second means by which NF-kappaB/Rel proteins could act differently in SOP and in non-SOP cells, may be the presence/absence of co-factors. It has been shown that Dorsal can be converted from an activator to a repressor by association with the co-repressor Groucho. This bi-functionality is attributable to the fact that Dl only weakly interacts with Gro. During embryogenesis both Cut and Dead ringer bind an AT-rich silencer sequence, AT2, present in target genes of Dorsal and both Dorsal and Dead ringer bind the co-repressor Groucho and recruit it to DNA. A similar AT-rich sequence (the β box) is present in the sc SOP enhancer. Furthermore repression of sc by the E(spl) proteins, targets of Notch signalling in non-SOP cells, is already known to require the activity of Groucho (Ayyar, 2007).
Transcripts for sc, ase and sens (and GFP) accumulate in Rel and Toll-8 mutants as a result of increased transcript stability. Transcript stability correlates with the presence of a six or seven nucleotide motif in the transcribed sequence of these genes. The motif is present in sc, ase and sens, but not ac the transcription of which is unaffected in Rel mutants. The motif is almost identical to the heptamer in MyoD and Sox9 that is associated with transcript stability after inhibition of NF-kappaB/Rel signalling in C2C12 cells. A sc mutant with a truncated sc transcript lacking one of the two motifs present in the coding sequence of this gene, has a phenotype similar to Rel and Toll-8 mutants and an increase in sc mRNA. It has been suggested that increased stability of the transcripts rather than increased transcription underlies this phenotype. The presence of the heptamer is noted in a number of genes involved in sensory organ patterning suggesting possible regulation by NF-kappaB/Rel of a battery of genes in the imaginal epithelium. A similar motif is present in other vertebrate targets of NF-kappaB/Rel. Post-transcriptional regulation of target genes by NF-kappaB/Rel could therefore be an ancient feature common to Drosophila and mammals and possibly even jellyfish. It has been suggest that an unknown factor, presumably a transcriptional target of NF-kappaB/Rel, regulates messenger turnover through association with this sequence. In Rel and Toll-8 mutants the accumulated transcripts are not translated. This must be an effect of the mutants because ectopic expression in wild-type flies allows translation and ectopic bristle formation (Ayyar, 2007).
Promotion of a rapid turnover of transcripts of neuronal genes presumably does not take place in the SOPs where high levels of the protein products of these genes are required. Accordingly Toll-8 expression is extinguished in the SOPs after their formation. Factors specific to the SOP presumably allow translation of the transcripts. It is therefore suggested that high levels of Relish provided by Toll-8 in non-SOP cells might be required for post-transcriptional regulation of neuronal genes (Ayyar, 2007).
In wild-type animals expression of neuronal precursor genes such as sens and ase is restricted to SOPs where they are activated by high levels of Ac and Sc. The results suggest that they are in fact expressed over the entire neuro-epithelium but that mRNA turnover is rapid due to NF-kappaB/Rel activity. Activation of ac-sc in proneural clusters would counteract the effects of NF-kappaB/Rel to allow selection of SOPs. After selection of SOPs for the large sensory bristles is finished, Toll-8 expression is maintained in the epithelium, suggesting that high levels of NF-kappaB/Rel are still required for continued transcript turnover. Continuous buffering of neuronal gene expression presumably continues until the next round of neurogenesis that takes place after pupariation when precursors for the small bristles form. Therefore it is hypothesized that NF-kappaB/Rel plays a subtle role in maintaining steady state levels of expression of many genes required for neural development. The maintenance of low levels of expression of neuronal genes would keep the tissue poised for neurogenesis that takes place in repeated rounds. Perhaps low levels of expression of neuronal genes are characteristic of neuro-epithelia in general (Ayyar, 2007).
The hypothesis concerning the dual role of NF-kappaB/Rel in neurogenesis in Drosophila is as follows. The neuro-epithelium of the imaginal discs expresses neuronal genes. Prior to development of SOPs, high levels of Toll-8 maintain high levels of Rel and result in nuclear accumulation of NF-kappaB/Rel. Through an unknown transcriptional target(s), Relish promotes rapid turnover of neuronal transcripts by a post-transcriptional mechanism. This might be mediated by a specific sequence in the coding regions of target genes. Activation of ac and sc in proneural clusters by regulatory proteins of the notal prepattern counteracts the effects of Relish. After singling out of SOPs by Notch-mediated lateral inhibition, Toll-8 expression ceases in the SOPs. Reduced levels of signal uncover a trans-activator function for NF-kappaB/Rel that, synergistically with Sc, helps to maintain high levels of sc expression in the SOP, possibly through direct binding to consensus sequences in the sc SOP enhancer. The NF-kappaB/Rel proteins may also directly repress sc in non-SOP cells of the proneural clusters. It remains to be seen to what extent each of the three proteins participates in these two processes (Ayyar, 2007).
Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side (right and left) of the neuroectoderm. In the intermediate column, the receptor tyrosine kinase Egfr represses expression of proneural genes achaete and scute, and is required for the formation of neuroblasts. Most of the early function of Egfr is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that of Egfr mutants. MAP-kinase activation is also observed in the medial column where escargot (esg) and proneural gene expression are unaffected by Egfr. The homeobox gene ventral nerve system defective (vnd) is required for the expression of esg and scute in the medial column. vnd acts through the negative regulatory region of the esg enhancer that mediates the Egfr signal, suggesting vnd's role is to counteract Egfr-dependent repression. Thus, the nested expression of vnd and the Egfr activator Rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains (Yagi, 1998).
