hedgehog
In Drosophila, the sine oculis (so) gene is
important for the development of the entire visual system, including Bolwig's
organ, compound eyes and ocelli. Together with twin of eyeless,
eyeless, eyes absent and dachshund, so
belongs to a network of genes that by complex interactions initiate eye
development. Although much is known about the genetic interactions of the
genes belonging to this retinal determination network, only a few such
regulatory interactions have been analysed down to the level of DNA-protein
interactions. An eye/ocellus
specific enhancer of the sine oculis gene has been identified that is directly regulated
by eyeless and twin of eyeless. This regulatory element has been further characterized
and a minimal enhancer fragment of
so has been identified that sets up an autoregulatory feedback loop crucial for proper
ocelli development. By systematic analysis of the DNA-binding specificity of
so the most important nucleotides for this interaction have been identified.
Using the emerging consensus sequence for SO-DNA binding a
genome-wide search was performed and eyeless has been identified as
well as the signalling gene hedgehog as putative targets of
so. These results strengthen the general assumption that feedback loops
among the genes of the retinal determination network are crucial for proper
development of eyes and ocelli (Pauli, 2005).
In-vitro data on the autoregulatory element with the known so
target sequence of lz and the AREC3/Six4-binding site, the consensus
sequence GTAANYNGANAYC/G was identified as necessary for SO binding to DNA. This consensus
sequence was taken as a basis for scanning the Drosophila genome for similar
sites. In total, 1632 putative so targets emerged
from this survey. Out of the affected genes several candidates are already known
to be involved in eye development (Pauli, 2005).
decapentaplegic (dpp) signalling plays an important role
in the complex regulatory network of eye development. In dpp mutant
eye discs, so, eya and dac are not expressed, whereas
dpp is able to initiate ectopic expression of so and
dac when expressed at the anterior margin of the eye disc.
Conversely, dpp expression is patchy in eye discs of eya and
so loss-of-function mutants, suggesting that eya and
so are required for either initiation or maintenance of dpp
at the posterior disc margin before MF initiation (Pauli, 2005).
hh is required for dpp expression at the posterior margin
before MF initiation, and dpp expression is induced by hh in
the MF, supporting the assumption that dpp is downstream
of hh signalling. Since dpp alone is not able to rescue
posterior margin clones of hh, there have to be more eye-relevant
target genes of hh signalling during third instar larval development.
dpp in combination with eya can restore photoreceptor
differentiation in posterior margin clones lacking smoothened
(smo) expression (smo is a cell-autonomous receptor of
hh signalling). This shows that dpp, in combination with
eya, is able to bypass the requirement of hh during eye
development. Taken together, it is evident that hh is necessary
for proper eya and dpp expression, both of which can induce
so, and it contains two so target sites. It is therefore
hypothesized that the transcriptional complex consisting of Eya and So, as
with ey, might also feed back on hh in order to drive the
furrow during late eye development. In this model the genetic cascade starts
with hh, which induces dpp and eya, moves on to
so and through the So/Eya complex feeds back to hh in order
to maintain hh expression as a driving force of the MF (Pauli, 2005).
The impact of these so-binding sites in the hh enhancer
on eye development becomes evident from the fact that hh1
(bar-3) mutant flies have smaller eyes. The severity of the
hh1 mutant phenotype is probably diminished by an
additional putative So-binding site that resides outside the area covered by
the hh1 deletion. If functional, this region (5' to
the hh1 deletion) might mediate a residual
hh-expression that overcomes the loss of the other sites to some
extent. Another possible explanation for the rather weak
hh1 phenotype might be that the feedback of so on
hh is not crucial for MF initiation but still might be of importance
for the well-balanced expression of hh during MF propagation (Pauli, 2005).
Drosophila development depends on stable boundaries between cellular territories, such as the embryonic parasegment boundaries and the compartment boundaries in the imaginal discs. Patterning in the compound eye is fundamentally different: the boundary is not stable, but moves (the morphogenetic furrow). Paradoxically, Hedgehog signaling is essential to both: Hedgehog is expressed in the posterior compartments in the embryo and in imaginal discs, and posterior to the morphogenetic furrow in the eye. Therefore, uniquely in the eye, cells receiving a Hedgehog signal will eventually produce the same protein. The mechanism that underlies this difference is the special regulation of hedgehog (hh) transcription through the dual regulation of an eye specific enhancer. This enhancer requires the Egfr/Ras pathway transcription factor Pointed. Recently, others have shown that this same enhancer also requires the eye determining transcription factor Sine oculis (So). These data are discussed in terms of a model for a combinatorial code of furrow movement (Rogers, 2005).
There are two known eye-specific hedgehog (hh) mutations:
hhbar3 (also known as hh1) and
hhfurrow stops early (or hhfse). Both
are associated with deletions in the first intron.
hhbar3 is a homozygous viable allele with a strong
recessive eye phenotype resulting from arrest of the morphogenetic furrow.
hhfse is a gamma-induced viable allele with a weaker eye
phenotype. PCR and direct sequencing were used to determine the precise end-points of the deletions. The hhbar3 deletion
is 1885 bp and the hhfse deletion lies within the span of
hhbar3, but is shorter. Both
hhbar3 and hhfse are viable and can be
maintained as homozygous stocks, although they are not as vigorous as wild
type. This is probably not due to second-site recessive lethal mutations, since
lines were derived that are isogenic for the X and major autosomes and they are
no more vigorous. The cuticles and nervous system (by anti-Elav
and anti-Futsch stains) of the isogenic hhbar3 and
hhfse embryos were examined, and no detectable phenotypes were found (Rogers, 2005).
To determine if either of these two eye-specific alleles are null for
hedgehog function in the eye, all viable pair-wise
combinations of these alleles, wild-type and two zygotic lethal alleles
(hhAC and hh8), were derived.
hhAC is a single gene deletion that removes both the start
sites for transcription and translation.
hh8 (also known as hh13C) is a
chain-terminating mutation in the coding sequence. Both
alleles are zygotic lethal with strong cuticle phenotypes.
hhAC is thought to be a null because of the strength of
its phenotype and the nature of its lesion. On
phenotypic grounds and comparison with other alleles, other groups have also
reported hh8 to be functionally amorphic (Rogers, 2005).
These alleles form a series for adult eye phenotype. This
was quantified by counting eye facets in adult females; hhfse, hhbar3 and hh8
heterozygotes are not significantly different from wild type. However,
hhAC is slightly dominant, with an eye that is about 10%
smaller than wild type (although this difference is not statistically
significant) (Rogers, 2005).
