Distal-less
There are independent enhancers, arrayed over 40 kb of DNA. Head and leg synthesis is independently regulated. One distal 5' enhancer sequence, an early enhancer regulating Dll expression in germ band extended embryos, is subject to repression by Ultrabithorax and abdominal-A (Vachon, 1992).
A new technique is described using fluorescent probes to simultaneously detect an mRNA and a protein in the Drosophila embryo. For in situ hybridization, 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR is used as a fluorescent substrate for alkaline phosphatase. It is possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. This technique was applied to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. Embryos were stained bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA is detectable in the ventral region of the limb primordium; beta-galactosidase protein is detectable in the dorsal region. In the middle, both mRNA and protein are detectable. These results suggest that the Dll enhancer is activated in the ventral region of the limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium (Goto, 1997b).
The closely related Hox transcription factors Ultrabithorax (Ubx) and Antennapedia (Antp) respectively direct first abdominal
(A1) and second thoracic (T2) segment identities in Drosophila. It has been proposed that their functional differences derive
from their differential occupancy of DNA target sites. A hybrid version of Ubx (Ubx-VP16), which possesses a potent activation domain from the VP16 viral protein, no longer directs A1 denticle pattern in embryonic epidermal cells. Instead, it
mimics Antp in directing T2 denticle pattern, and it can rescue the cuticular loss-of-function phenotype of Antp mutants. In cells
that do not produce denticles, Ubx-VP16 appears to have largely retained its normal repressive regulatory functions. These results suggest that the modulation of
Hox activation and repression functions can account for segment-specific morphological differences that are controlled by different members of the Hox family. These
results also are consistent with the idea that activity regulation underlies the phenotypic suppression phenomenon in which a more posterior Hox protein suppresses
the function of a more anterior member of the Hox cluster. The acquisition of novel activation and repression potentials in Hox proteins may be an important
mechanism underlying the generation of subtle morphological differences during evolution (Li, 1999).
Interestingly, although
Ubx-VP16 acquires an Antp-like ability in denticle patterning, it
preserves the Ubx ability to repress Keilin's organ development in
thoracic segments. Therefore, Ubx-VP16 displays a mix
of Antp-like and Ubx-like functions, dependent on tissue types and cell positions. Since development of Keilin's organs requires the appendage-promoting
gene Distalless (Dll), the regulation
of Dll by Ubx-VP16 was examined. The expression of Dll in
thoracic appendage primordia cells is repressed by Ubx by means of the
Dll304 element, presumably by eliciting the Ubx
repression function on the element. In ectopic Ubx-VP16 embryos, both
Dll expression and
the activity of the Dll304 element are partially repressed. However, unlike ectopic Ubx,
Ubx-VP16 is capable of activating Dll304 in other cells
outside the appendage primordia.
Thus, the Ubx-like function of Ubx-VP16 in repressing Keilin's organ
development stems from retaining the Ubx repressive function upon
Dll transcription. Since this repression appears specific for
appendage primordia cells, the repression function of Ubx-VP16 is not
constitutive but rather generated in a regulated manner. Taken
together, the above results suggest that Ubx-VP16 functions are due to
normal Ubx repressive effects on some targets (e.g., Dll),
despite the attached VP16 activation domain, as well as a novel
activation function on other targets (e.g., Antp) caused by
the VP16 domain. The mix of functions that Ubx-VP16 exhibits is also often observed for
natural Hox proteins (Li, 1999).
In these experiments the strength of activation function in Ubx is artificially varied. However,
the partial change in segmental identity conferred by the Ubx-VP16 protein suggests that regulating the activity state of Ubx may modulate its functional specificity in
denticle patterning. The fact that the Ubx-VP16 denticle patterning function is Antp-like suggests that the functional difference between the Ubx and Antp proteins in
diversifying denticle patterns may reside in differences in activation and repression strengths on similar target genes rather than in differences in target occupancy. This
suggestion is consistent with results indicating that Ubx and Antp recognize identical DNA sequences in vitro and regulate several common target genes
in embryos. This evidence indicates that the segment identity functions of Ubx and Ubx-VP16 are distinct, but it does not eliminate the possibility
that the VP16 domain increases activation function by altering the binding selectivity of the hybrid protein in developing embryos. This is thought to be unlikely because
the specific Ubx targets such as dpp, Antp, and Dll are all regulated, and thus presumably occupied at similar Hox sites, by both Ubx and Ubx-VP16 (Li, 1999 and references).
How can it be examined whether this process has occurred in evolution? In the embryo of the crustacean Artemia, the Antp, Ubx, and abd-A homologs are
coexpressed in a trunk region that is composed wholly of appendage-bearing segments. In contrast to Drosophila, the Artemia Ubx and Abd-A homologs do
not repress Dll transcription and do not repress appendage development. There are a variety of reasons why the Artemia Ubx and Abd-A proteins might be
incapable of repressing appendages, but one possibility is that sequence motifs within the proteins that would allow them to repress the appendage enhancer of Dll
are missing. This possibility may be testable by placing the Artemia versions of Ubx and Abd-A proteins in the context of Drosophila early embryonic cells and
assaying their effects on appendage development (Li, 1999 and references).