To investigate the involvement of Egfr in neurogenesis, mutant phenotypes of Efgr and its activator rho were examined at various stages of neurogenesis. The dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg, which is expressed in the lateral and medial columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of Egfr and a null mutation of rhomboid were used for analysis throughout this work. Egfr and rho mutations cause ectopic expression of esg in the intermediate column. Repression of esg in the intermediate column is likely to require a relatively high dose of Egfr signal. To examine the potential role of Egfr in neurogenesis, expression of the proneural genes ac and sc was carried out. These two proneural genes begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage. In Egfr and rho mutant embryos, ac and sc become ectopically expressed in the intermediate column. This phenotype is less penetrant and, occasionally, gaps of ac and sc expression are observed in the intermediate column. Since sc expression was similarly derepressed in Egfr mutant embryos, these phenotypes are likely to represent the near null phenotype of Egfr in the neuroectoderm. These data indicate that, in the intermediate column, the Egfr signal represses not only esg but also proneural genes, which are known to play key roles in neurogenesis (Yagi, 1998).
A study by S. D. Weatherbee (1998) is arguably the best study yet published about how gene regulation differs in homologous structures, and points to future studies for how differential gene regulation will be shown to account for the structural differences between species. The differentiation of the Drosophila haltere from the wing through the action of the Ultrabithorax (Ubx) gene is a classic example of Hox regulation of serial homology. This study reveals several features of the control of haltere development by Ubx which, in principle, are likely to apply to the Hox-regulated differential development of other serially homologous structures in other animals. The formation of margin bristles is regulated by Wingless via the induction of the proneural achaete (ac) and scute (sc) target genes and also requires the Cut transcription factor. In the haltere disc, Cut is expressed along the anterior DV boundary, whereas ac and sc are not induced. To determine if Ubx represses ac/sc activation by Wg, Ubx clones were examined. In the haltere disc, sc expression is derepressed in clones that touch or cross the anterior portion of the DV boundary. Conversely, sc expression is lost in anterior wing disc cells that ectopically express Ubx. This repression by Ubx is sensitive to the dosage of Ubx activity, since ectopic ac/sc expression is observed in Ubx/+ haltere discs. This ectopic expression corresponds with ectopic bristles found on the halteres of Ubx/+ adults. Further reductions of Ubx function in haltere discs cause greater derepression of sc on the DV boundary and a corresponding emergence of triple row bristles on the adult haltere. The haltere has several types of sense organs, including the proximally located pedicellular sensillae, which are not present on the wing. Correspondingly, sc is expressed in the presumptive pedicellar portion of the haltere disc but not in the equivalent part of the wing disc. In Ubx clones in this region of the haltere disc, sc expression is lost. Therefore, Ubx is required to positively regulate sc in this unique pattern in the haltere disc. Together with the repression of sc along the DV boundary of the haltere, these observations suggest that Ubx acts on two independent domains of the sc expression pattern, presumably via specific cis-regulatory elements controlling each aspect of sc gene expression (Weatherbee, 1998).
Ectopic expression of the scute gene in the developing haltere is sufficient to induce ectopic sensory organs. Interestingly, near the DV boundary, large bristles resembling those of the wing margin are induced, whereas in more proximal regions, sense organs form that are characteristic of the haltere. This result suggests that the repression of sensory organ formation by Ubx at the DV boundary is largely at the level of the sc gene, whereas the character of the proximal sense organs is modified by Ubx action downstream of or parallel to scute. Thus, all three outcomes outlined above are obtained in these ectopic expression experiments, which reveal that Ubx acts independently on the five genes identified as well as on genes further downstream of or parallel to these regulators in the wing patterning hierarchy (Weatherbee, 1998).
In developing organs, the regulation of cell proliferation and patterning of cell fates is coordinated. How this coordination is
achieved, however, is unknown. In the developing Drosophila wing, both cell proliferation and patterning require the secreted
morphogen Wingless (Wg) at the dorsoventral compartment boundary. Late in wing development, Wg also induces a zone of
non-proliferating cells at the dorsoventral boundary. This zone gives rise to sensory bristles of the adult wing margin. How Wg coordinates the cell cycle with patterning has been investigated by studying the regulation of this growth arrest.
Wg, in conjunction with Notch, induces arrest in both the G1 and G2 phases of the cell cycle in separate subdomains of the
zone of non-proliferating cells (ZNC). The ZNC is composed of three subdomains, each about four cells wide. Cells in the central domain express wg. This domain is flanked by dorsal and ventral domains, which, in the anterior compartment, express Achaete and Scute. Cells in the ZNC stop proliferating 30 h before most of the other cells in the disc but re-enter the cell cycle for two or three divisions after pupariation. This arrest is seen by an absence of cells in the S phase of mitosis. The domain architecture of the ZNC is suggested by the expression of string and the G2 cyclins A and B. In the anterior compartment, cells in the dorsal and ventral domains do not express STG messenger RNA but accumulate high leves of G2 cyclins in the cytoplasm. Since Stg is required for mitosis and Stg and the G2 cyclins are degraded at cell division, these patterns are indicative of arrest in G2. In contrast, in the central domain CycA and CycB proteins are undetectable, but STG mRNA is expressed. This indicates that these cells may be arrested in G1. G1 arrest may be due to inactivation of dE2F, a factor required to activate the transcription of genes needed for DNA replication (Johnston, 1998).