By facet number, hhbar3 is a strong, eye-specific
hypomorph. It is fully recessive in trans to wild type, has a severely reduced
eye when homozygous (68% smaller than
hhbar3/hh+) and in trans to the null
hhAC it is smaller still (82% smaller than
hhbar3/hh+). This suggests that
hhbar3 is not an amorph for eye size by Muller's test: the
phenotype becomes stronger in trans to the null.
hhfse is similar to but weaker than
hhbar3: the hhfse homozygous eye is
only 32% smaller than hhfse/hh+ and in
trans the null (hhAC), it is further reduced to 78%. Thus,
by both measures (phenotype as a homozygote and in trans to a null),
hhbar3 is a strong hypomorphic allele and
hhfse is a weaker hypomorph. From the 95% confidence
limits, all these results are statistically significant (Rogers, 2005).
Probably hhbar3 and hhfse affect a
transcriptional enhancer and not the protein itself or the gene promoter,
because neither lesion directly affects the coding sequence. In sequencing 23
cDNAs from eye-imaginal discs, no alternative first exon or start
site was found in the region of the two mutations (Rogers, 2005).
These two eye-specific alleles of hedgehog were characterized and they were found to delete elements that are specifically necessary for expression
in the developing eye, posterior to the morphogenetic furrow. This
hedgehog eye enhancer drives expression in all of the developing
ommatidial cells except the R8. This element was reduced to a 203 bp minimal
fragment that is sufficient for reporter expression. The
hedgehog eye enhancer is regulated by pointed in vivo and
bound by Pointed in vitro. Since Egfr/Ras-driven Pointed activates reporters in
all the cells except the R8, it is suggested that the hedgehog expression
in the developing eye is driven by this enhancer and that Hedgehog is
expressed in the developing ommatidial cells excepting the R8 (Rogers, 2005).
It is proposed that hhbar3 is indeed null for
hedgehog expression in the developing eye, consistent with the loss
of detectable antigen. This appears to contradict facet count data, which
show that hhbar3 is not null for eye size. It is suggested that
hedgehog functions elsewhere (probably in the eye disc margin),
expressed at some lower level, and acts redundantly with Decapentaplegic to
drive the early phases of furrow progression. This is consistent with data
from others for an early role for hedgehog in the eye margin for
furrow initiation, and with a proposed redundancy between
hedgehog and dpp in the furrow.
The enhancement of the hhbar3 phenotype when it is placed
in trans to a null (hhAC) suggests that hhbar3 may reduce, but not eliminate this early function (Rogers, 2005).
Several examples of eye-specific transcriptional enhancers have been
characterized. A number of these are in genes that act early in retinal
determination (eyes absent, dachshund and sine oculis), and
are not directly involved in the morphogenetic furrow. Some
enhancers that function in and posterior to the morphogenetic furrow have also
been studied. One example is the atonal gene, which has been shown to
have two regulatory enhancers with specific and different activities in the
furrow.
Interestingly the atonal enhancers produce almost the reciprocal
expression pattern of the hedgehog eye enhancer described here:
hedgehog is expressed in all cells except the R8 and atonal
expression is in only the R8, posterior to the furrow. Furthermore,
atonal mutations can affect hedgehog signaling, although
this may be indirect, and indeed, hedgehog is also known to regulate
atonal. Other enhancers that act posterior to the furrow have been
characterized in the rough, sevenless and prospero genes,
but none of these appears to show the particular type of regulation
described in this study (Rogers, 2005).
A similar DNA fragment from the hhbar3 region confers post-furrow, eye-specific expression on a lacZ reporter (Pauli, 2005). The consensus binding site for another transcription factor was characterized: the retinal determination protein Sine oculis (So). Two So-binding sites were found in the hhbar3 region, and it was shown that these are necessary for the normal function of the hedgehog eye enhancer. A So site tetramer is
sufficient to drive reporter expression in the entire presumptive eye field in
the third instar disc. One of the two So sites lies
within the 203 bp minimal element (Rogers, 2005).
Taken together, both sets of data suggest that Pointed and So activation
at the minimal element are each necessary, but that neither is sufficient for
the specific activation of the hedgehog eye enhancer posterior to the
furrow. It is proposed that they act together to confer this dual regulation. This
is consistent with the following model: that special dual regulation of hedgehog is the mechanism which makes the morphogenetic furrow move, unlike the stable compartment boundaries. It is suggested that this dual regulation depends on one 'selector' signal that is eye specific (So), to differentiate the furrow from boundaries in other organs. The second component must act to close a loop such that cells which receive the furrow inducing signal will later send it, after a delay, to make the boundary move forward. This 'signal' component is Pointed, acting downstream of Egfr/Ras signaling in the assembling ommatidia. This may be a case of 'selector' and 'signal' transcriptional integration. Indeed, pointed itself has been shown to integrate 'selector' factors in muscle development. It is proposed that by this dual regulatory mechanism, a system that first evolved to divide the bauplan into metameric parasegments has been co-opted to drive a moving wave of differentiation in the developing eye (Rogers, 2005).
The Drosophila wing is formed by two cell populations, the anterior and posterior compartments,
distinguished by the activity of the selector gene engrailed (en) in posterior cells. EN governs growth and patterning in both compartments by controlling the expression
of the secreted proteins HH and Decapentaplegic (DPP) as well as the response of cells
to these signaling molecules. EN activity programs wing cells to express
hh, whereas in the absence of EN activity they are able to respond to HH by expressing dpp. As a
consequence, posterior cells secrete HH and induce a stripe of neighboring anterior cells across the
compartment boundary to secrete DPP (Zecca, 1995).
The normal growth of the wing disc requires
that posterior-specific genes, such as hedgehog and engrailed are not expressed in cells of the
anterior compartment. Hedgehog has the capacity to activate engrailed in the anterior
compartment but both hedgehog and engrailed are specifically repressed in anterior cells by
the activity of the neurogenic gene groucho. In groucho mutant discs, hedgehog and engrailed are
expressed at the dorsoventral boundary of the anterior compartment, leading to the ectopic
activation of decapentaplegic and patched and to a localised increase in cell growth associated with
pattern duplications. The presence of Engrailed in the anterior compartment causes the
transformation of anterior into posterior structures (de Celis, 1995).
The identity of anterior cells in the wing imaginal disc requires cubitus interruptus function. Anterior cells lacking ci express hedgehog and adopt posterior properties without expressing engrailed. Most clones cause an up-regulation of CI protein levels in surrounding cells, in a manner that is similar to that of CI along the A/P compartment boundary. Increased levels of CI can induce the expression of the HH target gene decapentaplegic in a HH-independent manner, suggesting that dpp is a target gene of CI. Thus, expression of CI in anterior cells controls limb development by restriction HH transcription to posterior cells and by conferring competence to respond to HH by mediating the transduction of this signal. The multiple role of CI in the anterior compartment suggests that anterior cell identity is not a default fate that imaginal cells adopt in the absence of engrailed (Domínguez, 1996).
wingless and hedgehog are not regulated by pair rule genes in the head as they are in the trunk. Instead they are regulated by head gap genes. Both wg and hh are normally expressed at blastoderm stage
in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage,
each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic
region: huckebein, orthodenticle and buttonhead regulate the anterior blastoderm expression of wg, while tailless and empty spiracles
regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe
(mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the
cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap
of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage (Mohler, 1995a).