The 26 bp bx1 element from the regulatory region of Distal-less is capable of imposing control by the homeotic genes
Ultrabithorax and abdominal-A on a general epidermal activator in Drosophila. This provides an assay to analyze the sequence
requirements for specific repression by these Hox genes. Both the core Hox binding site, 5'-TAAT, and the adjacent Exd 5'-TGAT core site
are required for repression by Ultrabithorax and abdominal-A. The Distal-less bx1 site thus fits with the model of Hox protein binding
specificity based on the consensus PBX/HOX-family site TGATNNAT[g/t][g/a], where the key elements of binding specificity are proposed
to lie in the two base pairs following the TGAT. A single base pair deletion in the bx1 sequence generates a site, bx1:A-mut, which on the
consensus PBX/HOX model would be expected to be regulated by the Deformed Hox gene. It has been observed, however, that the bx1:A-mut site
is regulated predominantly by Sex combs reduced, Ultrabithorax and abdominal-A. The analysis of this site indicates that the specificity of
action of Hox proteins may depend not only on selective DNA binding but also on specific post-binding interactions (White, 2000).
A homeotic response element that mediates
repression by the Hox genes Ubx and abd-A has been built. The element is based on two short sequence modules; the 21 bp binding site Grainyhead binding site element
(Gbe) for the transcription factor GRH and the 26 bp UBX/
ABD-A bx1 footprint site from the Dll regulatory
sequences. On its own the Gbe mediates uniform epidermal
activation. However, the combination of the Gbe and the
bx1 element produces a homeotically modulated response.
Specifically in the domain of expression of the Hox genes
Ubx and abd-A the epidermal expression is repressed. This
homeotic element thus successfully recapitulates the
features of endogenous regulatory elements from homeotic
target genes giving tissue-specific regulation by multiple
homeotic genes (e.g. connectin). However, while endogenous target
gene regulatory elements tend to be large (typically covering several kb) and are correspondingly difficult to analyze,
this constructed element is appealingly simple and provides
a manageable system for analyzing the specificity of the
homeotic response (White, 2000).
The bx1 element contains the notable sequences 5' -
TAAT (the core Hox binding site) and 5' -TGAT (the core
Exd binding site). These two sequences are contained
within the 5' -TGATTTAATT which is similar to the
proposed PBX/HOX consensus site 5' -TGATNNAT[g/t][g/a]. Mutation of the 5' -TAAT
site within bx1 abolishes the abdominal repression
conferred by this element, presumably by reducing the affinity of the site for Ubx and Abd-A proteins. Thus, both Ubx and Abd-A appear to bind in vivo to the same site in the
bx1 element. Mutation of the 5' -TGAT putative Exd binding site also abolishes the abdominal repression, suggesting
that both Ubx and Abd-A require the binding of the cofactor Exd in order to function as repressors on this construct (White, 2000).
The basis of Hox target specificity has been proposed to
depend on the nucleotides immediately following the Exd
core site in the 5' -TGATNNAT[g/t][g/a] PBX/HOX
consensus target site. In vivo
studies have indicated that NN=GG produces a lab-specific response whereas NN=TA elicits a response to Dfd. Previous in vitro studies on the optimum
target sequences for the whole range of PBX/HOX heterodimers have emphasized the importance of the nucleotide at position 7 in the PBX/HOX consensus sequence 5' -
ATGATTNATGG. Preference for a
particular nucleotide at N7 varies across the Hox complex.
The most anteriorly-expressed proteins, HOXB1 and 2,
prefer G at this position, the next more posteriorly-expressed Hox proteins allow N7=A, and from
HOXB6-10 preference grows for N7=T. The optimum
consensus for binding site for the PBX/HOXB7 heterodimer, equivalent to Ubx or Abd-A/Exd in Drosophila, is
5' -ATGATTTATGG. This is similar to the sequence
ATGATTTAatt (differences in lower case)
in the bx1 element. In particular, the nucleotide at N7 is T
and thus fits with the predictions from the in vitro binding
data. Taking the two elements previously studied in vivo
into account, N7=G is in a lab (Hoxb1) element;
N7=A in a Dfd (Hoxb4) element, and now N7=T is present in the bx1 element, which responds to Ubx and abd-A.
Thus, the specificity of regulation of the bx1 element fits
well with the expectations of the PBX/HOX model where
the nucleotides immediately 3' of the TGAT PBX core
provide the specificity for a particular subset of Hox
proteins through interactions with the specific residues of
the N-terminal arm of the homeodomain (White, 2000).
The bx1 element on its own does not drive reporter gene
expression. It acts as a control module that together with the
Grh activation module constitutes a homeotic response
element. This may represent a general model for a target
gene regulatory element, where proteins bound to a PBX/
HOX site interact with neighbouring regulatory complexes
to modulate their activity. The PBX/HOX modules may act
to overlay homeotic control on a whole variety of tissue-specific, signal transduction pathway-specific, temporal or
other regulatory elements. It is worth noting that the regulatory regions that have been identified in homeotic target
genes all drive highly tissue-restricted patterns, indicating
the collaboration between tissue-specific and homeotic
control. These observations give an insight into the modular
construction of complex enhancers and also have important
implications for the specificity of homeotic gene function. If
the PBX/HOX complexes in target gene regulatory
sequences act by interacting with other regulatory
complexes, it seems very likely that different Hox proteins
will vary in their ability to mediate specific interactions.
Thus, there is clearly the potential for much of the specificity of responses to homeotic gene control to reside in post-binding interactions (White, 2000).
In Drosophila, differences between segments, such as the presence or absence of appendages, are controlled by Hox transcription factors. The Hox protein Ultrabithorax (Ubx) suppresses limb formation in the abdomen by repressing the leg selector gene Distalless, whereas Antennapedia (Antp), a thoracic Hox protein, does not repress Distalless. The Hox cofactors Extradenticle and Homothorax selectively enhance Ubx, but not Antp, binding to a Distalless regulatory sequence. A C-terminal peptide in Ubx stimulates binding to this site. However, DNA binding is not sufficient for Distalless repression. Instead, an additional alternatively spliced domain in Ubx is required for Distalless repression but not DNA binding. Thus, the functional specificities of Hox proteins depend on both DNA binding-dependent and -independent mechanisms (Gebelein, 2002).
