Dichaete/Sox box protein 70D
Pair-rule gene expression is disrupted in Dichaete mutants. Expression of the gap genes Krüppel, knirps, and giant are normal, indicating that Dichaete acts in parallel or downstream of these gap genes. The so-called primary pair-rule genes even-skipped, Hairy, and runt each show reductions in levels of expression in Dichaete mutants, with variable stripe specific effects on eve, fushi tarazu, hairy and runt. Since the stripes of pair rule genes generally occur in the correct anterior-posterior position in Dichaete mutants, the gene is unlikely to provide key positional information; it is more likely to be required in the maintainance or establishment of appropriate levels of pair-rule gene expression in the central region of the embryo (Russell, 1996 and Nambu, 1996).
There are also clearly segment-specific defects in wingless expression. The loss of wg stipes in the ventral regions of the maxillary and labial segments is independent of the corresponding defects in ftz or eve expression (Nambu, 1996).
Ectopic expression of Dichaete disrupts pair-rule gene expression. There is no consistent effect on the expression of eve or ftz. With runt and hairy, however, there are reproducible alterations in expression. In both cases, there is a precocious expression of normal wild-type expression features. In the case of runt, the transition from seven to fourteen stripes occurs earlier than in wild-type controls. In addition there is a high level of ectopic expression of both hairy and runt between the second and third Dichaete stripes, as well as other, more variable patches of expression between other stripes (Russell, 1996).
An investigation was carried out of the gene regulatory functions of Drosophila Sox box protein 70D (also known as Dichaete or Fish-hook), a high mobility group (HMG) Sox protein that is essential for embryonic
segmentation. The Dichaete HMG domain binds to the vertebrate Sox protein
consensus DNA binding sites, AACAAT and AACAAAG, and this binding induces
an 85 degrees DNA bend. A heterologous yeast system has been used to show that
the NH2-terminal portion of Dichaete protein can function as a transcriptional activator. The HMG and C-terminal regions may partially mask the transcriptional activation function of the N-terminal region. Dichaete
directly regulates the expression of the pair rule gene even-skipped (eve) by binding to
multiple sites located in downstream regulatory regions that direct formation of eve
stripes 1, 4, 5, and 6. Dichaete may function along with the Drosophila POU domain proteins
Pdm-1 and Pdm-2 to regulate eve transcription, since genetic interactions are detected
between Dichaete and pdm mutants. In the blastoderm embryo, pdm-1 and pdm-2 are both expressed in wide posterior bands of cells that are completely contained within the Dichaete expression domain. In double Dichaete/pdm mutants there is a complete loss of eve stripe 5, and fusions between stripes 3 and 4 as well as stripes 6 and 7. This pattern of defects is never observed in mutants for only one or the other of the two genes. The downstream region contains a perfect octamer POU domain consensus binding site. Dichaete protein is expressed in a
dynamic pattern throughout embryogenesis, and is present in nuclear and cytoplasmic
compartments. The protein is first detected in embryos during nuclear cycle 12. At this time Dichaete is present in a wide stripe that encompasses most of the trunk domain, extending from eve stripes 2-7. It is suggested that the DNA-bending properties of Dichaete could enhance or stabilize interactions between regulatory complexes present at distant downstream eve regulatory regions and upstream regulatory complexes including those at the eve promoter. Sox proteins are known to interact with POU domain proteins in vertebrates (Ma, 1998).
During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the
axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent
on Single-minded, a basic-helix-loop-helix (bHLH)-PAS transcription factor. Fish-hook/Dichaete, a Sox
HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene
expression. single-minded, Dichaete, and drifter are all expressed in developing midline cells, and both loss- and
gain-of-function assays reveal genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the
slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Dichaete directly associates with the PAS domain of Single-minded
and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Dichaete can form homodimers and also associates with
other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription
factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes (Ma, 2000).
To address whether the sim, Dichaete, and
dfr genes might functionally interact to regulate
development of the embryonic CNS midline, whether
they exhibit overlapping expression in developing midline cells was examined. This was accomplished using anti-Dichaete and anti-Dfr sera, as well as a
P[3.7sim-lacZ] marker that mimics sim midline expression. P[3.7sim-lacZ] embryos were immunostained using anti-ß-gal and either anti-Dichaete or anti-Dfr sera. Prominent overlapping expression was detected between Sim and Dichaete in developing CNS midline cells from stage 8 throughout the remainder of germ band extension. Overlap was also detected in a subset of prospective foregut cells. Similar overlapping expression was also detected between Sim and Dfr. Midline coexpression of Dichaete and Dfr was detected by immunostaining wild-type embryos with anti-Dichaete and anti-Dfr sera. Both genes are expressed together in the CNS midline throughout germ band extension. In germ band-retracted embryos, Dichaete exhibits overlapping expression with Sim and Dfr in the midline glia. Dichaete and Dfr are also detected together in lateral cells of the thoracic ganglia and a subset of ventral epidermal cells. These analyses indicate that sim, Dichaete, and dfr are coexpressed in developing CNS midline cells. The midline expression of these three genes also overlaps that of the slit gene, which is a downstream target of Sim (Ma, 2000).
