osa/eyelid
The distribution of Eld is ubiquitous in early embryos, showing no hint of a striped pattern. It is also ubiquitious in wing discs. Although the protein is present everywhere in eye discs, its strongest expression occurs in a band just anterior to the morphogenetic furrow, in the postion where cells respond to Hedgehog and Decapentaplegic signaling (Treisman, 1997).
Very few eyelid mutant cells are found when clones mutant for eld are analyzed in adult eyes. However, such clones are associated with scars, suggesting that eld mutant cells are present at a certain stage but interfer with normal develpment. The eld mutant clones are relatively small compared with clones mutant for other genes, suggesting that eld is required for cell proliferation and/or survival. Exceptions are clones that include the posterior margin of the disc, which were frequently much larger than internal clones. The difference might lie in the fact that development at the posterior margin is driven by dpp whereas the internal propagation of the furrow depends on hedgehog (Treisman, 1997).
eld also has an affect on neuronal differentiation. Most photoreceptor clusters that form within eld clones contain fewer neuronal cells than normal; therefore, there may be a partial, though not absolute, requirement for eld for neuronal differentiation. The lack of differentiation is not simply attributable to poor cell viability, as all the cells in eld clones can be induced to differentiate as neurons by removing the function of Enhancer of split complex genes. The block to differentiation caused by loss of eld function in the eye resembles the effect of loss of shaggy or ectopic expression of wingless (Treisman, 1997).
Clones of eld mutant cells induced in the wing disc also produce pattern alterations suggestive of antagonism to wingless. One effect of clones produced early in development is the transformation of the posterior notum into a partial second wing. These wings have a reversed anterior-posterior polarity; their most clearly differentiated structure is an alula produced consistently at their anterior margin. This transformation is the reverse of that produced by the wingless1 mutation, which transforms the wing into a duplicated notum, and is similar to that produced by overexpressing wingless, decapentaplegic or optomotor-blind in the notum. Clones induced later in wing development are associated with ectopic wing margin bristles. Many or all of these ectopic bristles are not mutant for eld but they are sometimes seen to form adjacent to eld clones. Ectopic bristle formation is restricted to the dorsal surface of the wing within the anterior compartment, and is observed most commonly near the wing margin in tufts of the bristle type appropriate to their postion along the anterior-posterior axis. Ectopic wing margin bristles are also produced in clones mutant for shaggy. However, shaggy clones show neither the non-autonomy nor the positional restrictions observed for eld clones. These results suggest a cell non-autonomous role for eld in wing patterning (Treisman, 1997).
The activity of the E2F transcription factor is regulated in part by pRB, the
protein product of the retinoblastoma tumor suppressor gene. Studies of tumor
cells show that the p16ink4a/cdk4/cyclin D/pRB pathway is mutated in
most forms of cancer, suggesting that the deregulation of E2F, and hence the cell
cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or
suppress E2F activity are likely to alter cell-cycle control and may play a role
in tumorigenesis. An E2F overexpression phenotype in the Drosophila eye was use
to screen for modifiers of E2F activity. Coexpression of dE2F and its
heterodimeric partner dDP in the fly eye induces S phases and cell death. Thirty
three enhancer mutations of this phenotype were isolated by EMS and X-ray
mutagenesis and by screening a deficiency library collection. The majority of
these mutations sorted into six complementation groups, five of which have been
identified as alleles of brahma (brm), moira (mor),
osa, pointed (pnt), and polycephalon (poc).
osa, brm, and mor encode proteins with homology to SWI1, SWI2, and
SWI3, respectively, suggesting that the activity of a SWI/SNF
chromatin-remodeling complex has an important impact on E2F-dependent phenotypes.
Mutations in poc also suppress phenotypes caused by p21CIP1
expression, indicating an important role for Polycephalon in cell-cycle control
(Staehling-Hampton, 1999).
The molecular basis of the interaction between E2F and a BRM/MOR/OSA
chromatin-remodeling complex is not yet clear and a range of possibilities exists.
The genetic interaction may result from a direct physical interaction between
RBF/E2F complexes and chromatin-remodeling machinery. In support of this idea the
human homologs of BRM, hBRM, and BRG1 have been found to physically associate
with pRB. This raises the possibility that
BRM/MOR/OSA may help E2F/RBF repressor complexes bind to their target sites. This
interpretation is supported by experiments from Trouche and co-workers who used
transient transfection of mammalian cells to demonstrate that BRG1 can cooperate
with pRB to repress E2F-dependent transcription (Trouche, 1997). Consistent with this model,
the
introduction of two copies of GMR-RBF into a GMR-dE2FdDPp35/+;
brm-/+ background suppresses the enhancement by brm. Thus
the effect caused by low levels of brm can be overcome by increasing the
dosage of RBF. Additional evidence has been sought that
would be predicted by this model; to date, however, these experiments have been
inconclusive. BRM lacks the LXCXE motifs found in hBRM and BRG1, which have been
suggested to mediate the interaction with pRB. To date no physical interaction between BRM and RBF
or between BRM and dE2F has been detected. The interaction between endogenous pRB and hBRM or BRG1
proteins is hard to detect even in mammalian cells, and the failure to find
BRM/RBF complexes may simply reflect difficulty in extracting
chromatin-associated proteins under conditions that maintain the interaction (Staehling-Hampton, 1999).