Loss of wingless function during disc development abolishes both the G1 and G2 arrests and allows string expression in the anterior dorsal and ventral domains. Four observations suggest that the proneural genes achaete and scute regulate the G2 arrest of the ZNC:
Together, these results indicate that Wg induces G2 arrest in two subdomains by inducing the proneural genes achaete and scute,
which downregulate the mitosis-inducing phosphatase String (Cdc25). Notch activity creates a third domain by preventing
arrest at G2 in wg-expressing cells, resulting in their arrest in G1. To test whether Notch directly regulates the G1 arrest, discs were constructed lacking Wg activity, but expressing activated Notch in the ZNC. These discs do not form a ZNC at all. Thus, in the absence of Wg activity, Notch is not sufficient to induce a G1 arrest. It is noted that the string promoter contains putative Ac/Sc-binding sites, indicating that these basic helix-loop-helix proteins can repress string expression directly (Johnston, 1998).
The Bar homeobox genes function as latitudinal prepattern genes in the developing
Drosophila notum. In Drosophila notum, the expression of achaete-scute
proneural genes and bristle formation have been shown to
be regulated by putative prepattern genes expressed
longitudinally. The two Bar locus genes may belong to a different
class of prepattern genes expressed latitudinally: it is
suggested that the developing notum consists of checker-square-
like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate
the formation of microchaetae within the region of
BarH1/BarH2 expression through activating achaete-scute.
Presutural macrochaetae formation also requires Bar
gene activity. Bar gene expression is restricted in dorsal and posterior regions by Decapentaplegic
signaling, while the ventral limit of the expression domain
of Bar genes is determined by wingless, whose
expression is under the control of Decapentaplegic signaling (Sato, 1999).
The two closely related species of Drosophila, D. melanogaster and D. simulans, display an identical bristle pattern on the
notum, but hybrids between the two are lacking a variable number of bristles. The loss is temperature-dependent
and evidence is provided for two periods of temperature sensitivity. A first period of heat sensitivity occurs during
larval development and corresponds to the time when the prepattern of expression of genes is established. The products of these genes activate
achaete-scute in the proneural clusters, preceding bristle precursor formation. A second period of cold
sensitivity corresponds to the time of emergence of the bristle precursor cells and the maintenance of their neural fate, a
process requiring high levels of Achaete-Scute. Expression of achaete-scute at these two critical periods depends on
cis-regulatory elements of the achaete-scute complex (AS-C). The differences between males, which have only one copy of
the X-linked AS-C from D. simulans, and females, which have copies from both parental species, have been compared, together
with the effects of crossing in different rearrangements of the D. melanogaster AS-C that delete regulatory and/or coding
sequences. Evidence indicates that bristle loss in the hybrids may result from a decrease in the level of transcription at
the AS-C and argues that interaction between trans-acting factors and cis-regulatory elements within the AS-C have diverged
between the two species (Skaer, 2000).
An exogenous supply of Scute delivered with a
heat shock construct can rescue missing bristles. Animals grown at 18ƒC were heat shocked
for 3 h between 12 and 24 h BPF. There is a significant
recovery of bristles (a total loss of 23% bristles in experimental
flies compared to 38% in controls. All bristle types show some rescue from
this treatment though to varying extents.
Effects of heat shock at different
times before or after pupariation were examined. The best rescue was
obtained from heat shocks applied close to the time of
formation of the precursors for each bristle. For example,
the APA and PSC precursors form early between 24 and
12 h before pupariation. These bristles show some rescue
from early heat shocks but a much greater rescue from heat
shocks given 6-12 h before pupariation. They were not
rescued by heat shock after pupariation. In contrast the
precursor for the PPA bristle forms later, just after pupariation.
This bristle is rescued after heat shock at all stages
before pupariation but the greatest rescue is seen at pupariation
itself and some rescue is still possible after pupariation. It is concluded that an exogenous supply of Scute can
effect some rescue of the bristles in the hybrid (Skaer, 2000).
An early step in the development of the large mesothoracic
bristles (macrochaetae) of Drosophila is the expression of
the proneural genes of the achaete-scute complex (AS-C)
in small groups of cells (proneural clusters) of the wing
imaginal disc. This is followed by a much increased
accumulation of AS-C proneural proteins in the cell that
will give rise to the sensory organ, the SMC (sensory organ
mother cell). This accumulation is driven by cis-regulatory
sequences, SMC-specific enhancers, that permit self-stimulation
of the achaete, scute and asense proneural
genes. Negative interactions among the cells of the cluster,
triggered by the proneural proteins and mediated by the
Notch receptor (lateral inhibition), block this accumulation
in most cluster cells, thereby limiting the number of SMCs.
In
addition, proneural proteins trigger positive interactions among cells of the cluster
that are mediated by the Epidermal growth factor receptor
(Egfr) and the Ras/Raf pathway. These interactions,
which are termed 'lateral co-operation', are essential
for macrochaetae SMC emergence. Activation of the
Efgr/Ras pathway appears to promote proneural gene
self-stimulation mediated by the SMC-specific enhancers.
Excess Egfr signaling can overrule lateral inhibition and
allow adjacent cells to become SMCs and sensory organs.
Thus, the Egfr and Notch pathways act antagonistically
in notum macrochaetae determination (Culí, 2001).