Both loss-of-function and gain-of-function assays were used to
detect genetic interactions between sim, Dichaete,
and dfr. Mutants are known to show genetic interactions in CNS midline differentiation and in Slit protein expression. Potential cooperative interactions between sim, Dichaete, and dfr in
regulating slit gene transcription were examined through the use of a P[1.0slit-lacZ] marker. This reporter contains a portion
of a slit intron that drives lacZ expression mimicking that
of the native slit gene in developing midline glia;
P[1.0slit-lacZ] expression is first detected in germ
band-extended stage 11 embryos and is maintained throughout the
remainder of embryogenesis. Dichaete
null mutant embryos exhibit a misplacement and loss of midline glia,
as detected via anti-ß-gal immunostaining. P[1.0slit-lacZ] is expressed normally in stage 11 Dichaete mutant embryos, but during germ band retraction the number of midline glia becomes reduced from wild
type, and many cells are located at aberrant ventral positions within
the nerve cord. Similar, although less severe, defects are observed in
dfr mutant embryos, where some midline glia are displaced
from their normal positions. Notably, ß-gal-expressing midline glia are still detected in both Dichaete and dfr mutants, indicating that unlike Sim, Dichaete and Dfr are not absolutely required for
P[1.0slit-lacZ] expression or midline glial development (Ma, 2000).
A dfr-Dichaete double mutant strain was used to examine
whether Dichaete and dfr might act together to
regulate midline gene expression. Embryos mutant for both
Dichaete and dfr exhibit much more
severe defects in P[1.0slit-lacZ] expression than either Dichaete or dfr single mutants. Although P[1.0slit-lacZ] is activated normally in stage 11 dfr-Dichaete double mutant embryos, there is a striking loss of midline P[1.0slit-lacZ] expression during germ band
retraction. This synergistic effect strongly suggests that Dichaete and Dfr function together to regulate slit transcription. These functions may be mediated directly through Dichaete and Dfr binding sites present in the slit 1 kb regulatory region. Another, nonexclusive possibility is
that Dichaete and Dfr might indirectly control slit
transcription by regulating the expression of sim. To
address this possibility P[3.7sim-lacZ] expression was examined in wild-type and dfr-Dichaete embryos. Compared with
wild-type embryos, dfr-Dichaete double mutants exhibit a
severe decrease in P[3.7sim-lacZ] expression, a phenotype
that first becomes apparent during germ band retraction. Thus, Dichaete
and dfr also influence sim expression and hence may indirectly influence the expression of a wide array of midline genes (Ma, 2000).
Because homozygous sim mutants exhibit severe CNS midline defects, it is not informative to analyze the phenotypes of Dichaete-sim or dfr-sim double mutants. Instead, potential interactions between Dichaete and sim were examined via a gain-of-function approach using the Gal4/UAS targeted gene expression system. A P[GMR-Gal4] strain that drives Gal4 expression in and behind the morphogenetic furrow in the developing eye imaginal disc was crossed to P[UAS-Dichaete] and P[UAS-sim] strains. P[GMR-Gal4]/+;P[UAS-Dichaete]/+ animals exhibit a moderate eye roughening with disruption of ommatidia organization and loss of mechanosensory bristles. In contrast, ectopic sim expression results in essentially normal eye morphology. The effects of Dichaete and sim coexpression reveal a nonadditive phenotype; there is a stronger disorganization of ommatidia and mechanosensory bristles than seen in flies expressing Dichaete or sim alone, and there is also a dramatic loss of eye pigmentation. These results indicated that ectopic expression of Dichaete and sim synergistically alters normal eye development, and supports the hypothesis that these genes can interact functionally (Ma, 2000).
Analysis of a 380 bp slit midline
regulatory fragment has indicated the presence of a single CNS midline element (CME), through which Sim::Tgo heterodimers act. The CME is located within 300 bp of the distal end (farther from the promoter in the native slit gene) of this fragment. An inverted TTCAAT repeat (TTCAATTTCATTGAA) is located 20 bp proximal to the CME. This sequence resembles a (A/T)(A/T)CAAT consensus binding site for Sox proteins, although binding of Sox proteins to a TTCAAT sequence has not been reported.
Because sequences present in an extended 1 kb slit DNA
fragment are required for normal levels of slit expression
in vivo, additional DNA sequences have been obtained. This analysis indicated that no other CMEs are present in the 1 kb slit DNA fragment. However, two perfect Dfr consensus binding sites, ATGCAAAT and
CATAAAT, located within 500 bp of DNA proximal to the CME were identified. These two Dfr binding sites are separated by ~150 bp and flank a consensus Dichaete binding site, TACAAT. These data suggest
that Dichaete, Sim, and Dfr may all bind to sites present in the 1 kb
slit regulatory DNA fragment. To test this possibility, DNA
gel mobility shift assays were performed using the Dichaete HMG domain and
full-length Dfr protein on double-stranded oligonucleotide probes
corresponding to sequences from the slit 1 kb fragment. The
Dichaete HMG domain binds strongly to a 26 mer probe containing the TACAAT
site. In contrast, Dichaete does not bind consistently to a 26 mer probe containing both TTCAAT sites, suggesting that Dichaete can distinguish between closely related DNA sequences. Dfr protein binds very strongly to a 33 mer probe that contains the ATGCAAAT site, and less strongly to a 32 mer probe containing the CATAAAT site. Dfr binds the ATGCAAAT site
both as an apparent monomer and a dimer, because two distinct bands
with reduced mobilities are detected. The 1 kb slit
fragment thus may integrate the actions of at least three different
types of regulatory proteins, represented by Sim, Dichaete, and Dfr (Ma, 2000).