An alternative possibility is that the BRM/MOR/OSA chromatin-remodeling
complex is an important regulator of the expression of some key E2F-target genes,
but this complex does not interact directly with either RBF or E2F. In this case
the functional interaction occurs because these proteins converge on overlapping
sets of promoters. This model is difficult to test because it is not yet clear
which, and how many, E2F target genes are functionally significant. RNR2,
one example of an E2F-dependent gene, is expressed normally in embryos mutant for
brm, osa, or mor; no
change in the expression of RNR2 in GMR-dE2FdDPp35 eye disks
heterozygous for brm, osa, or mor alleles could be detected. While RNR2
expression is often used to provide an in vivo readout of E2F activity,
experiments suggest that it is not a critical E2F target. The effects of brm, mor,
and osa may only be evident at a subset of E2F-regulated promoters and an
extensive screen of E2F targets will be necessary to find the appropriate gene (Staehling-Hampton, 1999).
It is possible that E2F and brm act in distinct pathways that
influence cell-cycle progression. In this model the activity of a
BRM/MOR/OSA-containing complex may have a function that influences the ability of
E2F or RBF to control S-phase entry. Several observations have linked BRM-related
proteins to cell-cycle control. brm null clones in the adult cuticle often
show duplications of bristle structures, suggesting a possible role for
brm in proliferation, and mice lacking the BRM homolog
SNF2alpha show evidence of increased cell
proliferation. Although brm, mor, and
osa have no effect on the GMR-p21 phenotype, both
brm and mor mutations have been isolated as suppressors of a
hypomorphic cyclin E eye phenotype, demonstrating that brm and
mor can affect other cell-cycle phenotypes in the eye. Other studies have shown that
the activity of hSWI/SNF complexes is itself cell-cycle regulated. Transformation by activated Ras
decreases the expression of the murine ortholog of hBRM in mouse fibroblasts, whereas growth
arrest leads to an accumulation of protein. Recently, BRG1 and BAF155, a human
ortholog of Moira, have been shown to associate with cyclin E and are suggested to be
targets for cyclin E-dependent kinases during S-phase entry (Staehling-Hampton, 1999 and references).
During this study it was observed that GMR-dE2FdDP p35/+;
brm-/+ eyes develop necrotic patches that increase in severity
with the age of the adult fly. This raised the possibility that brm
mutations might enhance the phenotype by promoting E2F-induced apoptosis.
However, further experiments have failed to support this hypothesis. brm
mutations fail to enhance the GMR-dE2FdDP phenotype, which has elevated
levels of apoptosis, or to modify a GMR-rpr phenotype. In addition,
brm mutations have no effect on the phenotype of animals in which
GMR-rpr and GMR-hid-induced apoptosis is blocked by GMR-p35.
No increase in the number of apoptotic cells is detected when
GMR-dE2FdDPp35/+; brm-/+ third instar eye disks are
stained with acridine orange (Staehling-Hampton, 1999).
The promoters of Drosophila genes encoding DNA
replication-related proteins contain transcription
regulatory element DRE (5'-TATCGATA) in addition to
E2F recognition sites. A specific DRE-binding factor, DNA replication-related element factor or DREF, positively regulates DRE-containing genes. In addition, it has been
reported that DREF can bind to a sequence in the hsp70 scs'
chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, transgenic flies were
established in which ectopic expression of DREF was
targeted to the eye imaginal discs. Adult flies expressing DREF
exhibited a severe rough eye phenotype. Expression of DREF induces
ectopic DNA synthesis in the cells behind the morphogenetic
furrow that are normally postmitotic, and abolishes
photoreceptor specifications of R1, R6, and R7.
Furthermore, DREF expression caused apoptosis in the imaginal
disc cells in the region where commitment to R1/R6 cells takes place,
suggesting that failure of differentiation of R1/R6 photoreceptor cells
might cause apoptosis. The DREF-induced rough eye phenotype is
suppressed by a half-dose reduction of the E2F gene, one of
the genes regulated by DREF, indicating that the DREF
overexpression phenotype is useful to screen for modifiers of DREF
activity. Among Polycomb/trithorax group genes, it was found that a half-dose reduction of some of the trithorax group
genes involved in determining chromatin structure or chromatin
remodeling (brahma, moira, and osa)
significantly suppresses and that reduction of Distal-less
enhances the DREF-induced rough eye phenotype. The results suggest a
possibility that DREF activity might be regulated by protein
complexes that play a role in modulating chromatin structure. Genetic
crosses of transgenic flies expressing DREF to a collection of
Drosophila deficiency stocks allowed identification of several
genomic regions, deletions of which caused enhancement or suppression
of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators (Hirose, 2001).
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. Indeed, ChipE flies
with only one copy of osa+
(ChipE;osa616/+) are weak and sterile
but show wild-type DC bristle pattern (Heitzler, 2003).