This work begins with a characterization of a Ubx binding site in the Dll gene that is critical for Dll repression; both Exd and Hth play a role in Ubx binding and repression. The Dll304 enhancer is sufficient to recapitulate the early expression pattern of Dll in the embryonic leg primordia. In addition to activation functions, Dll304 contains two Hox binding sites, Bx1 and Bx2, that repress enhancer activity in the abdomen and thereby restrict Dll expression to the thorax. Most of the repression activity is conferred by Bx1, a sequence bound by Ubx and Abd-A. In agreement with this result, a Distalless minimal element (DME) that lacks the Bx2 site accurately recapitulates the expression of Dll304 in the embryonic thorax. The DME enhancer also shows no derepression within the abdomen, suggesting that Bx1 is sufficient to fully repress Dll (Gebelein, 2002).
To better understand how Bx1 represses Dll, the presence of Exd and Hth binding sites were sought near the previously characterized Hox binding site. A consensus Exd site and a near consensus Hth site are in close proximity to the Hox site of Bx1. The Hox/Exd site (5'-AAATTAAATCA-3'), however, is unlike other previously characterized Hox/Exd binding sites because it contains an additional base pair in between the Hox and Exd half-sites. The Bx1 region containing this Hox/Exd/Hth site is referred to as the Distalless repression element (DllR). To determine whether DllR is required to repress DME expression in the abdomen, it was deleted from the DME enhancer (DMEact) and its ability to activate a reporter gene was tested in vivo. DMEact drives gene expression in all abdominal segments as well as in the thoracic region. Because the thoracic expression driven by DMEact is similar to that of DME, the DllR region is not required for DME activation but solely functions in the repression of Dll in the abdomen (Gebelein, 2002).
To determine whether Exd and Hth stimulate Hox binding to DllR, electrophoretic mobility shift assays (EMSAs) were performed with purified Ubx, Exd, and Hth proteins. Unless stated otherwise, all of these experiments were performed with UbxIa, the most widely expressed of several Ubx isoforms. By themselves, Ubx or an Exd/Hth heterodimer are capable of weakly interacting with DllR. The combination of all three proteins results in a slower migrating band indicating the formation of a Ubx/Exd/Hth/DNA complex. The formation of this protein/DNA complex is highly cooperative when compared to the amount of binding observed with Ubx or Exd/Hth alone. To test the contribution of each binding site, point mutations were introduced within the individual Hox, Exd, and Hth sites. Mutation of any one of these sites results in a decrease in the formation of the trimeric protein/DNA complex, suggesting that all three are required for optimal binding to DllR (Gebelein, 2002).
To test whether the Hox, Exd, and Hth binding sites are also required for Dll repression in vivo, reporter constructs were created containing the lacZ gene under the control of mutant versions of the DME enhancer. Mutation of the Hox site (DMEHox) results in a similar level of derepression of reporter gene expression throughout the abdomen, as does the the complete deletion of DllR. Mutation of the Exd (DMEExd) and Hth (DMEHth) sites individually also results in derepression, albeit slightly weaker than mutation of the Hox site. However, if both the Exd and Hth sites are mutated together, full derepression is observed. Taken together, these results demonstrate that the efficient formation of a Hox/Exd/Hth trimeric complex on DllR is required for Dll repression within the abdomen (Gebelein, 2002).
These above data support a model in which a Ubx/Exd/Hth complex bound to DllR is necessary for Dll repression. Whether a single copy of DllR is sufficient to repress a heterologous enhancer element was tested. An artificial enhancer, called fkh(250con), is activated by Scr, Antp, and Ubx (with Exd and Hth), and thus provides a useful heterologous activator to test for DllR function. A reporter construct under the control of both fkh(250con) and DllR was created. Unlike fkh(250con), which is expressed in parasegments (PS) 2-6, the composite enhancer (fkh250con-DllR) is not expressed in PS 6, where Ubx is expressed. Ubx-mediated repression of fkh(250con)-DllR is more obvious in embryos mutant for abd-A, which derepress Ubx and, consequently, fkh(250con) throughout the abdomen. In this genetic background, fkh(250con)-DllR is still active only in PS 2-5. Furthermore, misexpression of Ubx throughout the embryo activates fkh(250con) but represses fkh(250con)-DllR. Taken together, these results indicate that DllR is sufficient to confer Ubx-mediated repression of a heterologous enhancer. In addition, these results also illustrate that Ubx/Exd/Hth complexes can mediate repression through DllR in the same cells as it mediates activation through fkh(250con) (Gebelein, 2002).
A general question for all transcription factors is how they achieve specificity in vivo. For the Hox proteins, a large number of studies have implicated sequences both within and outside the homeodomain as being important for their in vivo specificities. But how do these sequences function? Because DNA binding domains, including homeodomains, can also be protein interaction domains, studies that map the domains necessary for target gene regulation cannot answer this question by themselves. Instead, direct transcriptional targets must be identified and, once binding sites are characterized, DNA binding, in addition to target gene regulation, must be measured. The results allow two steps to be discriminated in the repression of Dll by Ubx. First, Exd and Hth stimulate Ubx, but not Antp, binding to DllR. In contrast, Ubx/Exd/Hth and Antp/Exd/Hth have similar affinities for a different "consensus" binding site (5'-CCATAAATCA-3'), suggesting that subtle differences in the DNA sequence, in addition to differences between Ubx and Antp, contribute to specificity. A C-terminal peptide in Ubx stimulates this cofactor-dependent binding to DllR. DNA binding, however, is not sufficient for Dll repression. Instead, an additional linker domain included in only a subset of Ubx isoforms is required for repression. Thus, a second step, the recruitment of additional factors to the Ubx/Exd/Hth complex bound to DllR, is implied by these data. In addition to the UbxIa linker, this step also requires the specific sequences and conformation imposed on the Ubx/Exd/Hth trimer by DllR (Gebelein, 2002).