The ability of Dichaete, Dfr, Sim, and Tgo to directly
control slit transcription was examined using transient transcription assays in cultured Drosophila S2 cells. The P[1.0slit-lacZ] construct was used as a reporter with various combinations of plasmids
that express Dichaete, Dfr, Sim, or Tgo. Dichaete modestly activates P[1.0slit-lacZ] transcription, indicating that in both yeast and fly
cells, Dichaete can function as a direct transcriptional activator. Dfr
results in little if any activation of P[1.0slit-lacZ], and Dfr and Dichaete together do not exhibit any increased activation over the levels observed for Dichaete alone. Neither Sim nor Tgo alone is able to activate the P[1.0slit-lacZ] reporter, because only background levels of expression are detected. Furthermore, Sim and Tgo together yield only minimal activation. These results imply that although Sim::Tgo heterodimers strongly activate
expression of a P[6XCME-lacZ] reporter (>150 units) that contains
six multimerized CMEs, additional factors are
required to achieve high levels of reporter expression. Significantly, the combination of either Dichaete and Sim::Tgo or Dfr and Sim::Tgo both result in relatively high levels of activation. Thus, both Dichaete and Dfr strongly enhanced the ability of Sim::Tgo heterodimers to activate slit
transcription. Comparable levels of activation are observed when all four proteins are expressed together. Taken together, the DNA binding and transcriptional activation assays provide additional evidence that regulation of
slit expression in the midline glia requires functional
interactions between Dichaete, Dfr, Sim, and Tgo (Ma, 2000).
Functional interactions between Sim, Dichaete, and Dfr may also regulate
the midline expression of other genes, including sim and
breathless (btl). Thus, sim has
autoregulatory functions, and the combined functions of dfr and Dichaete are also required for sustained midline sim expression. In addition, a 2.8 kb interval in the P[3.7sim-lacZ] transgene used in this study contains six evolutionarily conserved CMEs as well as several consensus Dichaete and Dfr binding sites. btl encodes an FGF receptor homolog whose expression in the CNS midline and tracheal cells has been shown to depend, respectively, on Dfr as well as Sim and Tgo, or Trh and Tgo. A 200 bp btl midline/tracheal regulatory region contains three evolutionarily conserved CMEs. Inspection of this region also revealed the presence of a conserved consensus ATCAAT Dichaete binding site located in a 40 bp interval between CME2 and CME3, as well as a conserved consensus
GATAAAT Dfr binding site located 40 bp
downstream of CME3. Thus, functional interactions between Sim, Dichaete,
and Dfr could be a general mechanism to regulate gene transcription
during CNS midline development (Ma, 2000).
Trichomes are cytoplasmic extrusions of epidermal cells. The molecular
mechanisms that govern the differentiation of trichome-producing cells are
conserved across species as distantly related as mice and flies. Several
signaling pathways converge onto the regulation of a conserved target gene,
shavenbaby (svb, ovo), which, in turn, stimulates trichome
formation. The Drosophila ventral epidermis consists of the segmental
alternation of two cell types that produce either naked cuticle or trichomes
called denticles. The binary choice to produce naked cuticle or denticles is
affected by the transcriptional regulation of svb, which is
sufficient to cell-autonomously direct denticle formation. The expression of
svb is regulated by the opposing gradients of two signaling molecules
- the epidermal growth factor receptor (Egfr) ligand Spitz (Spi), which
activates svb expression, and Wingless (Wg), which represses it. It
has remained unclear how these opposing signals are integrated to establish a
distinct domain of svb expression. This study shows that the expression of the
high mobility group (HMG)-domain protein SoxNeuro (SoxN) is activated by Spi,
and repressed by Wg, signaling. SoxN is necessary and sufficient to
cell-autonomously direct the expression of svb. The closely related
protein Dichaete is co-regulated with SoxN and has a partially redundant
function in the activation of svb expression. In addition, SoxN and Dichaete function upstream of Wg and antagonize Wg pathway
activity. This suggests that the expression of svb in a discreet
domain is resolved at the level of SoxN and Dichaete (Overton, 2007).
In the embryonic ventral epidermis of Drosophila, two alternative
cell fates are specified: smooth cells and trichome-producing cells. These
binary cell fates are distinguished by the expression of svb, the
most-downstream effector of epidermal morphogenesis. svb is necessary
and sufficient to cell-autonomously direct trichome formation. The
expression of svb is regulated by the opposing gradients of two
signaling molecules: Spi, which activates, and Wg, which represses,
svb expression. svb is expressed in segmentally reiterated,
epidermal stripes, which invariantly encompass six rows of cells. This raises
the question of how is opposing extrinsic information integrated to establish
a distinct domain of svb expression with a sharp posterior
border (Overton, 2007)?
This study demonstrates that the HMG-domain proteins SoxN and
Dichaete represent a molecular link between the expression of svb and
the upstream Der- and Wg-signaling cascades. SoxN and Dichaete
are expressed in the ventral epidermis at the time when epidermal cell fates
are specified. The late phase of SoxN and Dichaete expression is stimulated by
Der- and repressed by Wg-pathway activity. These regulatory mechanisms result
in the expression of SoxN and Dichaete in those six rows of cells within each
abdominal segment that differentiate to produce trichomes. SoxN and, to a
lesser extent, Dichaete, are necessary and sufficient to activate the
expression of svb. Furthermore, these results show that the
well-described repression of svb by Wg is due to the repression of
SoxN, which, in turn, results in the loss of svb activation.
Likewise, the Spi-mediated activation of svb expression relies on the
activation of SoxN, which, in turn, activates svb. This indicates
that the competition of Der- and Wg-pathway activities for the specification
of trichome-producing versus smooth cell fates is resolved at the level of
SoxN and Dichaete (Overton, 2007).