These genetic interactions suggest that Osa can antagonize the function
of Pnr. Moreover, overexpressed Osa
(+/UAS-osa;Gal4-pnrMD237/+) induces a thoracic cleft
and the loss of DC bristles
similar to the loss-of-function pnr alleles. In contrast, loss-of-function
osa alleles display an excess of DC bristles similar to
overexpressed Pnr. For example,
(osa14060/+), (osa616/+), and
(osaE17/+) flies exhibit respectively
2.35 ± 0.12, 2.38 ± 0.12, and 2.43 ± 0.17 DC bristles per
heminotum (Oregon wild-type flies have 2.00 DC bristles/heminotum).
Furthermore, transallelic combination of osa14060
with the hypomorphic osa4H
(osa4H/osa14060) accentuates the excess of
DC bristles compared with (osa14060/+).
(osa4H/osa14060) flies display
4.17 ± 0.19 DC bristles per heminotum. In contrast,
(osa4H/osa4H) flies display 2.50 ± 0.11
DC bristles per hemithorax. The development of the extra DC bristles
revealed by phenotypic analysis was compared with the positions of the
DC bristle precursors detected with a LacZ insert, A101, in
the neuralized gene that exhibits
staining in all sensory organs. In
(osa14060/osa4H) discs, additional DC
precursors are observed that lead to the excess of DC bristles.
The pnrD alleles encode Pnr proteins carrying a
single amino acid substitution in the DNA binding domain that disrupts
interaction with the U-shaped (Ush) antagonist.
Consequently, PnrD constitutively
activates ac/sc, leading to an excess of DC bristles.
This excess is accentuated when osa function is simultaneously reduced (pnrD1/osa616) (Heitzler, 2003).
Since osa shows genetic interactions with trithorax
group genes encoding components of the Brm complex like moira
(mor) and brm, whether mutations in
mor and brm suppress the ChipE
phenotype was investigated. Loss of one copy of brm+ in
(ChipE; brm2/+) flies suppresses the lack
of DC bristles observed in ChipE flies,
similar to loss of one copy of osa+. This
shows that brm and osa both act during Pnr-dependent patterning, in agreement with the fact that they have been shown to be
associated in the Brm complex. In contrast, reducing the amount of Mor
by half [(ChipE;mor1/+) flies] is not
sufficient to modify the ChipE phenotype. This does not definitely exclude the possibility that
mor is directly or indirectly involved, via the Brm complex,
in Pnr-dependent patterning (Heitzler, 2003).
The complete osa open reading frame of 2715 amino acids and
the intronic splicing signals were PCR amplified from genomic DNA
prepared from homozygous embryos (osaE17 and
osa14060) and homozygous first instar larvae
(osa4H). For osa14060 and
osa4H, the sequence analysis revealed a single
mutation in the N terminus that causes a glutamine to stop codon
substitution. The conceptual translation of
osa14060 leads to a truncated Osa protein lacking both
functional domains, whereas Osa4H retains the ARID domain but
lacks the C-terminal EHD. Wild-type osa function is
necessary for patterning of the DC bristles. Although
osaE17 behaves as a stronger allele than
osa14060 and osa4H, molecular identity of the mutation is unknown.
Hence, the osaE17 phenotype may result from a mutation in
regulatory sequences that affects osa expression (Heitzler, 2003).
It has been shown that a complex containing Pnr, Chip, and the
(Ac/Sc)-Da heterodimer activates proneural expression in the DC
proneural cluster and promotes development of the DC macrochaetae.
Osa and Pnr/Chip have antagonistic activities
during development because loss of osa function
(osa4H and osa14060) displays
additional DC bristles. However, since the current study reveals that
osa genetically interacts with pnr and Chip,
it was asked whether Osa physically interacts with the Pnr and Chip
proteins. Immunoprecipitations of protein extracts made
from Cos cells cotransfected with expression vectors for tagged Osa and
either Pnr or tagged Chip were immunoprecipitated.
Because Osa is a large protein, several expression vectors
encoding contiguous domains of Osa were used. Osa
coimmunoprecipitates with Pnr and Chip and can be detected
on Western blots with appropriate antibodies. The interactions appear
to require the overlapping domains Osa E (His1733/Glu2550) and Osa F
(Ala2339/Ala2715) corresponding to the EHD.
Enhancer-promoter communication during proneural activation and
development of the DC bristles requires regulatory sequences scattered
over large distances and appears to be negatively regulated by
interaction of Pnr and Chip with Osa through the EHD. Interestingly,
the EHD is not conserved in yeast. In yeast, the UAS sequences are
generally close to the promoter and there is no requirement for
long-distance interactions. This observation could support the idea
that the EHD is essential for long-distance enhancer-promoter
communication. Alternatively, yeast may just lack proteins like Chip or Pnr (Heitzler, 2003).
The DNA-binding domain and the C-terminal region are essential for the
function of Pnr during development of the DC sensory organs. The pnrVX1 and pnrVX4
alleles (collectively pnrVX1/4) are characterized by
frameshift deletions that remove two C-terminal alpha-helices and result
in reduced proneural expression and loss of DC bristles (Heitzler, 2003).
The molecular interactions between Osa and
PnrD1 and between Osa and PnrVX1 were investigated.
PnrD1 protein interacts with the EHD as efficiently as
wild-type Pnr. In
contrast, the physical interaction is disrupted when the C terminus of
Pnr encompassing the alpha-helices is removed.