The earliest stage in macrochaetae development is the
formation of the proneural clusters of ac-sc expression. Accumulation of Sc in cells of proneural clusters located
at the more central positions of the wing disc decreases upon
reduction of the level of Egfr signaling. The effect is cell-autonomous,
which indicates that reception of the signal is
important for cells to express sc properly. In contrast, more
marginally located clusters, like the notopleural or scutellar,
are unmodified or slightly enhanced under conditions of
insufficient Egfr signaling. It is known that expression of
ac-sc in different proneural clusters depends on separate,
functionally independent enhancers which are thought to
respond to local, specific combinations of transcription factors
(prepattern). The different,
spatially restricted effects of the insufficiency of Egfr
function may thus be due to interference in the deployment or
function of particular factors expressed in the affected area.
Interestingly, the expression of the homeobox genes of the
iroquois complex, necessary for the expression of ac-sc in
many notum proneural clusters, is
especially sensitive to the expression of the Vein Egfr ligand
in the central region of the notum.
Alternatively, since Egfr function is a well known requisite
for growth and patterning of imaginal discs, the
reduced expression of sc may be due to a more general
impairment of the patterning of the central area of the disc (Culí, 2001).
The data support a key role for Egfr signaling in the
emergence of the notum macrochaetae SMCs from proneural
clusters. Indeed, expression of the Egfr inhibitory ligand Aos
exclusively in proneural clusters, a condition that permits
essentially wild-type Sc accumulation in these clusters, almost
completely suppresses the appearance of SMCs and SOs. SMC
emergence is also impaired in discs from heat-treated
temperature sensitive Egfr larvae and in clones of cells expressing
UAS-rafDN2.1. Moreover, when the cells that accumulate RafDN2.1 occupy positions where SMCs normally appear, wild-type
neighboring cells give rise to displaced SMCs. This
phenomenon is reminiscent of and in accordance with the
observation, made with mosaic individuals, that when the
position of a dorsocentral bristle is in ac minus territory, this bristle
does not develop, but a nearby ac plus cell can give rise to a
dorsocentral bristle displaced from its wild-type position. The cell-autonomous effect of RafDN2.1 indicates that reception of the Egfr signal, mediated by the Ras/Raf/MAP kinase cassette, is essential for notum
macrochaetae SMC determination. This was further
substantiated by the cell autonomous induction of SMCs and
bristles in clones of cells overexpressing a constitutively
activated form of Ras. Taken together, these results indicate
that reception of the Egfr signal promotes sc expression and
SMC determination (Culí, 2001).
In the notum anlagen the expression of rho/ve
occurs mainly in proneural clusters and this expression
is dependent on ac-sc. Rho/ve facilitates the processing of
Spitz, an activating ligand of Egfr. The soluble, active form of Spitz promotes
ectopic sc expression and SMC emergence. Hence, these data
suggest that, in proneural clusters, Ac-Sc promote expression
of rho/ve, which by activating Spitz, would stimulate Egfr
signaling in the cells of the cluster. (The Vein Egfr
ligand probably does not specifically act in proneural clusters,
because many of these lie outside of its expression domain). It is thus
proposed that Egfr mediates a mutual positive signaling
among cells of the proneural cluster, which promotes SMC
emergence by probably reinforcing ac-sc expression. This positive signaling is called lateral cooperation. Evidently, this does
not exclude an autocrine activation of the Egfr pathway in
the cells that express AS-C proteins, but the lateral
cooperation hypothesis is favored since it is well established in other
systems that the Egfr pathway is used mainly for intercellular
communication. This signaling should facilitate
the acquisition of the SMC state by one or a few cells of a
proneural cluster (Culí, 2001).
The SMC state is associated with greatly increased levels
of proneural protein. These are
accomplished by the self-stimulation of ac, sc and ase
mediated by AS-C enhancers that activate these genes
specifically in the cells that become SMCs. Since Ras1V12 elicits the
expression of both sc and SRV-lacZ, it is proposed that, in the
extant proneural clusters, the SMC-specific enhancers are
targets of Egfr signaling. Unidentified effector(s) of the
Egfr/Ras pathway should facilitate the self-stimulation of
the proneural genes mediated by the SMC-specific enhancers
by, possibly, binding to these enhancers. Conclusive evidence
in support of this role requires the identification of the
signaling effector(s) and of their interaction with the
enhancer. Interestingly, overexpression of the effector
Pointed P1 promotes development of many extra
macrochaetae on the notum and putative Ets-domain binding sites have been identified in the sc and ase SMC enhancers (GTGGAAAT and ACGGAAAC,
respectively) (Culí, 2001).
Egfr-mediated lateral cooperation should tend to activate the
SMC-specific enhancers in many cells of the proneural clusters. This, however, is prevented by N signaling, which is
activated by Ac and Sc in the cells of the cluster. This signaling, by means of the bHLH proteins of the E(spl)-C, blocks the ac-sc-ase self-stimulatory loop promoted by the SMC-specific enhancers. However, within a proneural cluster the cells of the proneural field accumulate greater amounts of Ac-Sc proteins. As it has been
hypothesized that cells that signal the most are the least
inhibited by their neighbors, eventually, a cell of the proneural
field will be released from the inhibitory loop and its levels of
E(spl)-C bHLH protein will become minimal. This cell will turn on the ac-sc-ase self-stimulation and become an SMC. The SMC signals maximally to its
neighbors and prevents them from following the same fate
(lateral inhibition). These results add to this scenario the requirement for Egfr-mediated signaling for one cell of the proneural field to turn
on the ac-sc-ase self-stimulatory loops and become an SMC. According to this model, Ac-Sc activate both the N-and Egfr-mediated signaling pathways, with their SMC-suppressing
and SMC-promoting abilities, respectively, and
both signaling systems appear to act on the same SMC-specific
enhancers (Culí, 2001).