These results do not provide much insight into the issue of how opposing
extrinsic information is integrated such that a sharp posterior border of
svb expression is achieved. Instead, they raise the question of how
is a sharp posterior border of SoxN and Dichaete expression
established/maintained? The findings suggest that this is achieved by a
combination of negative- and positive-feedback loops. (1) Evidence is provided that SoxN and Dichaete negatively regulate Wg pathway
activity. This negative-feedback loop provides a likely mechanism for the
establishment and maintenance of a sharp posterior border of SoxN and Dichaete
expression. The issue arises of how robust this system might be in the face of
fluctuating levels of Wg pathway activity. The efficiency with which SoxN and
Dichaete restrict Wg pathway activity will crucially rely on the levels of
SoxN and Dichaete protein. In this context, it is noteworthy that the levels
of SoxN protein, but not Dichaete, are several-fold higher in the two
posterior-most rows of the SoxN stripe compared with the anterior four rows. The regulatory mechanisms that underlie the different levels of SoxN expression are currently unclear. (2) Evidence is provided that the maintenance of SoxN and Dichaete expression is supported by a positive-feedback loop: svb, the
expression of which is activated by SoxN and Dichaete, is itself required for
the maintenance of SoxN and Dichaete expression. Together,
these mechanisms contribute to an invariant read-out of cell identity from
opposing Der- and Wg-pathway activities (Overton, 2007).
In Drosophila, SoxN and Dichaete are necessary and sufficient to
activate the expression of svb, which in turn directly regulates the
expression of genes involved in trichome morphogenesis.
Is a function in hair formation of the Sox proteins conserved in other
species, including vertebrates? A previous study has shown that the mouse Sox9
protein is required for the differentiation of hair-producing epidermal cells
and acts genetically downstream of sonic hedgehog pathway activity
(Vidal, 2005). This
study did not address whether Sox9 regulates the expression of movo1
(Ovol1), the mouse ortholog of svb.
Nevertheless, the demonstrated roles of SoxN, Dichaete and Sox9 raise the
exciting question of do Sox proteins have an essential function in the
activation of an epidermal differentiation program that is conserved across
species as distantly related as mice and flies (Overton, 2007).
Functional complexity of the central nervous system (CNS) is reflected by the large number and diversity of genes expressed in its many different cell types. Understanding the control of gene expression within cells of the CNS will help reveal how various neurons and glia develop and function. Midline cells of Drosophila differentiate into glial cells and several types of neurons and also serve as a signaling center for surrounding tissues. This study examined regulation of the midline gene, wrapper, required for both neuron-glia interactions and viability of midline glia. A region upstream of wrapper required for midline expression was identified that is highly conserved (87%) between 12 Drosophila species. Site-directed mutagenesis identifies four motifs necessary for midline glial expression: (1) a Single-minded/Tango binding site, (2) a motif resembling a Pointed binding site, (3) a motif resembling a Sox binding site, and (4) a novel motif. An additional highly conserved 27 bp are required to restrict expression to midline glia and exclude it from midline neurons. These results suggest short, highly conserved genomic sequences flanking Drosophila midline genes are indicative of functional regulatory regions and that small changes within these sequences can alter the expression pattern of a gene (Estes, 2008).
To facilitate the identification of sequences responsible for wrapper expression in the midline glia of Drosophila, the genomic region flanking the wrapper transcription unit was examined to determine the degree of conservation between the 12 available Drosophila species. The regions most likely to contain regulatory control elements (motifs) of wrapper are tractable; the genomic regions flanking the transcription unit and the first intron are relatively small. The results of this analysis highlighted a region between -492 and -326 upstream of the transcription start site of wrapper that is highly conserved in all Drosophila 12 species examined, particularly a 70-bp region. To test if these sequences are responsible for the wrapper expression pattern in embryos, this genomic region was amplified within a 884-bp fragment, and then fused it to the green fluorescent protein (GFP) reporter gene within the pHstinger vector, which contains a minimal Hsp70 promoter. This DNA construct (wrapper W:GFP) was injected into D. melanogaster embryos using P element-mediated transformation to generate stable fly lines. Embryos containing this construct express GFP in midline glia beginning at stage 12 of embryogenesis and throughout larval stages. It was confirmed that GFP was expressed in midline glia by staining embryos simultaneously with either (1) wrapper and GFP or (2) sim and GFP. Because wrapper protein is found at the surface of midline glial cells, but the GFP produced by pHstinger localizes to the nucleus, wrapper protein encircles the GFP in these cells. The wrapper W:GFP reporter construct also drives expression in a few additional cells within the lateral CNS and muscles, a pattern that differs from the endogenous wrapper expression pattern. This suggests that the W fragment, although sufficient to drive high levels of expression in midline glia, lacks certain sequences that exclude expression in lateral CNS cells. To confirm the midline expression pattern generated by the reporters, all subsequent experiments were performed by staining embryos with both sim and GFP at stage 16 of embryogenesis. These experiments revealed that GFP generated by the wrapper W:GFP reporter gene was indeed expressed in the midline glia, but not in the cells that develop into midline neurons (Estes, 2008).
Next, to determine the minimal sequences required to provide expression in midline glia, this 884-bp region was divided into several subregions, fused to GFP within the pHstinger vector and tested for the ability to drive midline expression in transgenic embryos. Region E, extending from sequences -756 to -286, is sufficient to drive high levels of GFP expression in midline glia. Moreover, a smaller 166-bp (-492 to -327) G fragment, and an even smaller 119-bp (-492 to -374) internal K fragment, that both include the highly conserved region, are also sufficient to drive GFP expression in midline glia, but the level of expression is reduced compared to that of the E fragment and the intact 884-bp W fragment. None of the other reporter constructs drove GFP expression in the midline. The K fragment is also expressed in a subset of midline neurons, including progeny of the median neuroblast, suggesting that the larger W, E, and G fragments contain a silencer, which is absent from the K fragment and normally represses expression in these midline neurons (Estes, 2008).