Although the Ubx C terminus plays an important role in cofactor-dependent binding to DllR, additional domains contribute to optimal binding. In the presence of Exd and Hth, the AAUU chimera, but not heterologous AAUA or AAAU, binds DllR, suggesting that both the Ubx homeodomain and C terminus are important for optimal DNA binding to this site. The C terminus is not absolutely required for binding because a Ubx protein that lacks this domain (UUU*) is still able to bind well to DllR. Last, the finding that UUU*, but not AAU*, binds DllR suggests that a domain N terminal to the homeodomain also enhances DllR binding. Based on the crystal structures of Hox/Exd/DNA complexes, this difference could be due to the YPWM motif. Taken together, the data suggest that multiple regions of Ubx contribute to binding DllR and that no one domain is sufficient for full binding activity. This finding may be understood in light of the fact that the entire Ubx coding sequence has been constrained over millions of years of insect evolution to maintain leg (and Dll) repression in the abdomen (Gebelein, 2002).
How might the Ubx C terminus and YPWM motifs contribute to DNA binding? It is suggested that these regions could make additional protein-DNA contacts and/or protein-protein interactions that help stabilize the DllR-bound form of the trimeric complex. In support of this idea, the C termini of other homeodomain proteins also contribute to DNA binding. The Exd C terminus, for example, consists of an alpha helix that packs against its homeodomain and contributes to DNA binding. The C terminus of the MATalpha2 protein from yeast forms an alpha helix that contacts the MATa1 homeodomain to stabilize heterodimer formation on DNA. Interestingly, the two Hox proteins that repress Dll expression, Ubx and Abd-A, share sequence homology in their C termini, and are the only Drosophila Hox proteins predicted to form an alpha helix after their homeodomains (Gebelein, 2002).
The Ubx YPWM motif may also help stabilize complex formation on DllR. In the Hox/Exd/DNA crystal structures, this motif, together with flanking amino acids, directly contacts a hydrophobic pocket within the Exd homeodomain. These protein-protein contacts are thought to stabilize protein-DNA contacts made by the complex. The amino acids surrounding the YPWM motifs are different in Ubx and Antp and thus could contribute to DNA binding specificity by such an indirect mechanism (Gebelein, 2002).
The finding that UbxIa, but not UbxIVa, is able to repress Dll suggests that the linker region in UbxIa is required for repression. In addition, these results suggest that alternative splicing has the potential to modulate Ubx's control of gene expression. In support of this view, the expression of Ubx isoforms is temporally and spatially regulated. In addition, misexpression experiments using UbxIa and UbxIVa have shown that while both perform many of the same functions, only UbxIa efficiently transforms the peripheral nervous system. The finding that UbxIa and UbxIVa have different transcriptional regulatory properties provides a possible explanation for their distinct abilities to transform this tissue (Gebelein, 2002).
One argument against the idea that the different Ubx isoforms have distinct functions is that flies containing a genetic inversion that prevents the inclusion of the second microexon are, for the most part, normal. Although this mutation prevents the expression of UbxIa, it is unclear which other Ubx isoforms are expressed in this mutant because the inversion does not include both microexons. Furthermore, the effect that this mutation has on Dll expression has not been examined. A definitive test of the idea that Ubx isoforms have unique functions will require determining whether a Ubx allele in which both microexons are eliminated can provide all Ubx functions in vivo (Gebelein, 2002).
As in Drosophila, Dll expression is a marker for leg primordia in many animal phyla. Animals with appendages on their abdominal segments, such as crustaceans and onychophora, coexpress Ubx with Dll, demonstrating that Ubx is not a repressor of Dll in these species. The ability of Ubx to repress Dll probably arose in a subset of arthropods, the hexapods. Consistent with these findings, two recent studies suggest that one relevant difference between Ubx orthologs that repress Dll (for example, Drosophila Ubx) and Ubx orthologs that do not repress Dll (for example, onychophoran Ubx) maps to the C-terminal regions of these Hox proteins (Galant, 2002; Ronshaugen, 2002). These two groups, however, propose different mechanisms for how these sequences function. Galant suggests that the Drosophila Ubx C terminus actively represses transcription via a polyalanine motif that is present in the Ubx orthologs from all hexapods. Ronshaugen suggests that the Drosophila Ubx C terminus is only permissive for repression. Instead, they argue that crustaceans, which have abdominal legs, evolved a C-terminal sequence that inhibits Dll repression. However, neither study analyzed the binding of these proteins to the relevant binding sites in Dll, leaving open the possibility that the effects they observe could also be due to effects on DNA binding (Gebelein, 2002).
The data provide additional insights into how repression mechanisms may have evolved in these different species. It was found that the Drosophila Ubx C terminus contributes to DllR binding but is not sufficient for Dll repression in vivo. Thus, the positive role, observed by Galant, that the Drosophila sequence plays in Dll repression, could be due to an effect on DNA binding. These experiments also implicate the linker region of UbxIa as important for repression, but not DNA binding. Because some of the onychophora/ Drosophila and crustacean/ Drosophila chimeras lack this linker but are able to repress Dll, the crustacean and onychophoran Ubx orthologs must have repression domains that are different from the one identified in Drosophila Ubx (Gebelein, 2002).