If Senseless is required for proneural expression, ectopic expression of Sens may induce proneural gene expression. Indeed, ectopic expression of Sens using the dpp-GAL4 driver causes robust expression of Sens in the expected wing stripe. This expression causes the formation of numerous bristles and sensilla campaniforma in the adult wing in proximity of the third wing vein where dpp is normally expressed. Similarly, ectopic expression of Sens in the leg disc causes many supernumerary bristles in the sternopleural area as well as in more distal portions of the leg. Ectopic bristles are observed with all UAS-sens reporters. Some UAS-sens transgenes driven by dpp-GAL4 cause very severe tufting in the adult notum, wings, and legs, and loss of tissues in other portions of imaginal discs, e.g., wing margins and distal leg structures. It is concluded that ectopic expression of Sens is sufficient to initiate ectopic external sensory organ development (Nolo, 2000).
To determine the molecular cascade underlying the formation of the extra external sensory organs, wing discs of UAS-sens; dpp-GAL4 larvae were stained with anti-Scute antibodies. Ectopic Sens expression causes ectopic activation of Scute and Asense. Hence, Sens is able to activate proneural gene expression. This provides a molecular basis for the generation of additional external sensory organs, since ectopic proneural gene expression has previously been shown to be sufficient to induce ectopic PNS organ formation (Nolo, 2000).
If Sens induces proneural gene expression and proneural genes are required for Sens production, a super-additive or synergistic interaction between sens and proneural genes may occur. Therefore, the weakest UAS-sens transgene (C1) was expressed in combination with an UAS-scute and an UAS-atonal transgene under the control of dpp-Gal4. Overexpression of Scute or Atonal alone causes a relatively mild phenotype with relatively few extra bristles. Scute expression induces Sens expression, but the expression levels of Sens are lower than those induced by dpp-Gal4; UAS-sens. Ectopic expression of Sens with the dpp-Gal4 driver causes a stronger phenotype when compared to ectopic expression of Scute or Atonal. However, simultaneous overexpression of Sens and Scute or Atonal causes severe tufting, including in many areas where Scute, Atonal, or Sens, when expressed individually, does not normally cause ectopic bristles. These areas do correspond to areas where the dpp-Gal4 driver has previously been shown to be expressed. It is therefore concluded that sens and the proneural genes can interact in a synergistic fashion (Nolo, 2000).
Each sensory organ of the Drosophila peripheral nervous system is derived from a single sensory organ precursor cell (SOP). These originate in territories defined by expression of the proneural genes of the Achaete-Scute complex (AS-C). Formation of ectopic sensilla outside these regions is prevented by transcriptional repression of proneural genes. The BTB/POZ-domain transcriptional repressor Tramtrack (Ttk) co-operates in this repression. Ttk is expressed ubiquitously, except in proneural clusters and SOPs. Ttk over-expression represses proneural genes and sensilla formation. Loss of Ttk enhances bristle-promoting mutants. Using neural repression as an assay, functional domains of Ttk have been dissected, confirming the importance of the Bric-a-brac-Tramtrack-Broad complex (BTB) motif. The Ttk BTB domain is a protein-protein interaction motif mediating tetramer formation (Badenhorst, 2002).
The ablation of SOPs is caused by the repression of proneural genes. Ectopic expression of Ttk69 under the control of a heat-shock promoter inhibits achaete and scute transcription. Accumulation of Asense protein is also blocked. Over-expression of Ttk88 also perturbs achaete, scute and asense expression showing that both isoforms of Ttk can repress the AS-C. Significantly, though, the extent of repression is lower. This could reflect differences in protein stability of the two isoforms. Both are targeted for ubiquitin-dependent proteolysis. However, Ttk69, unlike Ttk88, is post-translationally modified by the small ubiquitin-like molecule dSmt3. This modification has been shown to protect IkappaBalpha from ubiquitin-dependent degradation (Badenhorst, 2002).
Ttk blocks SOP recruitment by repressing transcription of the proneural genes. In the developing PNS, Ttk completely inhibits achaete and asense expression and blocks part of the scute expression profile. Surprisingly, in the embryonic central nervous system (CNS), Ttk over-expression only represses asense but has no effect on achaete. Inspection of the promoters of the proneural genes reveals that the immediate 5' promoter region of asense contains many clustered consensus Ttk69-binding sites, suggesting that Ttk inhibits asense by directly repressing the proximal promoter. In contrast, the upstream promoter region of achaete does not contain large numbers of consensus sites. A cluster of Ttk69-recogntion sites is found downstream of achaete. It is conceivable that specific repression of achaete in the PNS is achieved by blocking PNS-specific enhancers while not affecting regulatory elements required for expression in the CNS. The existence of separate enhancers directing expression of achaete and scute in the CNS and PNS has been inferred from deletions and inversions that affect subsets of the achaete expression profile (Badenhorst, 2002).
Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).
To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2–5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).
Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10–11 embryos, approximately 4–7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).
Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).
Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).
A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).
The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).
Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).
In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).
The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.
The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).
Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).
To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).