Next, to determine if the observed conservation at the sequence level between Drosophila species reflects conservation in function, the corresponding E region from D. virilis was tested to see if it could drive GFP reporter expression in the midline glia of D. melanogaster. The E region is also located upstream of wrapper in D. virilis and is 476 bp in length, while it is 462 bp in melanogaster. The entire E region is 58.4% identical in the two species, and the 70-bp highly conserved section differs by only six nucleotides. The midline expression pattern provided by the D. virilis wrapper E:GFP construct in D. melanogaster flies is indistinguishable from that of the corresponding D. melanogaster E region. These results suggest that the location and function of the regulatory sequences of wrapper have been conserved between D. melanogaster and D. virilis (Estes, 2008).
To determine if previously identified midline transcription factors affect wrapper through these regulatory sequences, the wrapper W:GFP reporter gene was tested in a number of mutant backgrounds. First, the effect of sim mutations on the reporter gene was tested by placing the 884-bp wrapper W:GFP transgene into a simH9 mutant background, a mutation that eliminates Sim protein expression. In this background, GFP expression was abolished in most cells, suggesting that sim expression is required for wrapper transcriptional activation in the midline. A few remaining cells did express GFP and these are likely lateral CNS cells also observed in wild-type embryos containing the wrapper W:GFP reporter (Estes, 2008).
Next, the reporter gene was tested in a spitz (spi) mutant background. Spi is a signaling molecule that plays multiple roles during Drosophila development. Wrapper protein is normally found on the surface of midline glia where it mediates direct contact with the lateral CNS axons that cross the midline and promotes survival of midline glia. In wrapper mutant embryos, this intimate interaction cannot occur and additional midline glia die. The amount of spi signaling provided by lateral CNS axons determines how many midline glia survive in each segment. The spi mutation severely disrupted CNS development so that the sim positive cells remained on the ventral surface of the embryo. Only a few of the sim positive cells also express GFP driven by wrapper regulatory sequences, suggesting these are the remaining midline glia. The cells expressing sim, but not GFP, are likely midline neurons, while cells expressing GFP and not sim are lateral glia, because they also express reversed polarity (repo), a marker of lateral CNS glia. These results indicate spi mutations reduce the number of midline glia in the embryo and also reduce expression of the wrapper W:GFP reporter gene (Estes, 2008).
In addition to sim and tgo, the transcription factors Dichaete (D), a Sox HMG protein, and Dfr, a POU domain protein, regulate genes expressed in midline glia. The D protein directly interacts with the PAS domain of Sim and the POU domain of Dfr and all three genes activate expression of slit in midline glial . The wrapper W:GFP construct was tested in both a D and dfr mutant background. In both cases, the number and behavior of midline cells was altered and they did not migrate to the dorsal region of the ventral nerve cord, as they normally do. While development of midline cells was disrupted in these mutant backgrounds and fewer midline glia were present, robust GFP expression was still observed from the reporter construct in the midline cells that remained, suggesting that (1) D and Dfr do not directly activate wrapper via these regulatory sequences, (2) additional, redundant factors exist that can substitute for them, or (3) they can substitute for one another (Estes, 2008).
In summary, midline cell development was disrupted in sim, spi, D, and dfr mutant backgrounds. The simH9 mutation eliminated midline glia and neurons, while a mutation in spi eliminated most midline glia. As predicted, both sim and spi mutations severely reduced the number of cells expressing GFP driven by the wrapper W:GFP reporter gene. In the D and dfr mutants, the number of midline glia was reduced and the remaining midline glia expressed high levels of GFP (Estes, 2008).
Ectopic sim expression converts neuroectodermal cells into midline cells and activates downstream, midline genes. To test the effect of ectopic sim on wrapper expression, sim was overexpressed using the UAS/GAL4 system and it was found that wrapper was expressed in neuroectodermal cells outside of the midline, but not in all cells that overexpress sim. In the UAS-sim/da-GAL4 embryos, wrapper is activated in cells that correspond to the lateral edges of the CNS and the cells in the anterior of each segment, with gaps in the expression pattern. Next, whether overexpression of the secreted form of spi could expand wrapper to cells outside the midline was tested. Ectopic expression of secreted spi with the da-GAL4 driver also expanded wrapper expression. To determine if it is possible to expand the expression domain of wrapper further, sim was overexpressed together with spi. This caused additional expansion of the wrapper domain into broad stripes within ectodermal cells. In addition, overexpression of either sim or spi causes severe disruption in embryonic development (Estes, 2008).
Next, the ability of sim and spi, either alone or together, to expand expression of the wrapper reporter genes was tested. Expression from both the full-length reporter construct, wrapper W:GFP, and the smaller wrapper G:GFP construct expanded in the UAS-sim/da-GAL4 embryos to a greater extent than the endogenous wrapper gene. The expression pattern provided by the reporter constructs differs from the endogenous wrapper expression pattern, suggesting that either (1) some of the sequences that normally repress wrapper in tissues outside the midline glia may be missing in these wrapper W and G constructs, or (2) ectodermal cells overexpressing sim may undergo cell death and the GFP marker may be more stable in these dying cells compared to wrapper. Overexpression of spi alone also expanded reporter gene expression driven by both the wrapper W:GFP and wrapper G:GFP constructs. The GFP expression domain was expanded to a greater extent in embryos overexpressing sim together with spi compared to those overexpressing either gene alone. Taken together, the results indicate that (1) limiting the wrapper regulatory sequences and (2) increasing the cells that express sim and spi converts the highly specific expression pattern of wrapper from a single strip of CNS cells to a more general pattern throughout the ectoderm of the embryo. In addition, these results suggest that both the sim transcription factor and spi signaling molecule can activate transcription through these sequences derived from the regulatory region of wrapper (Estes, 2008).