Ronshaugen suggests that the phosphorylation of serine and threonine residues in the crustacean Ubx C terminus is necessary for it to prevent Dll repression (Ronshaugen, 2002). This is an intriguing possibility in light of the fact that phosphorylation of a Hox C terminus can inhibit cooperative DNA binding with Exd. Taken together with the current data that the C terminus of Ubx enhances DNA binding to DllR, it is suggested that the inhibition of Dll repression by the crustacean C terminus may be due to a reduced ability to bind DllR with Exd and Hth. This model accounts for why a Drosophila UbxIa protein containing the crustacean C terminus is unable to repress Dll (Ronshaugen, 2002) and for the inability of onychophora Ubx, which also contains a putative phosphorylation site in its C terminus, to repress Dll. Taken together, it is suggested that the evolution of limb suppression by Hox proteins, and probably many other Hox functions, depended upon the modification of both DNA binding-dependent and -independent mechanisms controlling Hox specificity (Gebelein, 2002).
Although these experiments focused on understanding why Antp is different from Ubx, the results provide some insights into the mechanism of transcriptional repression. The data strongly argue that a DNA-bound Ubx/Exd/Hth complex is necessary, but not sufficient, for repression. First, in addition to repressing Dll, Ubx/Exd/Hth activates fkh(250con). When both fkh(250con) and DllR simultaneously regulate the same reporter gene, DllR is able to repress gene expression in the same cells in which fkh(250con) normally activates gene expression. This result suggests that the repressor proteins required for DllR activity are not cell type specific and are widely expressed in the embryo. Further, these results suggest that differences between the fkh(250con) and DllR sequences determine whether transcription is activated or repressed. These sequences may recruit additional DNA binding factors that interact with the trimeric complex. These factors, which have not yet been identified, might provide or reveal a latent activation or repression domain within the Hox/Exd/Hth complex. Alternatively, another DNA binding factor may not be needed. Instead, the unique arrangement or spacing of the Hox, Exd, and Hth sites in these two elements may result in distinct conformations of the trimeric complex that recruit different coactivators or corepressors. Such a mechanism has been suggested for the nuclear receptor family of transcription factors and for the POU domain protein Pit-1, where a difference in spacing in a Pit-1 dimer binding site regulates the recruitment of a corepressor. Consistent with such a mechanism, it was found that the DllRcon binding site, which has one less base pair between the Hox and Exd half-sites than the DllR binding site, fails to repress transcription despite having a higher affinity for Ubx/Exd/Hth complexes. In addition, although repression activity for the UbxIa linker and C terminus in S2 cells can be measured, the experiments suggest that their activities are context dependent. The abdominal expression of DMEcon-lacZ suggests that the mere presence of these domains is not sufficient for repression. Thus, the data suggest that transcription factor domains have distinct properties when assayed by themselves versus when they are part of a multiprotein complex. Further, it is concluded that the unique architecture of the complex assembled on DllR is necessary for efficient repression (Gebelein, 2002).
During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).
The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior-posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior-posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior-posterior axis (Gebelein, 2004).
This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior-posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).
Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior-posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).
Exd and Hth, as well as their vertebrate counterparts, are used as Hox
cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).
To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).
One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).
To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).
Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).
Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).
On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).
To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).
On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).
Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).
Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).
The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).
Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).
According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).
Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).
Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).
The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior-posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).
These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).
These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).
Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).
The development of the Drosophila leg requires both Decapentaplegic (Dpp) and Wingless (Wg), two signals that establish the proximo-distal (PD) axis by activating target genes such as Distalless (Dll). Dll expression in the leg depends on a Dpp- and Wg-dependent phase and a maintenance phase that is independent of these signals. This study shows that accurate Dll expression in the leg results from the synergistic interaction between two cis-regulatory elements. The Leg Trigger (LT) element directly integrates Wg and Dpp inputs and is active only in cells receiving high levels of both signals. The Maintenance (M) element is able to maintain Wg- and Dpp-independent expression, but only when in cis to LT. M, which includes the native Dll promoter, functions as an autoregulatory element by directly binding Dll. The 'trigger-maintenance' model describes a mechanism by which secreted morphogens act combinatorially to induce the stable expression of target genes (Estella, 2008).
This study provides evidence that Dll expression during Drosophila leg development is controlled by separate, synergistically interacting cis-regulatory elements. The first element, LT, activates transcription only in response to high levels of Wg and Dpp signaling. The second element, M, includes the Dll promoter and has the ability to activate transcription in a Wg- and Dpp-independent manner, but only when in cis to LT. Together, these results fit well with previous genetic experiments showing that the Wg and Dpp inputs into Dll are only required transiently, prior to ~60 hr AEL. Based on the data, it is hypothesized that LT, and perhaps other elements with similar properties, is responsible for activating the Wg- and Dpp-dependent phase of Dll expression. Further, the data suggest that the combination of LT+M executes the Wg- and Dpp-independent phase of Dll expression. The existence of a two-component cis-regulatory system for Dll expression has several interesting implications and provides a mechanistic understanding of how Wg, Dpp, and Dll inputs are integrated into Dll expression (Estella, 2008).
The requirement for multiple inputs for gene activation is a common theme in transcriptional regulation. Enhancer elements can be thought of as 'logic integrators' that are only active in the presence of the correct activators and in the absence of repressors. The LT element defined here behaves as such a logic integrator. To be active, at least three conditions must be met. (1) LT must be bound to a transcriptionally active form of Tcf, a condition which indicates high levels of Wg signaling. (2) LT must be bound to a transcriptionally active form of Mad, and, (3) LT must not be bound to Brk. The second and third of these three conditions both indicate high levels of Dpp signaling. This combination of inputs ensures that LT is triggered only only where Wg and Dpp signaling are both active. In addition, it is hypothesized that there must be another input that restricts LT's activity to the ventral discs (e.g., it is not active in other tissues where Wg and Dpp signaling intersect such as the wing disc). Such a ventral-specific input could be Dll itself, which is expressed before LT is active via the Dll304 enhancer, and/or another ventral-specific factor such as buttonhead (btd), which is also required for Dll expression. Consistent with this idea, LT-lacZ is lost in Dll– clones and in Dll hypomorphic discs, suggesting that Dll input, in addition to Wg and Dpp, is required for its activity (data not shown) (Estella, 2008).