To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).
In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).
Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).
The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).
The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).
Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).
S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).
To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).
Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).
Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).
Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).
Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).
Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).
The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).
It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).
During early Drosophila and C. elegans development, the germ cell precursors undergo a period of transcriptional quiescence. Germ cell-less (Gcl), a germ plasm component necessary for the proper formation of 'pole cells', the germ cell precursors in Drosophila, is required for the establishment of this transcriptional quiescence. While control embryos silence transcription prior to pole cell formation in the pole cell-destined nuclei, this silencing does not occur in embryos that lack Gcl activity. The failure to establish quiescence is tightly correlated with failure to form the pole cells. Furthermore, Gcl can repress transcription of at least a subset of genes in an ectopic context, independent of other germ plasm components. These results place Gcl as the earliest gene known to act in the transcriptional repression of the germline. Gcl's subcellular distribution on the nucleoplasmic surface of the nuclear envelope (Jongens, 1994) and its effect on transcription suggest that it may act to repress transcription in a manner similar to that proposed for transcriptional silencing of telomeric regions (Leatherman, 2002).
gcl is required to repress transcription during the establishment of the germ cell lineage. To determine if this activity is dependent or independent of other germ plasm components, the effect of ectopically localizing Gcl on transcription was examined. Replacement of the 3'UTR of the gcl transcript with the 3'UTR of bicoid results in the anterior localization of gcl mRNA and protein to the anterior pole of the embryo. In these 'hgb' embryos, a slightly variable but consistent decrease was found in the intensity of H5 staining (H5 is a monoclonal antibody that recognizes a phosphorylated form of RNA polymerase II that is associated with active transcription) in the anterior nuclei compared to control embryos throughout the syncytial blastoderm stage, and this decrease indicates that Gcl is sufficient to repress transcription ectopically. However, the anterior expression of Gcl clearly does not lead to complete silencing of the anterior nuclei, since some H5 staining persists (Leatherman, 2002).
The reduced H5 staining observed in the anterior of the hgb embryos could be due to global partial repression of all genes, or it could result from a specific subset of genes being severely repressed while others are unaffected. To distinguish between these possibilities, the expression was examined of specific genes whose expression pattern includes the anterior of the embryo, including sisA, sisB (scute), tailless, huckebein, hunchback, and knirps. These genes are all independently activated by maternally contributed factors, so any effects on their transcription are likely to be direct rather than a consequence of an earlier defect. By using in situ hybridization, it was found that the early anterior expression domains of sisA, sisB, tailless, and huckebein are severely repressed in all of the hgb embryos examined, but no effect was seen on hunchback and knirps expression. These data suggest that the transcriptionally repressive effect of Gcl is not global, but rather specific to a subset of genes. Gcl is also present in a variety of tissues later in development, at times when transcription is active, which further suggests a non-global mode of silencing (Leatherman, 2002).
In the wing discs of Drosophila, the mechanosensory precursor cells are singled out from clusters of cells blocked at the G2 phase of the cell cycle. This mitotic quiescence and the selection of the precursors are under strict spatio-temporal control. G2 cells were forced to enter mitosis by overexpression of string, the Drosophila homolog of the cdc25 gene. Premature entrance in the cell cycle is associated with a loss of precursor cells. Precursors are lost consecutively to a transcriptional down-regulation of the determinant proneural achaete/scute genes. This down-regulation results from an over-activation of the Enhancer of Split genes, known as effectors of the Notch signalling pathway. It is concluded that exit from the cell cycle is required for proper neural cell fate determination (Nègre, 2003).
Thus, forcing G2 arrested cells into mitosis results in a loss of adult sense organs. The corresponding precursors are also lost. This result was obtained by using two distinct transgenic systems to control the timing and spatial location of stg-overexpression. In both cases, precursors are not selected because ac/sc proneural expression is repressed. This repression occurs at a transcriptional level. Noteworthy is the fact that bristles are lost using either the sca-Gal4 driver to overexpress stg, or the klu-Gal4 driver; this demonstrates that overexpression of stg not only prevents the early accumulation of Ac/Sc (klu-Gal4 driver), but can also downregulate Ac/Sc after the levels of these proteins have started to rise (sca-Gal4 driver). Thus, it is concluded that the arrest in G2 is necessary for proper determination of precursor cells. The complexity of the 5' regulatory sequences of stg indicates that this mitotic regulator might itself integrate information from patterning genes. For instance, the regulatory regions of the stg gene possess putative recognition sites for Achaete and Scute transcription factors. Here, it has been shown that stg can itself control the expression of developmental genes. The effect of stg on cell determination is unlikely to be direct, however, since the only known function of stg is to dephosphorylate the CDK1- cyclin B mitotic kinase. Future genetic approaches may reveal whether or not String has other biochemical targets (Nègre, 2003).