Because the C terminus of Pnr is required for the Pnr-Osa interaction
in transfected cells extracts, the abilities of in vitro
translated 35S-labeled Osa domains to bind to GST-CTPnr
attached to glutathione-bearing beads were investigated.
Only Osa E and Osa F interact with the C terminus of Pnr. The
interaction between Chip and Osa, and it was found that Osa associates with
the N-terminal homodimerization domain of Chip,
also required for the interaction between Chip and Pnr, was investigated. Furthermore,
Osa E and Osa F also bind to immobilized GST-Chip.
Deletion of the alpha helix H1 disrupts the interactions
between Pnr and Osa. Interestingly, the same deletion
also disrupts the interaction with Chip.
Therefore, the functional antagonism between Chip and Osa during neural
development may result from a competition between these proteins for
association with Pnr. Alternatively, the deletion of H1 may affect the
overall structure of the C terminus of Pnr and disrupt the physical
interactions with Chip and Osa. To discriminate between these
hypotheses, immunoprecipitations of protein extracts
containing a constant amount of Pnr, a constant amount of the tagged
Osa E domain, and increasing concentrations of Chip were performed.
Pnr immunoprecipitates with
immunoprecipitated tagged Osa E and the amount of Pnr
immunoprecipitated increases in the presence of increasing
concentrations of Chip. The presence of increasing amounts of Chip does
not inhibit the Osa-Pnr interaction as would be expected if Osa and
Chip were to compete for binding to Pnr. In contrast, it suggests that
Chip and Pnr act together to recruit Osa and to target its activity and
possibly the activity of the Brm complex to the ac/sc promoter
sequences (Heitzler, 2003).
Using expression vectors encoding contiguous domains of Osa, it was shown
that the EHD of Osa mediates interactions with Pnr and Chip. Because
the EHD is lacking in the truncated Osa14060 and
Osa4H, it is hypothesized that the loss of interaction with Pnr
and Chip are responsible for the excess of DC bristles observed in
osa4H and osa14060 (Heitzler, 2003).
To investigate whether these interactions between Osa, Pnr, and Chip
function in vivo during DC bristle development, the
effects of both loss of function and overexpression of osa were examined on
the activity of a LacZ reporter whose expression is driven by
a minimal promoter sequence of ac fused to the DC enhancer (transgenic line DC:ac-LacZ).
It was found that expression of the LacZ transgene is
increased in osa14060/osa4H wing discs
when compared with the wild-type control. For
overexpression experiments, the UAS/GAL4 system was used, using as a driver the pnrMD237 strain
that carries a GAL4-containing transposon inserted in the pnr
locus (driver: pnr-Gal4). This insert gives an expression pattern of
Gal4 indistinguishable from that of pnr. It was found that overexpressed Osa
leads to a
strong reduction of LacZ staining in the DC area, consistent with
the lack of DC bristles. Thus, overexpressed Osa represses activity of the
ac promoter sequences required for DC ac/sc
expression and development of the DC bristles. It has been previously
reported that wingless expression is also required for
patterning of the DC bristles. However, the
repressing effect of Osa on development of the DC bristles is unlikely
to be the result of an effect of Osa on wingless expression
because overexpressed Osa driven by pnrMD237 has no
effect on the expression of a LacZ reporter inserted into the
wingless locus. Thus, Osa acts through the DC enhancer of the
ac/sc promoter sequences to repress ac/sc and neural
development (Heitzler, 2003).
ChipE disrupts the enhancer-promoter communication
and strongly affects expression of the LacZ reporter driven by
the ac promoter linked to the DC enhancer.
Because null alleles of osa suppress the loss of
DC bristles displayed by ChipE, the
consequences of reducing the dosage of osa was examined in
ChipE flies. The expression of the
LacZ reporter is not affected in ChipE
flies when Osa concentration is simultaneously reduced (Heitzler, 2003).
In conclusion, Pnr function during
proneural patterning is regulated by interaction with several transcription factors.
Pnr function is negatively regulated by Ush, which interacts with its DNA-binding domain.
Chip associates with the C terminus of Pnr, bridging Pnr at the
DC enhancer with the AC/Sc-Da heterodimers bound at the proneural
promoters, thus activating proneural gene expression.
The current study reveals that Pnr function can also be
regulated by interaction with Osa. Thus, Osa activity is specifically
targeted to ac/sc promoter sequences and the binding of Osa
therefore has a negative effect on Pnr function, leading to reduced
expression of the proneural ac/sc genes. Osa belongs to Brm
complexes, which are believed to play an essential role during
chromatin remodeling necessary for gene expression. For example, in
vitro transcription experiments with nucleosome assembled human
beta-globin promoters have shown that the BRG1 and BAF155 subunits of
the mammalian SWI/SNF homolog are essential to target chromatin remodeling and promote
transcription initiation mediated by GATA-1. In contrast to what was observed in vitro, the current
results suggest that in vivo the SWI/SNF complexes can also act to
remodel chromatin in a way that represses transcription. Alternatively,
the observed repression of proneural genes may simply define a novel
function of Osa, independent of chromatin remodeling (Heitzler, 2003).