As noted above, Dpp signaling uses two mechanisms (Mad binding and absence of Brk) to control LT's activity. Because Brk, a transcriptional repressor, binds directly to LT, it restricts the domain in which Wg signaling can activate this element. This conclusion is best supported by the expression pattern of the LT reporter gene in which the Brk-binding sites were mutated. Specifically, the expression of this reporter (LTBrk–-lacZ) was expanded ventrally, indicating its potential to be activated more broadly by Wg signaling in the absence of this repressor. Thus, it is suggested that the primary role of Brk is to provide spatial information to LT activation. The absence of Brk, however, is apparently not sufficient for LT activation; Mad input into LT appears also to be essential. Several experiments support this conclusion. Most informatively, LT-lacZ was not expressed in Mad–; brk– clones, and LT-lacZ reporter genes with either Mad site mutated were not expressed in brk– clones. Thus, even in the absence of Brk, LT requires Mad input. It is suggested that in contrast to providing spatial information, the Mad input into LT is important for boosting the level of its activation, together with Tcf, by providing an additional potent transcriptional activator. Further, LT is unlikely to be the only Dll cis-regulatory element that integrates Wg plus Dpp signaling during leg development. Although LT was the only fragment within the 14 kb of 5' DNA that drove strong expression in the leg disc in a standard reporter gene assay, thus allowing the dissection of Wg and Dpp signal integration, a second fragment was identified that was able to synergize with M to produce a Dll-like expression pattern. In summary, these data suggest that during the Wg- and Dpp-dependent stage, Dll expression is regulated by the direct binding of Tcf, Mad, and Brk to LT and, perhaps, additional regulatory elements (Estella, 2008).
As is the case for Dll, there are examples of other genes that have separable initiation and maintenance phases of expression. For many of these examples, expression is maintained by the trxG and PcG of epigenetic regulators. There are also examples of genes that require enhancer-promoter communication for maintenance. For example, a regulatory element from the Hoxb4 gene requires sequences from its own promoter for stable expression in the mouse hindbrain. In this case, a key input into the promoter-proximal sequences is the PcG protein, YY1. Dll expression is unaffected in trx mutant clones but is lost in a subset of Pc– and Scm– clones, raising the possibility that PcG functions play a role in maintenance. However, PcG functions are more typically associated with maintaining genes in a repressed state, not an expressed state. Moreover, because of PcG's widespread role in gene silencing, many genes are likely to be derepressed in these clones. In fact, the Hox gene Abd-B is derepressed in these clones, and Abd-B has the ability to repress Dll. Thus, on balance, it seems more likely that the loss of Dll expression observed in some Pc– clones is an indirect effect. In contrast, the results strongly argue that positive autoregulation, by direct binding of Dll to the M element, plays an important role in Dll maintenance (Estella, 2008).
One conclusion drawn from these observation that both LT and M are required for maintenance is that LT requires the Dll promoter to be fully active. Such promoter-specific enhancer activation has been observed previously and is generally thought to be important for remote enhancers to stimulate transcription from the correct promoter in gene-dense regions of the genome. The LT+M synergy described in this study is distinct from these other examples. In this case, although enhancer-promoter compatibility may be part of the reason that LT works better with M (and over large distances), the results show that the combination of the two has properties that are not exhibited by either element on its own. Specifically, while M-lacZ is very weakly expressed in leg discs, and LT-lacZ requires continuous Wg and Dpp inputs, the combination of LT+M allows Dll autoregulation to occur in a Wg- and Dpp-independent manner. Moreover, LT+M is not simply a Dll autoregulatory element: even though Dll is expressed in the wing disc, transcriptional activation by LT+M remains restricted to the ventral imaginal discs. This observation implies that the Dll input into LT+M can only occur in cells where LT was activated, which itself only happens in ventral discs. Thus, LT+M is not only a two-component Dll autoregulatory element, but is an autoregulatory element that requires the prior Wg- and Dpp activation of LT (Estella, 2008).
These observations lead to the suggestion of two classes of models by which maintenance may occur. In one, an activated LT element changes the chromatin structure of M, for example, by changing the position of a repressive nucleosome so that it can function as an autoregulatory element. According to this model, the continued presence of LT is required to maintain this chromatin structure. A second model that would also accommodate these data is that the combination of LT plus M is required to increase the efficacy of transcriptional activation by, for example, providing additional Dll (or other activator) binding. According to this scenario, LT activation by Wg and Dpp triggers the initial interaction between the LT and M elements, which would then be stabilized in a Wg- and Dpp-independent manner. These models are not mutually exclusive and both can be tested by analyzing the chromatin status at the M and LT elements (Estella, 2008).
The results also raise the question of what purpose this two-step trigger-maintenance mechanism may serve. One possibility is that, by having only a transient requirement for Wg and Dpp, these morphogens are available for carrying out completely different tasks, without affecting Dll expression. In support of this idea, in addition to working together to create the PD axis, Wg and Dpp function independently to instruct ventral and dorsal leg fates, respectively. Some of these late Wg and Dpp patterning functions may also require Dll input. The trigger-maintenance logic described in this study in principle allows Wg and Dpp to execute functions in collaboration with their own downstream target, Dll (Estella, 2008).