After stg overexpression using the klu-Gal4 driver, it was observed that E(Spl) expression is maintained in proneural regions in absence of Ac/Sc. It was also observed that stg can cause accumulation of the E(Spl) bHLH genes outside of proneural clusters, in a cell-autonomous mode. Maintenance of expression of E(Spl), a transcriptional repressor of the ac/sc expression, is relevant. It can functionally justify the loss of precursor cells. Nevertheless, it has been reported that E(Spl) transcription is dependent on the ac-sc genes in the proneural clusters. One explanation could be that deregulation of the cell cycle directly or indirectly increases transcription of the E(Spl) genes by modifying activity of upstream activators of the E(Spl) expression. Considering this hypothesis, E(Spl) should sometimes be expressed in incorrect positions compared to its wild-type expression. On the contrary, because E(Spl) genes are expressed at the exact positions for proneural clusters, it is suggested that forcing cell cycle more likely affects E(Spl) expression at a post-translational level rather than at a transcriptional level. In the mutants, initial transcription of E(Spl) genes would still have been dependent on Ac/Sc, which begin to accumulate in proneural domains. But, it is known that at least E(Spl) m5, m7 and m8 isoforms contain a PEST-rich motif that harbors an invariant Serine residue, which is phosphorylated by the casein kinase II. Casein kinase II is a ubiquitous serine/threonine kinase whose activity fluctuates with cell cycle progression. Phosphorylation usually regulates protein stability via activation of PEST motifs. Modification in the phosphorylation status of some E(Spl) proteins could exhibit a longer half-life in vivo, thus leading to their predominance over the proneural proteins, and therefore to an inhibition of neurogenesis. In other words, premature entry in the cell cycle would introduce an external bias in the highly dynamic process that opposes the antagonistic E(Spl) and Ac/Sc proteins and which normally occurs in cells of proneural clusters. It would confer an advantage to E(Spl) over proneural activity and would explain persistence of E(Spl) proteins after proneural products have disappeared (Nègre, 2003).
Altogether, these results suggest that proneural competence can only develop in mitotically arrested cells. The programmed incompatibility between cell cycling and proneural product accumulation may have several general, and not mutually exclusive, functional correlates. In proneural clusters, keeping cells together in a continuous group may be necessary. Indeed, cell interactions could be required to maintain Ac/Sc levels via indirect autoregulation through cell- cell signalling. Furthermore, a G2 arrest may be necessary to preserve a balance between the levels and/or activities of E(Spl) and Ac/Sc products that could directly or indirectly be dependent on post-transductional modifications. The relative strength of the signal impinging on a given cell determines whether products of the proneural genes or products of the E(Spl) become finally predominant. Changing the cell cycle phase could disrupt this equilibrium. Finally, divisions that underly normal cell proliferation and those involved in the fixed lineage of the precursor cell, make different demands on the cytoskeletal machinery. The asymmetric divisions of the precursor cell are strictly controlled in orientation and in time. These controls are presumably essential to realize a correct lineage. A period of mitotic quiescence may give the precursor cell the time and/or conditions required to reorganize its cytoskeleton in order to shift to an asymmetric mode of division. Although a quiescent period systematically precedes the emergence of neural precursors, re-entry into mitosis is independently controlled in the precursor and the surrounding epidermis. This suggests that quiescence is a necessary step preceding the lineage of the precursor. Moreover, the decision of the precursor to enter in its lineage is made independently of the mitotic state of its surrounding cells (Nègre, 2003).
In this study, causal relationship has been demonstrated to exist between cell cycle and neural determination in an endogenous system: the Drosophila wing imaginal discs, in which E(Spl) effectors of the Notch pathway behave as integrative sensors of the cell cycle status (Nègre, 2003).
In flies, scute (sc) works with its paralogs in the achaete-scute-complex (ASC) to direct neuronal development. However, in the family Drosophilidae, sc also has acquired a role in the primary event of sex determination, X chromosome counting, by becoming an X chromosome signal element (XSE) -- an evolutionary step shown here to have occurred after sc diverged from its closest paralog, achaete (ac). Two temperature-sensitive alleles, scsisB2 and scsisB3, which disrupt only sex determination, were recovered in a powerful F1 genetic selection; these alleles were used to investigate how sc was recruited to the sex-determination pathway. scsisB2 revealed 3' nontranscribed regulatory sequences likely to be involved. The scsisB2 lesion abolished XSE activity when combined with mutations engineered in a sequence upstream of all XSEs. In contrast, changes in Sc protein sequence seem not to have been important for recruitment. The observation that the other new allele, scsisB3, eliminates the C-terminal half of Sc without affecting neurogenesis and that scsisB1, the most XSE-specific allele previously available, is a nonsense mutant, would seem to suggest the opposite, but housefly Sc is shown to be able to substitute for fruit fly Sc in sex determination, despite lacking Drosophilidae-specific conserved residues in its C-terminal half. Lack of synergistic lethality among mutations in sc, twist, and dorsal argue against a proposed role for sc in mesoderm formation that had seemed potentially relevant to sex-pathway recruitment. The screen that yielded new sc alleles also generated autosomal duplications that argue against the textbook view that fruit fly sex signal evolution recruited a set of autosomal signal elements comparable to the XSEs (Wrischnik, 2003).
Thus a lesion in a new sex-specific allele led to an analysis of a cis-acting regulatory region downstream of the transcription unit that is likely to help drive the extremely early expression specifically required for XSE function. Strong functional synergism was observed between a deletion of this 3' sequence and lesions engineered in a heptameric regulatory sequence upstream of sc as potentially important for XSE evolution (Wrischnik, 2003).
The recovery of two new mutations, scutesisB2 and scutesisB3, that affect only sex determination, stimulated experiments that not only constrain speculation about how this member of a neuronal patterning gene complex acquired a key role in sex determination, but also point to specific regulatory information likely to have been involved. This study also sheds light on the molecular nature of some older scute mutants and on the effects of temperature and autosomal genes on X chromosome counting (Wrischnik, 2003).