To both (1) identify functionally important motifs needed for wrapper expression and (2) determine if all the invariant nucleotides within the conserved 70-bp region of wrapper are essential for the observed midline glial expression pattern, effects of select mutations within the wrapper G region were tested. Previous studies have demonstrated the importance of sim/tgo, D, dfr, and spi for the expression of midline glial genes and, therefore, possible binding sites for these factors were sought. To examine both predicted binding sites, as well as other conserved sequences that may contain binding sites for novel factors, the region was divided into eight motifs that were tested for their effect on midline glia expression (Estes, 2008).
Each of these conserved motifs was tested by changing 2-3 nucleotides in the context of the D. melanogaster G fragment. The altered G fragments were then inserted independently into the pHstinger vector and injected into fly embryos to test their ability to drive midline expression (Estes, 2008).
Despite the high degree of conservation within this region, only four of the eight mutations that were tested (G1, G2, G5, and G7) caused a noticeable reduction in reporter expression. Two of the mutation sets destroyed midline expression of the G reporter construct. The putative Sim/Tgo binding site (G2: CACGT) was needed for midline expression, because changing this sequence to GAAGT eliminated midline glial expression. In addition, another sequence, ATTTTATC (G5), located upstream of the G2, was required for expression of the reporter gene in wild-type embryos and changing this sequence to ATTGGATC eliminated midline glial expression. Two additional sites within the G fragment of wrapper are needed for midline expression: CGGAGAG (G7) and CACAAT (G1). If either of these motifs is altered, midline glial expression is greatly reduced, but not completely eliminated (Estes, 2008).
Therefore, these experiments suggest that Sim/Tgo heterodimers may directly regulate wrapper gene expression. (1) Activity of the wrapper W:GFP reporter gene is severely reduced in a sim mutant background, suggesting sim is necessary for expression of this transgene and that sim regulates wrapper by activating transcription through these sequences. (2) Midline activity of the wrapper reporter gene is abolished by eliminating the single CME (CACGT) present within this region. (3) wrapper reporter gene expression is expanded in sim overexpression embryos. Future biochemical studies will determine if Sim/Tgo heterodimers directly interact with the wrapper regulatory motif identified in this study (Estes, 2008).
The studies described in this study demonstrate that the wrapper reporter genes are sensitive to levels of spi signaling. Mutations in spi reduce wrapper reporter gene expression and overexpression of the secreted form of Spi, together with Sim expands, not only the expression domain of the endogenous wrapper gene, but the wrapper reporter genes as well. Spi binds the Epidermal Growth Factor Receptor in midline glia, leading to MAPK activation and subsequent activation of the ETS transcription factor, pnt. Therefore, it may be Pnt that directly activates wrapper transcription through the regulatory sequences studied in this study. One of the identified motifs needed for transcriptional activity of wrapper is: CGGAGAG, which loosely conforms to the consensus binding site for ETS transcription factors (C/A)GGA(A/T)(A/G)(C/T). However, further experiments are needed to determine if Pnt directly interacts with these regulatory sequences, as well as the precise mechanism whereby spi signaling regulates wrapper. Taken together with previous studies, these results suggest that the spi signaling pathway may play at least two roles in promoting survival of midline glia: (1) activating wrapper, needed for neuron-glial interactions and (2) phosphorylating, thereby inactivating Head involution defective, which would otherwise cause programmed cell death in midline glia (Estes, 2008).
Many genes expressed in the CNS of metazoan organisms are regulated through synergistic interactions between Sox HMG-containing proteins and POU domain proteins. Recently, many vertebrate genes expressed in the developing CNS have been shown to contain highly conserved noncoding DNA regions enriched for binding sites for three classes of transcription factors: Sox, POU, and homeodomain proteins. Experiments indicated that Sox and POU proteins work together to activate, while homeodomain proteins repress and limit expression of CNS genes. Interestingly, several motifs identified in this study as important for regulation in midline glia of Drosophila resemble binding sites for Sox (G1: CACAAT), POU (G4: ATGCAAAT, G6: ATGCAACA, and G8: ATGCGTGG), and homeodomain proteins (G5: ATTTTATC) (Estes, 2008).
That the wrapper K:GFP, but not the wrapper G:GFP construct is expressed in certain midline neurons, identifies a midline neural silencer in the 43-bp region present in the G fragment, but absent in the K fragment. Within this region, 27 bp are highly conserved in all 12 Drosophila species and two of the three mutations in the G fragment that cause slight activation of reporter gene expression in midline neurons are found within the 43-bp region. All three sites that lead to activation in midline neurons, G4, G6, and G8, conform to a POU domain binding site, suggesting a POU domain protein expressed in midline neurons may bind to one or more of these sites to keep the wrapper gene silent (Estes, 2008).
One POU domain protein, Dfr, binds to the sequence ATGCAAAT in other gene regulatory regions to activate transcription, including those of two genes expressed in midline glia: dfr itself and slit. This sequence is found at site G8 in the wrapper regulatory region, but when changed to ATGCTAGC, caused a low level of activation in midline neurons, rather than reducing expression in midline glia. Although the number of midline glia is reduced in a dfr mutant background, those that remain express a high level of reporter gene expression driven by wrapper sequences and the results suggest dfr is not absolutely required for wrapper reporter gene expression in midline glia (Estes, 2008).