The establishment of the dorsal-ventral axis of the Drosophila wing depends on
the activity of the LIM-homeodomain protein Apterous. Apterous activity depends
on the formation of a higher order complex with its cofactor Chip to induce the
expression of its target genes. Apterous activity levels are modulated during
development by dLMO (Beadex). Expression of dLMO in the Drosophila wing is regulated by
two distinct Chip dependent mechanisms. Early in development, Chip bridges two
molecules of Apterous to induce expression of dLMO in the dorsal compartment.
Later in development, Chip, independently of Apterous, is required for
expression of dLMO in the wing pouch. A modular P-element
based EP (enhancer/promoter) misexpression screen was conducted to look for genes involved in
Apterous activity. Osa, a member of the Brahma
chromatin-remodeling complex, was found to be a positive modulator of Apterous activity in
the Drosophila wing. Osa mediates activation of some Apterous target genes and
repression of others, including dLMO. Osa has been shown to bind Chip. It is
proposed that Chip recruits Osa to the Apterous target genes, thus mediating
activation or repression of their expression (Milan, 2004).
This study presents evidence that Osa, a member of a subset of Brahma chromatin
remodeling complexes, behaves overall as a general activator of Apterous
activity in the Drosophila wing. Overexpression of Osa rescues and loss of Osa
enhances the Beadex1 phenotype. It does so by
modulating the expression levels of Apterous target genes, some of them being
activated (e.g. Serrate and probably other unknown target genes) and some
repressed (e.g. Delta, fringe). Chip has been shown to bind Osa.
The fact that Osa has different
effects on the transcription of Apterous target genes suggests that Chip
recruits Osa to the promoters and in combination with other unknown factors
mediates either transcriptional repression or activation. Osa mediates
repression of both Apterous dependent and independent expression of
fringe, suggesting a direct and probably Chip independent effect of Osa
on fringe transcription (Milan, 2004).
Apterous activity is regulated during
development by dLMO. Osa is required to mediate repression of dLMO expression.
Since both early and late expression of dLMO
depend on Chip, it is postulated that Chip forms a transcriptional complex with
Apterous in the D compartment and an unknown transcription factor expressed in
the wing pouch. Osa may interact with Chip thus recruiting the Brahma complex to
the dLMO locus and remodeling chromatin in a way that limits dLMO
transcriptional activation. High levels of dLMO protein reduce Apterous activity
and the Notch dependent organizer is not properly induced along the DV boundary.
Osa mediated repression of dLMO expression may ensure moderate levels of
expression of dLMO in the wing, thus allowing proper wing development. Gain of
function mutations that cause misexpression of vertebrate LMO proteins have been
implicated in cancers of the lymphoid system. Truncating mutations in the human
SWI-SNF complex, the human homologues of the Brahma complex, cause various types
of human cancers. The SWI-SNF complex may be required to mediate repression of LMO
expression in lymphoid tissues. Thus, it would be very interesting to analyze if
truncating mutations in members of the human SWI-SNF complex cause higher levels
of LMO expression and are associated with lymphoid malignancies (Milan, 2004).
It has been shown that the Brahma complex plays a general role in transcription by RNA Polymerase II. Then, is Osa having a general effect on the expression levels of every gene involved in wing patterning? Several observations indicate this is not the case. (1) Osa is a component of a subset of Brahma (Brm) chromatin complexes.
(2) Brahma and Polycomb were shown to have non-overlapping binding patterns
in polytenic chromosomes. Those
genes involved in wing patterning and regulated by Polycomb (i.e. Hedgehog) may not be
affected by overexpression of Osa. (3) Overexpression of Osa has different
effects on the expression levels of Serrate, Delta and fringe.
(4) Osa has been shown to specifically regulate
the expression of Wingless target genes and the Achaete-scute complex genes,
interestingly by restricting their expression levels (Milan, 2004).
Hematopoiesis occurs in two phases in Drosophila, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. This study used PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. The effect of abrogating the function of 820 genes was assessed as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, loss- and gain-of-function phenotypes were characterized. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the hedgehog (hh) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the Pten, TSC1, and TSC2 tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland (Tokusumi, 2012).
The discovery of a stem cell-like hematopoietic progenitor niche in Drosophila represents a significant contribution of this model organism to the study of stem cell biology and blood cell development. Extensive findings support the belief that the PSC functions as the niche within the larval lymph gland, with this cellular domain essential to the control of blood cell homeostasis within this hematopoietic organ. Molecular communication between the PSC and prohemocytes present in the lymph gland medullary zone is crucial for controlling the decision as to maintaining a pluri-potent progenitor state versus initiating a hemocyte differentiation program. This lymph gland cellular organization and the signaling pathways controlling hematopoieis therein have prompted several researchers in the field to point out its functional similarity to the HSC niche present in mammalian (Tokusumi, 2012).
As a means to discover new information on genetic and molecular mechanisms at work within a hematopoietic progenitor niche microenvironment, an RNAi-based loss-of-function analysis was carried out to selectively eliminate individual gene functions in PSC cells. The effect of knocking-down the function of 820 lymph gland-expressed genes was assessed as to their requirement for niche cell production and differentiation, and 100 of these genes were shown to be required for one or more aspects of niche development. The distinguishable phenotypes observed in these analyses included change in number of Hh-expressing cells, change in number of Antp-expressing cells, scattered and disorganized niche cells, rounded cells lacking extended filopodia, and lamellocyte induction in the absence of a normal PSC. The genes were placed into sub-groups based on their coding capacity and known genetic interactions, and the phenotypes associated with the functional knockdown of members of three of these gene regulatory networks were characterized (Tokusumi, 2012).