The discovery that houseflies have ac showed that the duplication event that separated ac and sc occurred long before sc acquired a role in X chromosome counting. Thus, a change in the mechanism of sex determination does not appear to have been a factor driving that duplication event. The question of whether the partial functional redundancy that exists between sc and ac in neurogenesis was relevant to recruitment remains open. Notwithstanding that redundancy, Ac and Sc proteins have unique protein sequence identities that extend across species regardless of whether those species use sc as a sex signal (Wrischnik, 2003).
The discovery that eliminating the C-terminal half of Sc strongly interferes with sex determination but not bristle formation led to a search for other indications that the distal half of Sc might have become uniquely specialized for sex determination. Residues were found that clearly distinguish Sc in fly species that use it as a sex signal (the family Drosophilidae) from Sc in those that do not (other higher Diptera such as the housefly); however, the significance of this fact was undermined by the observation that the frequency of such residues was no higher for Sc than for the closely related proteins Ac and L'sc, which seem to not have important roles in X chromosome counting. Direct evidence that these Drosophilidae-specific conserved Sc protein sequences were not an important factor in the evolution of its sex determination was given by the demonstration that Sc from the housefly could substitute for D. melanogaster Sc in a transgenic assay for sex-determination activity. The fact that melanogaster Ac substituted only poorly for melanogaster Sc in the same assay showed that conserved residues distinguishing these two paralogs in all species examined are likely to be important for sex-determination function, but those differences evolved before sc became an XSE (Wrischnik, 2003).
Sc and Ac behave differently in their ability to regulate Sxl, but the biological significance of the transgene results presented in this study is far more certain because the proteins whose sex-determination activities were compared were produced at the wild-type time, place, and level. Moreover, large numbers of independent transgene lines were assayed, all of which had been backcrossed many generations to a standard line to ensure similar genetic backgrounds. Meaningful comparisons can be made in sex-determination signal studies only when genetic background is carefully controlled. The large differences in XSE activity that was observed among transgene lines show that chromosomal position effects can have a significant influence on gene expression even prior to the blastoderm stage (Wrischnik, 2003).
Given that the C-terminal half of Sc protein seems to not be uniquely devoted to sex determination, one might ask whether the sex-determination-specific mutant phenotype of scsisB3 can be explained simply by the hypothesis that loss of this region reduces all sc activities to the same extent, but the minimum activity that suffices for normal neurogenesis is very much lower than that for sex determination. Greater sensitivity of sex determination to disruption would not be surprising in view of the striking gene dose sensitivity that is an intrinsic part of X chromosome counting and the fact that ac seems likely to take up part of the slack for proneural targets when sc activity is reduced. The nearly normal bristle phenotype of the duplicated nonsense mutant allele scsisB1 supports the view that low levels of sc activity do suffice for neurogenesis but not sex determination. Nevertheless, a strong genetic argument can be made that loss of the C-terminal half of the protein does preferentially interfere with sex-determination activity, not just with general function. The sensitive emc suppression assay of sc proneural activity showed that even scsisB3, the more mutant of the two new alleles and the one that eliminates the C terminus, must be at least 50% as active as the wild type in this respect, while the female-lethal phenotype of both new alleles shows that they must have considerably < 50% of normal XSE activity, since a deletion of sc has no dominant-lethal female-specific effect. The selective forces that have shaped and maintained the distal half of Sc protein are simply likely to be too subtle to be apparent in the functional assays used in this study -- a caveat for anyone hoping to make functional predictions regarding mutant phenotype from striking patterns of sequence conservation (Wrischnik, 2003).
This study provides the first direct evidence that the heptamer sequence CAGGTAG found clustered upstream of all scute and other XSEs is functionally relevant to sex determination. The importance of this sequence might have been underestimated had the strong interaction between heptamer knockout mutations and deletion of 3' regulatory sequences not been observed. Further study will reveal whether these synergistic 5' and 3' lesions are truly specific for sex determination, affecting only preblastoderm stage expression of sc. The fact that the scsisB2 lesion had no effect on neurogenesis favors specificity (Wrischnik, 2003).
The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression
during development of the sensory organs of Drosophila through enhancer binding.
Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the
promoter and facilitates the enhancer-promoter communication required for
proneural development. This communication is regulated by Osa,
which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin
remodeling complexes, and this study shows that Osa negatively regulates ac/sc.
Consequently, Pannier and Chip also play an essential role during repression of
proneural gene expression. This study suggests that altering chromatin structure
is essential for regulation of enhancer-promoter communication (Heitzler, 2003).
ChipE is a viable allele of Chip that
is associated with a point mutation in the LIM-interacting domain
(LID), which specifically reduces interaction with the bHLH proteins
Ac, Sc, and Da. As a consequence, the ChipE mutation
disrupts the functioning of the proneural complex encompassing Chip,
Pnr, Ac/Sc, and Da. A homozygous ChipE mutant
shows thoracic cleft and loss of the DC
bristles, similar to loss of function pnr alleles (Heitzler, 2003).
To identify new factors that regulate this proneural complex, a
screen was performed for second-site modifiers of the ChipE
phenotypes. One allele
of osa (osaE17) was found among the putative mutants.
OsaE17 corresponds to a loss-of-function allele, and
homozygous embryos die with normal cuticle patterning. Both
osaE17 and null alleles of osa
(osa616 or osa14060) enhance the
cleft but suppress the loss of DC bristle phenotypes of
ChipE flies. Inde