Segment polarity genes are not activated in the anterior by pair-rule genes, as they are in the trunk but instead they are activated by gap genes. The segment polarity genes hedgehog and wingless are two important targets of cap'n'collar and forkhead, expressed in the anterior and posterior gut anlagen. cnc is expressed in the labral region of the foregut, fated to give rise to the dorsal pharynx and fkh is expressed in the adjacent esophagus. fkh is responsible for the maintenance but not the initiation of wg synthesis in the invaginating esophageal primordium. cnc is responsbile for the maintenance of wg in the dorsal pharyngeal domain of wg expression. Expression of hedgehog is similarly affected in cnc and fkh mutants. It is not known whether the actions of cnc and fkh on hh and wg are direct or indirect (Mohler, 1995 b).
Whereas the segmental nature of the insect head is well
established, relatively little is known about the genetic and
molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in
collier (col), which encodes the Drosophila member of the
COE family of HLH transcription factors and is activated
at the blastoderm stage in a region overlapping a
parasegment (PS0: posterior intercalary and anterior
mandibular segments) and a mitotic domain, MD2. col
mutant embryos specifically lack intercalary ectodermal
structures. col activity is required for intercalary-segment
expression both of the segment polarity genes hedgehog,
engrailed, and wingless, and of the segment identity gene
cap and collar. The embryonic head phenotype of col1 hemizygous mutant
embryos indicates a loss of skeletal structures derived from the
intercalary, and possibly mandibular, segments without transformation toward another
segment identity. To investigate this segmentation phenotype
in more detail, col expression was compared with that of
the segment polarity genes hh and wg. At the blastoderm stage,
the posterior limit of col expression is parasegmental
(PS0/PS1), since it precisely abuts the mandibular stripe of hh-expressing
cells. Whether its anterior limit is also
parasegmental cannot be answered at this stage because the
expression of segment polarity genes in pre-gnathal segments is not yet
established at this stage. Examination of early stage 11 embryos
shows that col expression overlaps the intercalary hh stripe and
abuts the intercalary Wg spot, indicating a parasegmental
anterior border for col expression. At this stage however, col
expression has been lost from the posterior part of PS0, since it
does not overlap mandibular Wg expression. The
cnc gene, which codes for a b-ZIP transcription factor, has been
postulated to act as a segment identity gene in the mandibular
segment. Consistent
with col being expressed in PS0, col and cnc expression only
partly overlap, in the region corresponding to the anterior
mandibular segment. Together, these data indicate a
parasegmental register of col expression at the blastoderm
stage, which is subsequently restricted to anterior PS0 (Crozatier, 1999b).
A determination was made of whether col mutations affect the
expression of wg and En, which mark the anterior and posterior
compartments of each segment, respectively. In col1 hemizygous embryos, both the
intercalary stripe of En and the spot of wg expression are
missing. Since col expression does not overlap the
intercalary Wg spot, the loss of this spot in col mutant
embryos suggested that col does not regulate wg expression
directly but possibly by an hh-dependent mechanism. It has indeed been found
that in col mutant embryos, the intercalary stripe of hh is also
absent, or much reduced. Together, these results show
that col controls hh, en and wg expression in the intercalary
segment and is required for establishing the PS(-1)/PS0
parasegmental border. The head skeleton structures ventral arm (VA) and lateral-gr”ten (LG), which are, respectively, either missing or
reduced in col mutant embryos, are also affected
in two other head mutants: crocodile (croc), which codes for a
forkhead-domain protein, and cnc. These
structures are also affected in embryos mutant for the homeotic
genes Dfd and lab, which are expressed, respectively, in the mandibular and
maxillary segments, and in the intercalary segment. col expression was examined in embryos
mutant for croc, cnc, Dfd or lab. In none of these embryos was there a change in col transcription. Conversely, no
changes could be detected for croc, Dfd or Lb expression in
col1 hemizygous embryos, indicating that expression of each
of these three genes is independent of col. In
contrast, col is required for cnc transcription in the posterior
intercalary segment at stage 9-10. Because this region
is anterior to the region of overlap between col and cnc
expression at the blastoderm stage, it is concluded that
this region corresponds to a secondary site of cnc expression initiated at
stage 9, under control of col activity. In cnc mutant embryos,
intercalary hh expression is normal,
indicating that hh and cnc are regulated by col, independent
of one another (Crozatier, 1999b).
forkhead is required for the activation of wingless, hedgehog and decapentaplegic in both the foregut and hindgut, considered to be ectodermal tissues. wingless is expressed initially in the whole hindgut primordium, but becomes restricted to a ring in the small intestine anterior to the outgrowing Malpighian tubules, and to a ring in the posterior region of the rectum. hedgehog is also expressed in the hindgut primordium but becomes restricted to a ring of cells posterior to the outgrowing Malpighian tubules in the future small intestine of the hindgut. A second hh expression domain is located in the anterior portion of the rectum. These two expression domains are adjacent to the wg expression domains. dpp is expressed in the hindgut primordium and later on one side in the large intestine of the hindgut tube, in between the small intestine and the rectum. Thus the expression domains of wg, hh and dpp subdivide the hindgut tube into a central portion (the large intestine) where dpp is expressed, and two flanking regions (the small intestine and the rectum) where wg and hh are expressed. In fkh mutant embryos, the foregut, the midgut and the hindgut epithelia are disrupted, and fkh is required for the activation of each of these genes in the fore- and hindgut primordia. fkh is expressed in the entire foregut and hindgut, whereas wg, hh and dpp are expressed only in restricted domains. Since the expression of these genes appear not to be established through cross-regulatory interactions, there must be other factors which act to spatially regulate wg, hh and dpp expression along the hindgut (Hoch, 1996).
Single-minded represses wingless, hedgehog and vnd gene expression in developing midline cells. By doing this sim plays a key role in proper patterning of the neuroetoderm by helping to generate the boundary between mesectoderm and ventral ectoderm. This process likely requires simultaneous function of
SIM as both a transcriptional activator (of slit and Toll) and transcriptional repressor within the developing midline cells (Xiao, 1996).