Previous studies have demonstrated that the PSC-specific ablation of srp function resulted in a lack of expression of the crucial Hh signaling molecule in these cells, the inactivity of the hh-GFP transgene in the niche, failure of niche cells to properly differentiate filopodial extensions, and the loss of hematopoietic progenitor maintenance coupled with the abundant production of differentiated hemocytes. Thus it was intriguing when it was observed that RNAi function knockdown of several members of the BAP chromatin-remodeling complex resulted in the identical phenotypes of lack of hh-GFP transgene expression and absence of filopodia formation in PSC cells. A convincing functional interaction was observed between srp encoding the hematopoietic GATA factor and osa encoding the DNA-binding Trithorax group protein in the inability of niche cells to express hh-GFP in double-heterozygous mutant lymph glands. Thus one working model is that the BAP chromatin-remodeling complex establishes a chromatin environment around and within the hh gene that allows access of the Srp transcriptional activator to the PSC-specific enhancer, facilitating Hh expression in these cells. It will be of interest to determine if there exists a direct physical interaction between Osa and Srp in this positive regulation of hh niche transcription and if so, what are the functional domains of the proteins essential for this critical regulatory event in progenitor cell maintenance. It is also likely that these functional interactions are important for Srp's transcriptional regulation of additional genes needed for the formation of niche cell filapodia (Tokusumi, 2012).
In this study, a total of 33 gain- or loss-of-function genetic conditions were analyzed that enhanced or eliminated the function of various positive or negatively-acting components of the insulin-like growth factor and TOR signaling pathways. A conclusion to be drawn from these analyses is that genetic conditions that have an end effect of enhancing translation activity and protein synthesis result in supernumerary PSC cell numbers in disorganized niche domains, while conditions that promote growth suppression lead to substantially reduced populations of niche cells. The same conclusion was obtained from recent studies performed by Benmimoun (2012). The Wg and Dpp signaling pathways have also been shown to be important for the formation of a PSC niche of normal size and function, and it is possible that the insulin-like growth factor and TOR signaling networks regulate the translation of one or more members of the Wg and/or Dpp pathways. These analyses have also shown that mutation of the Pten, TSC1, and TSC2 tumor suppressor genes results in severely altered blood cell homeostasis in lymph glands and in circulation, including the prolific induction of lamellocytes. A recent report demonstrated that in response to larval wasp infestation, the PSC secretes the Spitz cytokine signal, which triggers an EGFR-mediated signal transduction cascade in the generation of dpERK-positive lamellocytes in circulation. As dpERK activity is known to inhibit TSC2 function, inactivation of the TSC complex may be a downstream regulatory event leading to robust lamellocyte production in larvae in response to wasp immune challenge (Tokusumi, 2012).
To summarize, an RNAi-based loss-of-function analysis has been undertaken to identify new genes and their signaling networks vital for normal PSC niche formation and function. While information has been gained on the requirements of three such networks for PSC development and blood cell homeostasis within the lymph gland, numerous other genes have been discovered that likewise play key roles in these hematopoietic events. Their characterization is warranted as well to further enhance knowledge of genetic and molecular mechanisms at work within an accessible and easily manipulated hematopoietic progenitor niche microenvironment (Tokusumi, 2012).
Methyl-CpG-binding protein 2 (MECP2) is a
multi-functional regulator of gene expression. In humans, loss of
MECP2 function causes classic Rett syndrome (RTT: see Rett Syndrome in 'Drosophila as a Model for Human Diseases'), but
gain of MECP2 function also causes mental retardation.
Although mouse models provide valuable insight into Mecp2
gain and loss of function, the identification of MECP2 genetic
targets and interactors remains time intensive and complicated.
This study takes a step toward utilizing Drosophila as a
model to identify genetic targets and cellular consequences of MECP2
gain-of function mutations in neurons, the principle cell type
affected in patients with Rett-related mental retardation. It was
shown that heterologous expression of human MECP2 in Drosophila
motoneurons causes distinct defects in dendritic structure and
motor behavior, as reported with MECP2 gain of function in humans
and mice. Multiple lines of evidence suggest that these defects
arise from specific MECP2 function. First, neurons with
MECP2-induced dendrite loss show normal membrane currents. Second,
dendritic phenotypes require an intact methyl-CpG-binding domain.
Third, dendritic defects are amended by reducing the dose of the
chromatin remodeling protein, osa, indicating that MECP2 may act
via chromatin remodeling in Drosophila. MECP2-induced
motoneuron dendritic defects cause specific motor behavior defects
that are easy to score in genetic screening. In sum, this study
shows that some aspects of MECP2 function can be studied in the Drosophila
model, thus expanding the repertoire of genetic reagents that can
be used to unravel specific neural functions of MECP2. However,
additional genes and signaling pathways identified through such
approaches in Drosophila will require careful validation
in the mouse model (Vonhoff, 2012). Methyl-CpG-binding protein 2 (MECP2) is a multifunctional
transcriptional regulator involved in chromatin remodeling. Loss
of MECP2 function mutations cause classic Rett Syndrome (RTT), an
X-linked, dominant, progressive, neuro-developmental disorder.