Mutations in the POU domain motifs within the wrapper regulatory sequences suggest a notable difference between the CNS genes studied previously in vertebrates and the midline glial gene studied here. The POU domain binding sites appear to limit expression in midline neurons (rather than activate expression as in vertebrate CNS genes), and it is the Sox and homeodomain binding sites that are needed for activation. This may reflect a key difference in regulatory control of glial vs. neural genes and it is plausible that other midline glial genes excluded from midline neurons will contain silencer elements similar to the one identified in this study, but further experiments are needed to confirm this (Estes, 2008).
At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation. However, evidence for direct interactions and transcriptional activation is lacking. This study presents molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions (Yu, 2005; full text of article).
Mechanisms regulating CNS pattern formation and neural precursor formation are remarkably conserved between Drosophila and vertebrates. However, to date, few direct connections have been made between genes that pattern the early CNS and those that trigger neural precursor formation. Drosophila has been used to link directly the function of two evolutionarily conserved regulators of CNS pattern along the dorsoventral axis, the homeodomain protein Ind and the Sox-domain protein Dichaete, to the spatial regulation of the proneural gene achaete (ac) in the embryonic CNS. A minimal achaete regulatory region that has been identified that recapitulates half of the wild-type ac expression pattern in the CNS; multiple putative Dichaete-, Ind-, and Vnd-binding sites have been found within this region. Consensus Dichaete sites are often found adjacent to those for Vnd and Ind, suggesting that Dichaete associates with Ind or Vnd on target promoters. Consistent with this finding, Dichaete can physically interact with Ind and Vnd. Finally, the in vivo requirement of adjacent Dichaete and Ind sites in the repression of ac gene expression has been demonstrated in the CNS. These data identify a direct link between the molecules that pattern the CNS and those that specify distinct cell-types (Zhao, 2007).
Sox-domain proteins physically associate with other transcription factors to regulate gene transcription. Thus, the identification that Dichaete genetically interacts with Vnd and Ind suggested that Dichaete associates with Vnd and Ind to regulate gene expression in the CNS. To test this model, it was asked whether Dichaete can interact with Ind or Vnd in the yeast two-hybrid assay. Control experiments revealed that the full-length Dichaete protein as well as the region C-terminal to the high-mobility-group (HMG) DNA-binding domain (amino acids 221–384) activate transcription on their own when fused to the Gal4 DNA-binding domain, suggesting that the C-terminal region contains transcriptional activation activity. As a result, a number of distinct Dichaete fusion constructs were tested for self-activation of transcription and four were identified that were transcriptionally inert. One of these contained the HMG domain and the C-terminal region, indicating that the presence of the HMG domain may mask the transactivation properties of the C-terminal region. A prior study mapped a transactivation domain to the N-terminal region of Dichaete (Ma, 1998), yet no transactivation properties of this domain were identified in this study. Consistent with a transactivation domain residing in the C-terminal region of Dichaete, all other identified transactivation domains in Sox-family proteins map C-terminal to the HMG domain (Zhao, 2007).
By using the four Dichaete bait constructs, it was found that the N-terminal region of Dichaete (amino acids 1–141) specifically interacted with full-length Ind protein. In a reciprocal manner, the ability of the Dichaete N-terminal region to interact with two different regions of Ind was tested: the region N-terminal to the homeodomain (amino acids 1–302) and the region including the homeodomain and all residues C-terminal to it (296–391). Both regions of Ind interacted strongly with the Dichaete N-terminal region, suggesting that this region of Dichaete can interface with two distinct regions of Ind (Zhao, 2007).
In a similar manner, two distinct regions of Dichaete, the regions N-terminal (amino acids 1–141) and C-terminal (amino acids 221–384) to the HMG domain, interact with the full-length Vnd protein. Three different Vnd prey constructs were used to localize the regions of Vnd that interact with Dichaete. It was determined that the region of Vnd located between the TN domain (a domain common to Tinman/NK-2 proteins) and the homeodomain (amino acids 217–536) interacts with the Dichaete N-terminal domain. This result confirms and extends those of Yu (2005) who found that Vnd and Dichaete coprecipitate and that a Vnd deletion lacking the first 408 amino acids interacts with Dichaete. It was not possible to define the region of Vnd that interacts with the Dichaete C-terminal region, perhaps because the constructs interrupt the domain to which the C-terminal region of Dichaete binds or disrupt the general topology of this domain. Nonetheless, the yeast two-hybrid results indicate that Dichaete can interact with Ind and Vnd consistent with the model that Dichaete complexes with Ind and Vnd on target gene promoters to regulate transcription in the CNS (Zhao, 2007).
A molecular understanding of how Dichaete, Ind, and Vnd pattern the CNS requires the identification and characterization of the regulatory regions of candidate direct target genes. One such candidate is the ac gene. Prior studies on ac suggested that regulatory regions important for its spatial regulation exist both 5' and 3' to the ac gene. Thus, an 8.15-kb minigene was generated that contains the ac transcription unit as well as ~4.8 kb of DNA 5' to the transcription start and
~2.4 kb of DNA 3' to the polyadenylation site and its ability to drive ac expression in an In (1)y3PLsc8R mutant background was tested. This genetic background carries a deletion of ac and also deletes the regulatory regions necessary to drive sc expression in row 3. Thus, it allows visualization of ac expression as driven by the minigene in the absence of endogenous ac/sc gene expression in row 3. The ac minigene drives ac expression in half of its wild-type CNS pattern because ac is expressed normally in the medial and lateral clusters of row 3 but is not expressed in row 7. The dynamics of ac expression as driven by the minigene in row 3 mirror those of endogenous ac expression because ac expression in each cluster quickly becomes restricted to a single cell, the presumptive neuroblast, which then delaminates into the interior of the embryo and extinguishes ac gene expression before its first division. Thus, the DNA contained within the minigene is sufficient to activate ac in its wild-type expression pattern in row 3 and to mediate the Notch-dependent restriction of ac to the presumptive neuroblast (Zhao, 2007).