The regulation and function of the Hedgehog pathway activity has been compared in eye and wing discs, and there are significant differences. Whereas in the wing disc, engrailed function is required for hedgehog expression, in the eye disc activation and maintenance of hedgehog expression is achieved independently of engrailed. Nevertheless, engrailed functions in the eye disc, as elevated engrailed expression represses dpp, patched and cubitus interruptus in the eye disc, but does not disrupt morphogenesis. Regulation of decapentaplegic expression also differs: in the wing disc it is repressed in the anterior compartment by patched and in the posterior compartment by engrailed. In the eye disc, however, it is repressed posterior to the morphogenetic furrow in the absence of either patched or engrailed activity (Strutt, 1996).
Anterior terminal development is controlled by several
zygotic genes that are positively regulated at the anterior
pole of Drosophila blastoderm embryos by the anterior
(bicoid) and the terminal (torso) maternal determinants.
Most Bicoid target genes, however, are first expressed at
syncitial blastoderm as anterior caps, which retract from
the anterior pole upon activation of Torso. To better
understand the interaction between Bicoid and Torso, a
derivative of the Gal4/UAS system was used to selectively
express the best characterized Bicoid target gene,
hunchback, at the anterior pole when its expression should
be repressed by Torso. Persistence of hunchback at the pole
mimics most of the torso phenotype and leads to repression
at early stages of a labral (cap'n'collar) and two foregut
(wingless and hedgehog) determinants that are positively
controlled by bicoid and torso. These results uncovered an
antagonism between hunchback and bicoid at the anterior
pole, whereas the two genes are known to act in concert for
most anterior segmented development. They suggest that
the repression of hunchback by torso is required to prevent
this antagonism and to promote anterior terminal
development, depending mostly on bicoid activity (Janody, 2000).
The results indicate that early anterior expression of a labral
determinant, cnc, and of two foregut determinants, wg and hh,
is repressed when zygotic expression of hb is allowed to persist
at the anterior pole of the Drosophila blastoderm embryo.
Expression of cnc, wg and hh is under the positive regulation
of bcd and torso but no zygotic gene has yet been implicated
in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and
that torso-induced anterior repression of hb is necessary for
their positive control by torso. To determine whether the
positive control of cnc, wg and hh by torso could be the result
of a double negative control involving hb, expression of these
genes was analysed in hb zygotic mutant embryos derived from
torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb
at the pole, expression of these genes should be recovered in
hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is
not recovered in hb minus embryos derived from torso females
whereas it is normal in hb minus embryos. This indicates
that, although necessary, the anterior repression of hb is not
sufficient to mediate Torso positive control on cnc, wg and hh
early anterior expression (Janody, 2000).
A function of Gro in imaginal development has been investigated, namely the repression of hedgehog in anterior wing pouch cells. hh is repressed in anterior compartments at least partly via Ci[rep], a form of the multifunctional transcription factor Cubitus interruptus (Ci). Cells in the
wing primordium close to the AP boundary need gro activity to maintain repression of hh transcription, whereas in more anterior cells gro is dispensable. This repressive function of Gro does not appear to be mediated by Ci[rep]. Analysis of mutant gro transgenes has revealed that the Q
and WD40 domains are both necessary for hh repression. Yet, deletion of the WD40 repeats does not always abolish Gro activity. These findings
provide new insights both into the mechanisms of AP patterning of the wing and into the function of Gro (Apidianakis, 2001).
Although Ci[rep]-mediated repression can account for the lack of hh expression away from the AP boundary, it has not been firmly established that Ci[rep] is operational close to the AP boundary. These cells receive high Hh signal and as a result not only do they not process Ci to Ci[rep], but also they activate full-length Ci into a strong activator, Ci[act], by post-translational modification. There is indirect evidence that Hh-receiving cells do not contain sufficient Ci[rep] levels to repress hh: in posterior cells, ci is repressed by En; other than this, the cellular mechanism for Hh signal transduction is present. When full-length ci is provided by ectopic expression in the posterior compartment, hh-lacZ is not repressed. This suggests that these cells cannot produce appreciable amounts of Ci[rep], consistent with their responding to Hh signaling. That this is indeed the case was shown by the fact that ectopic expression of ci does repress posterior hh-lacZ in smo loss-of-function clones, where the Hh signal transduction has been disrupted. If anterior cells that are exposed to Hh behave similarly, then the lack of hh expression there cannot be attributed to Ci[rep]. It is proposed that a Gro-dependent repression complex supplies this function, since gro- clones exhibit strong derepression of hh-lacZ near the AP boundary. The Gro complex is not required in anterior cells far from the boundary, because those receive no Hh signal and thus contain sufficient Ci[rep] to repress hh. Accordingly, by supplying increased levels of Ci[rep] near the AP boundary via the ciCe2 allele, the need for Gro-mediated hh repression is able to be largely abolished, with the exception of the DV boundary. Since Gro is a ubiquitous co-repressor, one has to postulate the existence of a DNA-tethering factor, which will be referred to as 'X' for the purpose of this discussion, and some process of spatial regulation of the X-Gro complex activity. The possibility that X is a form of Ci itself was tested and the answer was negative: using three different assays -- GST pulldowns, yeast two-hybrid and transfection colocalization -- no interaction between Gro and either form of Ci could be shown. Most importantly, the fact that Ci[rep] does not require Gro to repress hh in anterior cells away from the boundary supports a model where Ci and Gro repress hh independently of each other (Apidianakis, 2001).
The quantitative aspect of hh derepression in gro- clones is intriguing: clones abutting the AP boundary (type I) express the highest hh-lacZ levels, which drop gradually as clones arise further from the P compartment. This might reflect the fact that Ci[rep]-dependent repression gradually increases away from the boundary, and this is independent of gro. This interpretation assumes that basal (unrepressed) hh transcription in the A compartment would be high and subject to the dual repressors (Ci and X-Gro). Alternatively, basal hh transcription could be low, but, in addition to the repression control, hh could display a positive response to Hh signaling at the AP boundary. The latter model is consistent with the fact that in ci- cells, basal hh expression appears to be low. It also agrees with the behavior of large type I gro- clones in the present study. In these clones, high levels of hh-lacZ could be observed throughout the clone, even at a distance from the AP boundary. This could be accounted for by Hh signaling, which, having risen over some threshold owing to hh derepression, further stimulates hh transcription to a high level. This effect would spread to the edge of the clone, beyond which activation of the X-Gro repressor would silence hh transcription. The putative inducer of hh by Hh signaling may be Ci[act], as with all other direct Hh target genes; alternatively, it may be another factor induced by Ci[act]. The hypothesis that Ci[act] itself can activate hh transcription is not unreasonable, since hh should contain a regulatory region(s) that bind(s) Ci[rep]. Ci[act] and Ci[rep] contain the same DNA-binding domain and recent work has shown that the two forms of Ci bind the same target sites, although some enhancers may be configured in such a way as to respond preferentially to either the activator or repressor form (Apidianakis, 2001).