Patients with RTT suffer from cognitive, language, motor
conditions, and seizures. However, MECP2 duplication is a frequent
case of mental retardation and progressive neurological symptoms
in males, and overexpression of MECP2 in the developing mouse
brain also causes progressive neurological disorder (Vonhoff,
2012). The MECP2 protein contains at least five distinct functional
domains (NTD, ID, MBD, TRD, and CTDα) which either bind DNA
autonomously or regulate MBD (methyl-CpG binding) function.
Historically, MECP2 is viewed as a transcriptional repressor that
localizes to chromatin by binding to CpG dinucleotides to regulate
gene expression through interactions with histone deacetylases and
other cofactors. However, MECP2 can also activate transcription,
associate also with un-methylated DNA, has chromatin compaction
and RNA splicing functions, and several MECP2 interacting proteins
are known. Therefore, multiple MECP2 functions might be mediated
by interactions with diverse co-factors and by binding to both
methylated and non-methylated DNA, consistent with the wide range
of phenotypes observed in patients with RTT (Vonhoff, 2012). Although Mecp2 mouse models recapitulate RTT phenotypes and
provide valuable mechanistic insight into neuronal defects caused
by Mecp2 mis-regulation, such as axon targeting, synaptic, and
dendritic defects, the identification of MECP2 functions and
target genes in this system is time intensive and complicated
(Vonhoff, 2012). The Drosophila genetic model system is increasingly
being used as a tool to analyze specific genetic and cellular
aspects of neurodevelopmental disorders. Short generation times,
high fecundity, high throughput screening techniques, facile
genetic tools, and relatively low costs have provided valuable
mechanistic insights into inherited diseases like Fragile-X,
Angelman syndrome, and neurofibromatosis. However, despite
considerable conservation in fundamental cell biological pathways,
the Drosophila genome encodes only about 75 percent of
human disease associated genes, and mecp2 is not among
these genes. Therefore, Drosophila can not be used to
study the pathophysiology resulting from loss of endogenous mecp2.
Instead, the Drosophila model relies on heterologous
expression of human MECP2 allele and consequential gain
of MECP2 function. Although classic Rett is mostly caused by
loss-of-function of MECP2, this is likely not an
artificial approach since in humans and in mouse models increased
levels of MECP2 also cause disease. Genetic and behavioral proof
of principle for the use of the Drosophila model to
address MECP2 gain-of-function has been provided earlier. In MECP2
transgenic flies, the MECP2 protein associates with chromatin,
interacts with homologs of known human MECP2 interactors, modifies
the transcription of multiple genes, and is phosphorylated at
serine 423, as in mammals. Most importantly, reported consequences
are developmental dysfunctions and motor defects, suggesting
parallels with RTT phenotypes. However, previous work on MECP2 in
the Drosophila CNS has not tested for cellular
phenotypes resulting from MECP2 over-expression in neurons,
although mouse models demonstrate that disease phenotypes result
from Mecp2 mis-regulation in postmitotic neurons. This
study presents the first data on cellular defects as resulting
from MECP2 gain-of-function in developing postmitotic Drosophila
neurons (Vonhoff, 2012). It was demonstrated that heterologous expression of human MECP2
in Drosophila motoneurons does not affect axonal
pathfinding, dendritic territory boundaries, or the neurons'
electrophysiology, but it causes a significant reduction in new
dendritic branch formation during development. Similarly, in the
mouse model Mecp2 mis-regulation results in pyramidal
neuron dendritic defects. This study provides four lines of
evidence that dendritic defects in Drosophila
motoneurons are caused by specific cellular functions that result
from MECP2 gain-of-function, and not from non-specific
over-expression or sequestering effects. First, MECP2 protein
specifically localizes to the nucleus of Drosophila
neurons, so that interactions of MECP2 with molecules in the
cytoplasm are unlikely. Second, targeted expression of MECP2 in Drosophila
motoneurons causes significant dendritic branching defects but
does not affect firing responses to current injections, voltage
activated potassium current, or firing frequencies during motor
behavior, indicating normal regulation of electrophysiological
properties. Although it was earlier demonstrated that Drosophila
motoneuron dendritic structure may undergo compensatory changes in
response to altered neuronal activity, and a link between
motoneuron activity and dendritic growth has clearly been
established, any evidence for homeostatic changes in motoneuron
excitability in response to developmental defects in dendritic
structure was not found in this study. Third, MECP2-induced
dendritic defects require intact MBD function of the MECP2 protein
because dendritic architecture is not affected following
expression of MECP2 alleles with non-functional MBD.