It is also noteworthy that the transient nature of the Wg and Dpp inputs into Dll is not the typical way these morphogens regulate their target genes in other tissues. In the Drosophila wing, for example, Dpp and Wg are required to continuously activate their targets, such as vestigial, optormotor blind, and spalt. One signficant difference between the regulation of wing and leg target genes by these morphogens is that in the wing Wg and Dpp generally act independently, whereas in the leg they act combinatorially to activate PD genes. Specifically, although they are expressed in ventral and dorsal sectors, respectively, Wg and Dpp activate Dll and dac in circular or nearly circular domains whose centers are located where the Dpp and Wg expression domains touch, in the middle of the leg disc. The trigger-maintenance mechanism defined in this study avoids the need for target genes such as Dll to continuously integrate Wg and Dpp inputs as the disc grows in size, and provides a mechanism to generate circular domains of gene expression using dorsal and ventral morphogen inputs (Estella, 2008).
Hox transcription factors are essential for shaping body morphology in development and evolution. The control of Hox protein activity in part arises from interaction with the PBC class of partners, pre-B cell transcription factor (Pbx) proteins in vertebrates and Extradenticle (Exd) in Drosophila. Characterized interactions occur through a single mode, involving a short hexapeptide motif in the Hox protein. This apparent uniqueness in Hox-PBC interaction provides little mechanistic insight in how the same cofactors endow Hox proteins with specific and diverse activities. This study identified in the Drosophila Ultrabithorax (Ubx) protein a short motif responsible for an alternative mode of Exd recruitment. Together with previous reports, this finding highlights that the Hox protein Ubx has multiple ways to interact with the Exd cofactor and suggests that flexibility in Hox-PBC contacts contributes to specify and diversify Hox protein function (Merabet, 2007).
The current view of Hox-PBC interactions is that they all occur through a single mode, involving a short hexapeptide (HX) motif. The importance of the Hox HX motif in mediating interaction with PBC proteins is extensively supported by its requirement in in vitro interaction assays and by crystallographic studies that showed that the HX provides most if not all major contacts. In contrast, in vivo functional support for a role of the HX in mediating interaction with PBC proteins is still limited, mainly because effects of HX mutations during development have only been assessed for two vertebrate Hox proteins, Hoxa-1 and Hoxb-8, and for three Drosophila proteins, Labial (Lab), Ubx, and Abdominal-A (AbdA) (Merabet, 2007 and references therein).
Mutation of the HX in Hoxb-8 results in dominant phenotypes, which are at present difficult to interpret with regard to Pbx recruitment. Hoxa-1 HX mutation mimics Hoxa-1 loss of function, including defects in the hindbrain that could relate to loss of Pbx recruitment because inactivation of Pbx2 and Pbx4 in the zebrafish affects hindbrain patterning. However, addressing in vertebrates whether phenotypes resulting from HX mutations are consequences of defects in Pbx interaction will require examination of combinations of Hox-1 paralogous and Pbx gene mutations. In Drosophila, mutation of the HX in Lab, the only representative of Hox-1 class genes, results in an hyperactive protein when assayed for its potential to activate transcription through an evolutionarily conserved Hoxb-1 autoregulatory element. This hyperactivity results from the loss of an inhibitory action of the HX on Lab DNA binding. In this context, it was proposed that HX-mediated recruitment of Extradenticle (Exd) acts to mask the DNA-binding inhibitory activity of the HX motif (Merabet, 2007 and references therein).
Although the Hoxa-1 and Lab studies support, yet not exclusively, a role of the HX in mediating recruitment of PBC class proteins during development, work on Drosophila Ubx and AbdA has provided evidences for HX-independent mode of Exd recruitment. Regarding Ubx, a truncated protein lacking N-terminal sequences (including the HX) was shown in vitro to retain Exd recruitment potential and to interact weakly with Exd in yeast two-hybrid assays. More specifically, mutation of the HX does not affect the capacity to recruit Exd on a Hox/Exd consensus target sequence in vitro and to repress in an Exd-dependent manner the limb-promoting gene Distalless (Dll), which has served as a paradigm to study Hox-Exd interactions. Concerning AbdA, the HX-deficient protein was shown to recruit Exd on the Dll regulatory element that mediates Dll repression, consistent with its retained ability to repress Dll (Merabet, 2007).
Thus, Hox-PBC interactions are not limited to HX-mediated interactions, highlighting that another Hox protein motif, yet to be identified, may also assume this function. The Ubx C terminus [sequences downstream of the homeodomain (HD), UC], important for Ubx segment identity functions and shown to increase the ability of the Ubx HD to associate with Exd in yeast two-hybrid assays, harbors an 8-aa peptide previously termed UbdA as well as a QA repression domain responsible for changes in Ubx activity. Although evolutionarily conserved, the precise function of the UbdA motif, only present in Ubx and AbdA proteins but absent from any other Drosophila Hox protein, is not known. This work reports on the function of the UbdA motif in the context of the Ubx protein. Because this motif is specific to Ubx and AbdA, which share the HX-independent mode of Exd recruitment, the analysis focused on the possible implication of this motif in mediating Exd recruitment (Merabet, 2007).
The results strongly support that the UbdA motif mediates Exd recruitment by the Ubx protein. This finding is first established by the requirement of the motif for Exd recruitment in the process of Dll regulation: mutation of the motif impairs the capacity of Ubx to mediate interaction with Exd on Dll regulatory sequences in vitro, which correlates with the reduced ability of UbxUbdA to perform Exd-dependent repression of Dll in vivo. Evidence is provided that mutation of the UbdA motif does not result in a globally defective protein: the UbdA mutated protein still binds DNA with appropriate affinities as a monomer, still represses the wing promoting genes dSRF, sal, and vg and still activates the dpp target gene in the visceral mesoderm. Thus, mutating the UbdA motif selectively affects a subset of Ubx functions. Importantly, the conclusion that the UbdA motif mediates Exd recruitment is also supported by the demonstration that the motif provides de novo Exd recruitment potential to a Hox protein that has been rendered deficient for this function. This finding is shown both in vitro by the potential of the motif to confer Exd recruitment to Antp on a Hox/Exd consensus sequence and on a cis-regulatory sequence of the Antp/Exd target gene tsh, and in vivo by its potential to restore Exd-dependent activation of tsh.