For the sake of simplicity, the existence of a low level ubiquitous activator of hh (basal levels) with a stronger activator located in P cells is postulated to account for the high levels of hh expression in P cells. In A cells that do not receive the Hh signal, the basal activity of hh is repressed by Ci[rep] and gro is not required. In A cells close to the Hh source, the basal transcription of hh would be enhanced by positive autoregulation; however, the presence of the repressive X-Gro complex does not allow this activation to take place. Implicit in this model is that X is itself activated by Hh (e.g. transcriptionally induced via Ci[act]), so that it only functions in Hh-receiving cells. In addition X production/activity should be spatially limited to the A compartment (e.g. repressed by En), since ectopic expression of full-length ci in the posterior cannot induce X-Gro activity to repress endogenous hh. According to this model, ci- clones close to the AP boundary express basal hh levels, since they lack both the X-Gro repressor (no activation of X in the absence of Ci[act]) and the activator of hh transcription (Ci[act] itself or a downstream target). By contrast, gro- clones in the same region only lack the repressive X-Gro complex and thus actively transcribe hh in response to Ci[act]; the high levels of hh produced are sufficient to initiate Hh signaling, which can propagate this effect of hh derepression throughout the clone (Apidianakis, 2001).
gro- clones near the DV boundary behave somewhat aberrantly. hh-lacZ derepression there is more efficient, observable in further anteriorly arising clones, compared with equivalent clones away from the DV boundary -- it even occurs in the presence of increased Ci[rep]. Although the mechanism remains to be discovered, one way to account for this special behavior, without invoking additional regulators, is that Ci[rep] is less active near the DV boundary and/or Ci[act] is more active, and this modulation of Ci activity in favor of the activator form allows high level hh expression at a greater distance from the Hh source and even in the ciCe2/+ background. Interestingly, ci- clones show little or no hh-lacZ derepression at the DV boundary, consistent with Gro, rather than Ci[rep], being the major hh repressor there (Apidianakis, 2001).
The model put forward here is perhaps the simplest, but by no means the only one that fits the existing data. For example, Gro might interact with Ci[act] itself, switching it from an activator into a repressor, given the right enhancer context, much like the effect Gro has on other activators, such as Dorsal. This interaction may be weak and/or require additional factors, accounting for the inability to detect it. To resolve the mechanism of hh repression at the AP boundary will necessitate detailed molecular dissection of the hh regulatory regions and characterization of relevant trans acting factors. Whatever the mechanism, it appears that a Gro-containing complex is deployed in the wing to block the spread of hh expression anteriorly from the AP compartment boundary. This should ensure a spatially fixed organizer (dpp expression stripe), in contrast to a moving one, as found in the fly retina (Apidianakis, 2001).
Gro is the founder of a family of transcriptional co-repressors encountered in invertebrates and vertebrates. Gro proteins are multipurpose co-repressors, since they can interact with a good number of DNA-binding repressors. A number of Gro mutants were tested both for subcellular localization. Grocdc2- and GroDeltaQ show the same nuclear accumulation as wild-type Gro. GroDeltaWD40 is also nuclear, but it shows a striking departure from the rather uniform wild-type pattern, since it localizes predominantly to a small number of subnuclear particles. GroNLS- is both nuclear and cytoplasmic, whereas GroDeltaGCS is exclusively cytoplasmic. This suggests that the GP, CcN and SP domains contain at least two different regions needed for efficient nuclear accumulation, one of which is the canonical NLS. It can be speculated that other such regions might be those necessary for association with histones or with DNA-bound repressors, which might promote nuclear accumulation of Gro even in the absence of the NLS (Apidianakis, 2001).
In vivo activity was tested by assaying the ability of mutant Gro proteins to repress anterior hh-lacZ expression. GroDeltaQ and GroDeltaWD40 proteins were inactive in this assay. In contrast, Grocdc2- was as active as wild-type Gro. The inability of GroDeltaQ to function as a co-repressor is expected, since the Q domain is the strongest repression domain and is needed both for tetramerization as well as for histone interaction. The inactivity of the GroDeltaWD40 mutant might be accounted by its inability to interact with the X-factor tether. Or one could suggest an alternative explanation based on the localization data: that GroDeltaWD40 is retained in subnuclear particles and as a result cannot gain access to target genetic loci. Whether the aberrant subnuclear localization of GroDeltaWD40 is a cause or a consequence of its inactivity is a matter for further study. Despite its aberrant localization, GroDeltaWD40 is as active as wild-type Gro and Grocdc2- when overexpressed by omb-Gal4: all three transgenes results in abnormal leg development. Gro-DeltaQ, -NLS- and -DeltaGCS did not have such an effect. This shows that GroDeltaWD40 retains some activity, although in the absence of data regarding the cause of defects in leg patterning, the function of the mutant protein is as yet unknown. 'Short' Gro family proteins that lack WD40 repeats exist in vertebrates. These, human AES and mouse Grg5, contain only Q and GP domains, thus they are not entirely equivalent to the DeltaWD40 mutant. It has been shown that these proteins are cytoplasmic, although they are readily transported to the nucleus upon interaction with a Tcf partner. Their role in transcription seems to be context dependent, since they can act as co-repressors in some cases, whereas in others they might counter repression by 'long' Gro proteins. One study suggests that this anti-repression effect is not necessarily due to the absence of the CcN/SP/WD40 domains, but rather due to the inability of the GP domain of the 'short' proteins to interact with HDAC1. In this study, GroDeltaWD40 was active in one assay and inactive in another. It will be interesting to determine its activity in additional biological contexts where Gro is required (Apidianakis, 2001).
Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by
the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs (hyd) gene misexpress both of these
signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and
overgrowth of surrounding tissue. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of
hedgehog expression for proteolysis (Lee, 2002).
Since hyd is expressed in the wing disc and is required for its
normal growth, whether its effects on wing development might
be mediated by alterations in hh expression and Ci levels was examined. In wild-type wing discs, hh is expressed uniformly throughout the
posterior compartment of the wing pouch, while dpp is expressed in
the anterior compartment in a stripe along the AP border.
Ci155 is present at high levels in a similar stripe at the AP
border and at lower levels elsewhere in the anterior compartment.
Expression of hh, dpp and Ci155 in hyd clones
remains restricted to the correct compartment. However, some hyd
mutant clones in the posterior compartment express elevated levels of
hh-lacZ. This misexpression of hh is correlated with a rounded shape and
apparent overgrowth of the clones. The only known regulator of hh
expression in the wing disc is Ci, which is restricted to the anterior
compartment by En-mediated repression; Ci76 represses hh
there. These results suggest that a Ci-independent activator of hh expression must be present in the posterior compartment and kept in check by Hyd activity (Lee, 2002).