This indicates that human MECP2 exerts specific action in Drosophila
neurons via chromatin remodeling. Fourth, MECP2-induced dendritic
phenotypes can be ameliorated by reducing the dose of osa, a
member of the SWI/SNF complex. This genetic interaction is
consistent with the hypothesis that human MECP2 may exert specific
action in Drosophila motoneurons via chromatin
remodeling. It also indicates that MECP2 gain-of-function
activates specific cell signaling pathways in Drosophila,
and may not cause unspecific over-expression effects. Therefore,
the study concludes that Drosophila neurons can serve as
a valuable model system to identify some cellular mechanisms by
which MECP2 gain-of-function affects neuronal development
(Vonhoff, 2012). It was shown that dendritic defects, as induced by heterologous
expression of MECP2 in Drosophila motoneurons, require
an intact MBD domain, because expression of MECP2 with a point
mutated or truncated MBD domain does not affect dendritic
structure. However, each UAS-MECP2 transgene is likely
inserted into a unique site in the Drosophila genome,
and therefore, the possibility that different UAS-MECP2
transgenes may yield different expression levels or other genetic
interactions can not be excluded. The finding that dendritic
defects as caused by the expression of full length UAS-MECP2,
but not by the expression of UAS-MECP2 transgenes with
defective MBD domain, are a result of the unique insertion sites
of the UAS-MECP2 constructs into the Drosophila
genome, is unlikely for two reasons. First, both UAS-transgenes
with defective MBD do not cause dendritic defects. Second, similar
dendritic defects are observed following the expression of the
full length MECP2 construct inserted in the second or in
the third chromosome (Vonhoff, 2012). MBD domains recognize two key mechanisms of chromatin regulation
in eukaryotes, C5 methylations of DNA at cytosines and
post-translational histone modifications. Although the existence
of DNA methylation was demonstrated earlier in the fly genome,
methylation levels are several orders of magnitude lower than in
mammals. The fly genome contains only one methylated DNA binding
protein (dMBD2/3) and only one DNA methyltransferase (dDNMT2),
which shows highest affinity to t-RNA. Consequently, Drosophila
DNA is only sparsely methylated, so that MECP2 interactions with
modified histone tails seem the more parsimonious scenario. This
is consistent with the finding in this study that MECP2-dependent
dendritic defects are suppressed in an osa heterozygous mutant
background. Osa is a member of the SWI/SNF complex (human homolog
is BAF250), a class of trithorax proteins involved in chromatin
remodeling which are highly conserved between flies and humans.
This indicates that human MECP2 may exert specific action in Drosophila
motoneurons via chromatin remodeling. In fact, it was previously
suggested that MECP2 associates with human Brahma, a catalytic
component of the SWI/SNF chromatin remodeling complex to regulate
gene repression, although this finding is disputed. Nevertheless,
the Drosophila system provides some unique advantages to
study possible interactions of MECP2 and members of the SWI/SNF
chromatin remodeling complex with genetic tools (Vonhoff,
2012). The finding in this study that flies with MECP2 over-expression
in motoneurons show normal take-off likelihoods as well as normal
motoneuron firing and wing beat frequencies but can not sustain
flight is in accord with specific MECP2 effects on dendrite
development in otherwise normal motoneurons. In Drosophila,
take-off can be mediated by the escape response neural circuitry.
This circuitry bypasses flight motoneuron dendrites by synapsing
directly on MN5 axon, but it relies on normal synaptic
transmission and flight motoneuron physiology. Therefore, initial
take-off and initial motoneuron firing are not affected by
dendritic defects. In Drosophila motoneuron, firing
frequencies are directly proportional to wing beat frequency, and
thus, these are also not affected. By contrast, flight can not be
sustained because the significantly reduced dendritic surface
likely reduces the excitatory synaptic drive to motoneuron
dendrites that is necessary to stay in flight. Therefore, flies
with MECP2-caused motoneuron dendritic defects show a 30- to
60-fold reduction in flight duration. This behavioral phenotype is
obvious, and thus, useful for screening. Although the
quantification of flight durations and take-off likelihoods does
not allow for rapid genetic screening, high throughput screening
can easily be developed based on the observed reduction in flight
duration by more than 30-fold. Moreover, high throughput assays
which utilize Drosophila behavior for rapid screening
have been developed by others. Such approaches may help the future
identification of candidate MECP2 targets or interactors (Vonhoff,
2012). Identification of genetic interactors and modifiers of MECP2
function in neurons will be imperative toward developing future
treatment strategies. MECP2 itself is not a promising
treatment target because the X-linked MECP2 gene is
mosaic regulated in the human brain. Furthermore, both loss and
gain of function cause disease phenotypes. The sparse methylation
landscape in Drosophila may offer unique promise of
identifying non-methylated DNA-dependent functions of MECP2 in
neurons, the cell type that is most relevant to Rett syndrome.
Since known binding partners of MECP2 are conserved in flies (e.g.
YB-1, mSin3A etc.), it seems plausible that gain-of-function of
human MECP2 may affect neural development via a cellular machinery
that is partly conserved between flies and humans (Vonhoff, 2012). MECP2-induced dendritic phenotypes in flight motoneurons cause a
severe motor behavioral phenotype in that flight bout duration is
reduced approximately 30- to 60-fold. Rapid screening assays for Drosophila
behavioral phenotypes are available. Combined with the fast
generation times, high fecundity and facile genetic tools
available in Drosophila, this offers a powerful tool to
identify molecules that interact with MECP2 in neurons. However,
potential MECP2 candidate target genes or genetic modifiers of
MECP2 function that can readily be identified in the Drosophila
system will then have to be further evaluated in the existing
mouse models of RTT (Vonhoff, 2012).
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osa/eyelid:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 3 January 2020
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