By creating a series of 5' and 3' deletions of the initial minigene, the regulatory regions sufficient to drive ac expression in row 3 was delimited to a 2.84-kb genomic fragment (pG7), which is referred to as the row 3 element. This element contains the ac transcription unit, 1.34 kb of DNA 5' to the start of transcription and 542 base pairs of DNA 3' to the end of the transcription unit. ac minigenes were characterized for their ability to respond to the functions of Dichaete, ind, and vnd and for the presence and in vivo relevance of putative binding sites for these factors (Zhao, 2007).
In support of Dichaete, Vnd, and Ind acting directly on the row 3 element to regulate ac expression, loss of Dichaete, vnd, or ind function affects ac expression as driven by ac-pG4 or ac-pG7 in the same way, and these defects are identical to those observed for endogenous ac expression in these mutant backgrounds. For example, loss of ind or Dichaete causes, respectively, strong or modest derepression of ac expression in the intermediate column, whereas loss of vnd results in the absence of ac expression in the medial column (Zhao, 2007).
To see whether Dichaete, Ind, or Vnd act directly on the row 3 element to control ac expression, this element was searched for perfect matches to the consensus Vnd [CAAGTG], Sox-domain [(A/T)(A/T)CAA(A/T)G and homeodomain (TAATGG) binding sites. The canonical Sox-domain and homeodomain binding site sequences were used because the consensus sites for Dichaete and Ind have not been determined. This search identified one match for Vnd (V) and three each for Dichaete (S1, S3, and S4) and Ind (H1, H3, and H4). Notably, predicted Dichaete/Sox-binding sites tend to reside close to predicted Vnd or Ind sites, consistent with Dichaete acting with Vnd and Ind to regulate ac expression. The sole exception is the Ind site (H1) located upstream of the transcriptional start site of ac. However, gel-shift assays identify a Dichaete-binding site 11 bp 5' of this Ind site (S2) (Zhao, 2007).
Because the precise binding specificity of Ind is unknown, whether Ind can bind the predicted sites was tested by using gel-shift assays. Focused was placed on the predicted Ind site located upstream of the transcription start site because it is the only location where Dichaete and Ind sites are found adjacent to each other. It was found that Ind specifically binds this site in vitro. During these experiments, a second Ind-binding site (TAAATG) 8 bp 3' to this site was found, that differs slightly from the consensus homeodomain site. Thus, Ind can bind to two sites located within 1 kb of the ac promoter, suggesting a possible molecular mechanism for Ind-dependent repression of ac (Zhao, 2007).
The initial search for Dichaete-binding sites required a perfect match to the consensus Sox-binding site. However, bona fide transcription factor-binding sites often differ from the experimentally defined consensus by a few base pairs, indicating that the search likely underpredicted possible Dichaete-binding sites. Because of this, gel-shift assays were used to search for Dichaete-binding sites throughout the entire row 3 element (pG7). Three sites were identified to which Dichaete bound specifically. Two of these correspond to sites identified in the consensus sequence search (sites S1 and S3); whereas the third resides 11 bp 5' of the first of the two Ind sites near the transcriptional start of ac (S2); this site (GACAATG) differs from the consensus by one base pair. No binding was detected of Dichaete to one predicted Sox site (S4). Because Dichaete and ind are known to repress ac expression, the three binding sites for Ind and Dichaete upstream of the ac promoter identify a likely site of action through which these factors repress ac (Zhao, 2007).
The clustering of binding sites for Dichaete, Vnd, and Ind, together with the ability of Dichaete to interact with Vnd and Ind, supports the idea that Dichaete acts with these factors to regulate ac expression in the CNS. To test this model directly, the in vivo relevance was assayed of the adjacent Vnd and Dichaete sites as well as the adjacent Dichaete and Ind sites on ac expression. ac expression was unaltered when the Vnd-binding site, the adjacent Dichaete site, or both sites were mutated. Thus, vnd either does not regulate ac expression directly or other Vnd binding sites in the row 3 element compensate for the loss of this site (Zhao, 2007).
The relevance of the three Dichaete- and Ind-binding sites located ~850 bp upstream of the start of ac transcription was assayed. Mutating any single site or any combination of two sites had no effect on ac expression. However, mutating all three sites derepressed ac expression in the intermediate column, a phenotype similar to that found in embryos mutant for ind or Dichaete. This result provides direct link between genes that pattern the CNS and those that specify distinct cell types. Because the derepression of ac is less severe than that observed in ind mutant embryos, Ind and Dichaete likely act through additional sites in this element to repress ac expression fully in the intermediate column (Zhao, 2007).
Unexpectedly, derepression of ac expression posterior to row 3 was observed upon mutation of the three sites. This posterior expansion of ac mimics the effect that removal of gooseberry function has on the expression of ac, suggesting that Gooseberry, another homeodomain protein, may bind the same sites as Ind and act with Dichaete to repress ac expression in its expression domain (Zhao, 2007).
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