In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular
Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of
developmental regulators, like the homeotic genes, that is defined embryonically. It was asked whether such CMMs could also control
expression of genes involved in patterning imaginal discs during larval development. The results demonstrate that expression of the
hedgehog gene, once activated, is maintained by a CMM. In addition, the experiments indicate that the switching of such CMMs to an
active state during larval stages, in contrast to embryonic stages, may require specific trans-activators. Thes results suggest that the patterning of cells in particular
developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, since the control
by CMMs ensures a preformatted gene expression pattern (Maurange, 2002).
Immunoprecipitation using cross-linked chromatin (XChIP) allows the mapping of in vivo DNA target sites of chromatin proteins. Because one Polycomb (PC, a member of the PcG) binding site on polytene chromosomes coincides with the
cytological position of hh at 94E, this method was applied to ask whether there are PC and GAGA factor (GAF/Trl, a member of the trxG) binding sites in the hh genomic region. These two factors had previously been found to be hallmarks of CMMs, and the GAF has been shown to be associated with some PcG complexes and necessary for the silencing function of PREs. Initially the immunoprecipitated material was hybridized to a genomic stretch of 45 kb encompassing the hh gene. This led to the identification of PC/GAF-binding sites in regions close to the transcription unit. To
further fine-map the location of the PC/GAF-binding sites,
the region around the hh gene was subdivided into 1-kb-sized PCR fragments (from 4 kb upstream of the hh transcription start site according to the transcript CG4637 from Flybase, to
13.4 kb downstream to the end of the gene). Slot-blot hybridizations of
immunoprecipitated material revealed two main sites where PC
and GAF are strongly enriched. The first site (A) is located
in a region between 0.07 and 1.06 kb upstream of the transcription
start site, whereas the second binding site (B) is found in a region
spanning the second exon of the hh gene and spreading about
0.4 kb on both sides of the exon. On both sites a
substantial overlap was observed between PC- and GAF-binding sites. The presence of this particular arrangement of PC- and GAF-binding sites in the
hh genomic region suggests that these PcG and trxG proteins
directly control hh expression (Maurange, 2002).
To investigate this at the functional level, the
accessibility of the hh promoter region to a
trans-activating factor was assessed. It is known that a PRE placed in the vicinity of an Upstream Activating Sequence (UAS) is able to counteract
GAL4 binding, preventing expression of the reporter gene (Zink,
1995; Fitzgerald, 2001). Advantage was taken of the
availability of an EP line possessing a UAS site close to the
endogenous hh transcription start site to
test whether the hh-PREs could inhibit the activation of
transcription induced by GAL4. The EP3521 line (termed here
EP-hh) possesses an EP transposon containing several UAS
sites, and is inserted in the hh promoter region (-0.36 kb). The endogenous hh gene is not transcribed in
salivary glands. By using an hs-GAL4 line, which is known to
be leaky at 25°C, weak expression of GAL4 in larval salivary glands
is observed. When hs-GAL4 is crossed to a line containing
UAS-hh integrated randomly in the genome, in situ stainings reveal that at 25°C, by the action of GAL4, the hh mRNA is
present in high amounts in all the salivary gland cells. However, when hs-GAL4 is crossed to the EP-hh line, in which the UAS sites are
juxtaposed to the presumptive PRE, hh transcription was
observed in only a very few cells situated mainly at the base of the
glands. It was reasoned, because in most cells transcription is inhibited, that the PcG proteins binding the PREs in the vicinity of
the hh promoter block the accessibility of GAL4 to the UAS sites. Accordingly, reducing the amount of some of the PcG proteins in
the cells by repeating the experiment with flies heterozygous for the
Pc3 allele or with males hemizygous for
the ph409 allele induces partial
derepression of transcription of the endogenous hh gene in a
substantial number of gland cells. These results indicate that the
repression observed in most of the salivary gland cells in the EP line
is caused by the action of the PcG proteins through their binding to the identified PREs. As such, these experiments demonstrate that the transcription of hh is directly repressed by the PcG proteins (Maurange, 2002).
Having shown that the hh gene is controlled by the PcG
proteins, it was of interest to see whether the mapped PC/GAF-binding sites could function as CMMs. Transgenic flies were produced using the
vector that allows for a test of the maintenance of the reporter gene
expression through cell divisions. A 3.4-kb
fragment, starting from position -3760 to -402 bp upstream of the
hh transcription start site (according to transcript
CG4637 from Flybase), and containing the PRE identified in the
hh promoter region, was linked to a
GAL4/UAS-inducible lacZ gene (UAS-lacZ) and
miniwhite as a reporter and transformation marker. Most of the lines obtained (15/22)
exhibit pairing-sensitive silencing, a phenomenon often associated with
PREs, when homozygous for the construct, indicated by the variegated
expression of miniwhite in the eyes. A short GAL4 pulse
produced in these flies during embryogenesis by activation of the
hs-GAL4 driver leads to homogeneous expression of the
lacZ gene in the entire embryo. When these
embryos are transferred back to 21°C and are allowed to develop to
adulthood, >90% of the offspring of the two lines tested displayed
partial or homogeneous miniwhite derepression in the eyes. These results show that the upstream 3.4-kb fragment is able to maintain the initial state of transcription of the reporter gene throughout development and therefore exhibits CMM properties (Maurange, 2002).
Having shown that the hh gene is controlled by PcG proteins
and that a DNA fragment upstream of the hh transcription start site can function as a CMM in a transgenic assay, tests were performed to see
whether the hh gene itself, in its original chromatin
environment, is regulated by CMM activity during imaginal disc
development, when cells undergo a high number of divisions. It is known
that all wing pouch cells are progenies of the cells determined at the
dorso-ventral (D-V) boundary at early larval stages. It was
hypothesized that if the transcription of a gene possessing a CMM is
activated in cells during early larval development at the D-V boundary, then transcription should be inherited to daughter cells after mitosis, resulting in expression of the gene in all wing pouch cells (Maurange, 2002).
During embryonic and larval development, En induces transcription of
hh in the posterior compartment of leg and wing imaginal discs, where the two factors substantially colocalize. Even though it is not presently clear whether En directly activates hh expression, this regulatory feature provides a tool to test for CMM activity at the hh gene. UAS-en was expressed at the D-V boundary using a
vestigial-GAL4 driver (vg-GAL4). This transgene combination allows expression of GAL4 in a thin
stripe (1 or 2 cells thick) along the D-V boundary during wing disc
development. Double stainings of such late third-instar wing
discs reveal that, surprisingly, En not only induces a thin stripe
of hh-lacZ expression (reflecting the hh expression
pattern in the P30 enhancer trap line) in cells along the D-V boundary
as expected, but also in all the posterior and anterior wing pouch
cells (except in a stripe along the A-P boundary). Strong
UAS-en expression is detected in cells at the D-V boundary
and lower levels of En in some regions of the anterior wing pouch. The repression of the endogenous en observed in some
parts of the posterior compartment is explained by the fact that high
levels of En could cause repression of the endogenous en in
the P compartment. Strikingly, the overlay of Hh-LacZ and En stainings clearly reveals large domains, in both anterior and posterior wing pouch, with strong hh expression in the absence of En, suggesting that the transcription of hh in these cells becomes independent of En. Furthermore, it is
known that En represses cubitus interruptus (ci) expression, and it has been shown that clones of A cells lacking Ci express low levels of Hh protein. These observations suggest that hh expression is activated by En at the D-V boundary in early larval development, and is inherited, even in the absence of the initial trans-activator (En), through mitosis in the cells forming, in later stages, the wing pouch (Maurange, 2002).
Alternatively, hh inheritance of transcription to daughter cells could be
explained by the existence of a positive feedback loop
allowing continuous maintenance of hh expression. This
positive feedback loop would be activated once hh is
expressed, either by autoactivation or cross-activation with another
factor, like En, for instance. To investigate this possibility, hh was
misexpressed along the D-V boundary, using the
vg-GAL4 driver and a UAS-hh transgene. Although
UAS-hh is continuously strongly expressed at the D-V boundary
from the second instar larval stage, in situ stainings do not reveal
any inheritance of hh transcription to daughter cells, because the presence of hh mRNA is always restricted to a thin row of cells at the D-V boundary, even in late third-instar wing discs. This result demonstrates that the previously observed inheritance of hh expression in wing pouch cells of vg-GAL4; UAS-en flies is not caused by autoactivation by Hh itself nor by any positive feedback loop (Maurange, 2002).
Furthermore, antibody stainings in such discs display a progressive
activation of en expression along the D-V boundary during development. In late third-instar larvae, a strong En signal is observed, testifying to the functional activity of the protein produced
by UAS-hh. Higher magnification shows that in these discs, Hh
is able to induce en expression non-cell-autonomously in a stripe of ~7 rows of cells. However, the fact that at this
stage, hh expression is only limited to a stripe of 2 rows of
cells indicates that En is no longer able to induce transcription of
the endogenous hh gene, in contrast with early larval stages. It implies that the low levels of En protein observed in some of the
anterior wing pouch cells of vg-GAL4; UAS-en
third-instar larvae is most probably caused by a late
activation of en transcription by Hh. In addition, hh
expression in these cells cannot be due to activation by low or
undetectable levels of En protein, because even
strong doses of En do not activate hh transcription in this
region at this stage of development (Maurange, 2002).
When UAS-en is misexpressed at the D-V boundary in a wild-type genetic background using vg-GAL4, it induces hh expression in most of the cells of the wing pouch except in a stripe along the A-P boundary where hh seems to be repressed. Whereas UAS-en is strongly misexpressed at the D-V boundary, the endogenous en gene is weakly misactivated in some cells of the anterior wing pouch (Maurange, 2002).
Repeating the same experiment in a genetic background hemizygous mutant
for an hypomorphic allele of polyhomeotic (ph409) leads to a broader domain of expression of hh. Remarkably, the region along the A-P boundary seems to be less refractory to activation of hh transcription, given that the territory of the repressed domain is reduced. Endogenous en is itself overexpressed in the anterior compartment. This is consistent with the findings demonstrating that en expression can be derepressed in a PcG gene mutant background. In this case in the anterior wing pouch cells, the activation of en transcription by Hh is probably more efficient than in a wild-type background because en cannot be correctly silenced by PH (Maurange, 2002).
The same experiment repeated in a genetic background now doubly
heterozygote for the trxG genes trithorax (trxE2) and brahma (brm2) consistently shows that hh
expression is activated at the D-V boundary, but can hardly be
maintained through cell divisions in the anterior compartment, because
with in situ staining, the Hh signal progressively fades away from the
D-V boundary. As expected, in such a case, en
expression in the anterior compartment is restricted to the D-V
boundary, because Hh might not be present in a sufficient amount to
activate transcription of the endogenous en gene in the
subsequent wing pouch cells (Maurange, 2002).
Furthermore, it is known that PcG-mediated silencing is enhanced at
higher temperature, and this hyperrepressed
state can be inherited through cell divisions.
Based on these observations, it was reasoned that raising embryos at 28°C
instead of 18°C would make the Pc-mediated silencing more difficult
to derepress, and influence the activation of hh transcription
by En. vg-GAL4; UAS-en embryos were allowed to
develop at 28°C until the beginning of second instar larvae, when the
D-V boundary is established in wing discs and UAS-en is
expressed there. As expected, stainings on third instar imaginal discs
reveal ectopic clones of wing pouch cells expressing hh. However, the frequency of cells expressing hh is lower than in discs of larvae grown at 18°C, indicating that the Pc-mediated silencing was harder to erase at 28°C. Nevertheless, in contrast with trxG mutant flies, once the transcription has initially been activated in this case, it is maintained in the subsequent daughter cells as suggested by the presence of clones spreading from the D-V midline to the limits of the wing pouch (Maurange, 2002).
These experiments demonstrate that once initiated by En, the
maintenance of the transcriptional state of hh to the daughter cells can be attributed to the action of the PcG and trxG proteins. It is
concluded that the CMM activity of the hh upstream region described in the transgenic assay is also efficient when considered in its natural chromatin environment and is responsible for
the inheritance of the initial transcriptional state of hh from the initiation to the completion of the wing pouch development (Maurange, 2002).
In the GAL4/UAS system, a GAL4 pulse,
when provided in larval stages, is only able to transiently activate
transcription of the reporter gene, but no heritable switching of the
Fab7-CMM is observed because transcription is lost as soon
as the trans-activator (GAL4) is down-regulated. These observations led to the hypothesis that Pc-mediated
silencing might be more stable in larval stages than in embryonic
stages, and CMMs cannot be switched to mitotically heritable activity
at these later stages. Consistent with these data,
the upstream 3.4-kb fragment showing a CMM activity cannot not be
switched to an active state through a GAL4 pulse produced during larval
stages as demonstrated by the lack of miniwhite derepression
in the eyes of the adult flies (Maurange, 2002).
However, in contrast to these experiments, the
endogenous hh CMM can be switched to an active state in larval
wing pouch cells upon an En pulse. The switch occurs in second instar
larval stages, when the D-V boundary is established through the action
of the Notch pathway and GAL4 expressed
by the vg driver. At this moment, en misexpression induces a
switch of the endogenous hh CMM at the D-V boundary to an
active state, leading to maintenance of hh transcription in
all wing pouch cells. It was of interest to test whether GAL4 is also able to
directly switch the endogenous hh CMM, in its natural
chromatin environment, in larval stages or whether this feature is
restricted to specific trans-activators like En. To perform
this experiment, the previously described line containing an
EP-element inserted into the hh promoter region (EP-hh) was used. By inducing GAL4 in the cells it is possible to
activate expression of the endogenous hh gene. It was postulated
that, by promoting transcription of the endogenous hh gene,
the hh CMM may be switched to an active state in wing pouch
cells. As observed on in situ preparations of late third-instar discs,
endogenous hh transcription is activated by GAL4 at the D-V
boundary, but is not maintained through cell division in wing pouch
cells. In comparison, also the
well-characterized Fab7-CMM is itself not switched to the
active state after GAL4 induction at the D-V boundary because expression of the reporter gene is not maintained in daughter wing
pouch cells. It is concluded that the GAL4 trans-activator is not able to switch a CMM in larval stages, although this can be carried out by the action of a gene-specific trans-activator, alone or more likely in association with other factors (Maurange, 2002).
Initially, CMMs were found to maintain the embryonically defined
expression of selector genes encoding the HOX/HOM factors, used to
established long-term cellular identities. However, CMMs appear to be
also used to freeze developmental decisions taken at later stages.
Indeed, the expression pattern of hh is subject to substantial
changes over time, depending on the morphogenetic field needed to be
patterned. Yet, the finding that hh expression, once activated, is also maintained by CMM mechanisms suggests that this type of control through chromatin-based epigenetic features is much more widespread and influenced by external signals. The results indicate that CMMs, if controlled by the correct trans-activator, can be switched and maintained in the active state at any time during development (Maurange, 2002).
Very little is known about how the gene expression pattern of cells
building compartments in imaginal discs is inherited through cell
divisions. Except for some homeotic genes, it is generally assumed that
auto- and cross-regulations allow selector and segmentation gene
expression to be maintained until the adult stage. However, this study
shows that at least in the case of hh, a cellular memory system
can take over to carry out the maintenance. It had already been
proposed that trxG proteins might be needed to allow a proper inheritance of En expression in the cells of the posterior compartment. It was also suggested that with a positive feedback loop between en and hh, these genes could achieve their own maintenance. The results presented here indicate that
this does not seem to be the case because the windows of time in which
En can activate hh and Hh can activate en seem not to
overlap over the entire wing development. During embryogenesis and
early larval development (at least until the D-V boundary is
established in wing disc), En is able to activate hh. This competence disappears later, in particular in third instar larvae, when even high amounts of En cannot activate hh transcription in at least the anterior compartment of the disc. However, Hh seems to acquire the competence to activate en transcription in late larval stages. These results are consistent with the fact that in late larval stages, the Hh gradient is able to induce a stripe of en expression at the A-P boundary, whereas En
does not in turn induce hh expression in this domain. Thus, because no feedback loop seems to exist, the data suggest that the hh CMM has a role in maintaining hh expression in the posterior domain during late stages of development (Maurange, 2002).
A domain along the A-P boundary exists that
seems to be refractory to a switch of the hh CMM to an active
state. Interestingly, it appears that in this region
Groucho and PH contribute to a strong repression system preventing
hh expression from being activated in the anterior compartment
in wild-type flies. Thus, these proteins may counteract a stable switch of the CMM to an active state. Consistent with this result is the reduction of the thickness of this refractory domain in flies mutant for ph (Maurange, 2002).
Large clones lacking en/inv expression in the posterior compartment of wing discs show reduced or no Hh protein, although this was not a universal feature of small clones. Apparently, in this
situation the loss of en/inv in the cells, especially when induced early in development, might cause a substantial
reprogramming of the gene expression pattern leading to repression of
hh, perhaps owing to the appearance of new repressors. In this
case, the initially activated CMM would not be able to overcome the repression (Maurange, 2002).
From these results, it is likely that CMMs have major direct roles in the
inheritance of the expression of hh in the development of
wing imaginal discs (it could also be imagined that the well-defined en-PRE could also act as a CMM). Furthermore, hh
and its vertebrate homologs are expressed in many other tissues during
development, in which its activation and/or maintenance are
independent of En and not yet elucidated (i.e., eye, gut, lung). Further studies will help develop an understanding of how the hh CMM may be involved in regulating the gene in different tissues (Maurange, 2002).
The finding that genes necessary to pattern imaginal discs can be
regulated by CMMs is in disagreement with models in which the
elaboration of pattern in multicellular fields is solely based on
information conferred by the local concentration of secreted signaling
molecules (morphogen model). In addition to this, it is proposed that the
establishment of a specific gene expression program in cells at various
developmental stages depends on both the information conferred by the
morphogens surrounding the cell and its history. Thus, a cell fate will
be specified by the transcriptional activation or repression of new
genes, as a result of surrounding information, as well as by the
maintenance of old transcriptional states established earlier and
inherited by CMMs through the action of the PcG and trxG proteins. It
has already been suggested that the gene optomotor-blind could
be regulated by a cellular memory mechanism in imaginal discs, although it was not directly demonstrated which mechanism
could allow inheritance of transcription (Maurange, 2002 and references therein).
It is important to note that the state of activation of a CMM does not
have to be established, once and for all, during embryogenesis, but can
be modified or stably switched later in development. This may be
especially true for genes patterning imaginal discs for which the
expression pattern is established during larval development in contrast
to homeotic genes defining the A-P axis during embryogenesis. However,
it seems that general trans-activating factors like GAL4, which are able to establish the active state of a CMM during
embryogenesis, are not able to modify or switch the CMM state later in
development, suggesting that the chromatin state of a CMM is more
difficult to reprogram at late developmental stages. During larval
stages, many cell divisions have been accomplished and cells are
getting more and more restricted in their determination state. The
chromatin could then be in a 'mature' conformation stable enough to
transmit a previously established transcriptional state despite the
potentially contradictory actions of other transcription factors found
simultaneously in the nucleus. Nevertheless, other transcription
factors such as En (in the case where En directly activates
hh) seem to be able, alone or by recruiting cofactors, to
stably switch a CMM from a repressed to an active state during larval
stages. At these stages, the switching of CMMs could require specific
factors to set epigenetic marks. It could be envisaged that the En
complex is able to attract some kind of chromatin-remodeling machinery that would have the potency to erase the memory and leave the chromatin
competent to be reprogrammed (Maurange, 2002).
In this way, it seems that the cell memory system is a complex and
dynamic process during development, in which the role of CMMs is to
heritably maintain a previously established transcriptional state until
new specific patterning cues are able to redirect the epigenetic marks
of the CMMs. However, this also makes it quite clear that during the
establishment of a morphogenetic field, besides the local specifying
signaling events, the previous history of a determining gene should be
taken into account (Maurange, 2002).
Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers molting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homolog Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. TRR is a histone methyltransferase capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. It is proposed that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters (Sedkov, 2003).
Continued, see Hedgehog Regulation: part 2/2
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