Complexity in Hox-Exd Interactions (Merabet, 2007).
The Ubx protein provides a so far unique situation wherein two identified protein motifs within the same Hox protein have the potential to perform the recruitment of the Exd cofactor, which raises the question of whether these two motifs are effectively used for Exd recruitment by Ubx. Previous work has shown that an HX-deficient Ubx protein was altered in its segment identity specification: whereas Ubx specifies A1 segment identity, the mutated form specifies A2-like identity. Interestingly, in a context deficient for zygotic Exd contribution, Ubx also specifies A2-like identity, suggesting that the HX motif is required for Exd-dependent A1 specification. These observations support that within this context, the HX is the motif used to perform Exd recruitment, although definitive support awaits characterization of Ubx-Exd interaction on a Ubx downstream target gene involved in segment identity specification. Considering the finding that the UbdA motif mediates Exd recruitment in the process of Dll regulation, it is proposed that depending on the developmental context, i.e., on the target gene regulated, Ubx uses different protein motifs for Exd recruitment. The contextual (gene-specific) use of the HX and UbdA protein motifs introduce a first level of complexity in Ubx-Exd interactions (Merabet, 2007).
A second level of complexity in the Ubx-Exd relationship is illustrated by the regulation of the dpp target gene. In this case, it was found that neither the HX nor the UbdA motif was required for Exd-dependent activation by Ubx. The possibility that these two motifs were acting in a redundant way was excluded by the observation that a Ubx protein mutant for both motifs still activates dpp. Thus, other protein motifs, yet to be identified, could confer an additional mode of Exd interaction, further increasing the diversity by which Ubx could contact the Exd cofactor. Alternatively, the dispensability of the HX and UbdA motifs for dpp activation may also suggest that Ubx/Exd contacts are not required. The latter hypothesis is supported by the existence in dpp regulatory regions of Exd-binding sites that are not closely associated to Hox-binding sequences and by the previous observation that Exd can improve Ubx monomer binding to dpp regulatory sequences in a manner that does not require the formation of a Ubx-Exd-DNA tripartite complex. In any case, the regulation of dpp suggests further complexity in the Ubx-Exd relationship, which, by extension, highlights that the functional interplay of Hox-PBC proteins is likely to be more diverse than the current view (Merabet, 2007).
Although previous studies showed that HX-deficient Hox proteins retain the capacity to interact with Exd and to mediate Exd-dependent functions, motifs responsible for alternative modes of interaction were not identified. This work identifies a so far unique HX-alternative mode of PBC recruitment, introducing the notion of flexibility in Hox-PBC contacts. This interaction mode was not anticipated from previous crystallographic studies because the truncated Ubx protein used was lacking the UbdA motif. Given the divergence of the primary sequences of the HX and UbdA motifs, their distinct location in the protein, and the absence of functional redundancy, the UbdA- and HX-mediated interaction modes are likely to be structurally distinct (Merabet, 2007).
These findings have also implications with regard to Hox protein diversity and specificity. Flexibility in Hox-PBC interactions allows addressing of the issue of diversity from a mechanistic point of view: depending on the motif involved in the interaction, which likely relies on the target sequence, the Hox-PBC complex may adopt different conformations, which in turn set structural bases for distinct activities. This process therefore provides cues to explain how diversity can be generated through qualitatively distinct interaction modes involving the same protein partners. Furthermore, because the UbdA motif is only found in Ubx and AbdA, it likely endows these two proteins with a specific Exd interaction mode. This mode may serve to distinguish Ubx and AbdA from other Drosophila Hox proteins, therefore providing basis for Hox protein specificity. Finally, this study questions whether additional HX-independent modes of PBC interaction exist. It was reported previously that the HX-deficient Lab protein retains Exd interaction potential and in vivo Exd-dependent activity. As Lab does not bear a UbdA motif, it supports further flexibility in Hox-PBC interaction. Addressing the issue of diversity in Hox-PBC interaction thus appears as a necessary step to understand the mechanisms underlying Hox protein activity in development and evolution (Merabet, 2007).
Limb development requires the elaboration of a proximodistal (PD) axis,
which forms orthogonally to previously defined dorsoventral (DV) and
anteroposterior (AP) axes. In arthropods, the PD axis of the adult leg is
subdivided into two broad domains, a proximal coxopodite and a distal
telopodite. This study shows that the progressive subdivision of the PD axis into these
two domains occurs during embryogenesis and is reflected in the cis-regulatory
architecture of the Distalless (Dll) gene.
Dll protein in the thorax was first detected during embryonic stage 11, and continues to be visualized in this region until the end of embryogenesis. Early
Dll expression, governed by the Dll304 enhancer, is in cells
that can give rise to both domains of the leg as well as to the entire dorsal
(wing) appendage. A few hours after Dll304 is activated, the activity
of this enhancer fades, and two later-acting enhancers assume control over
Dll expression. The LT enhancer is expressed in cells that
will give rise to the entire telopodite, and only the telopodite. By contrast,
cells that activate the DKO ("Distalless Keilin Organ") enhancer will give rise to a leg-associated larval sensory structure known as the Keilin's organ (KO).
Cells that activate neither LT nor DKO, but had activated
Dll304, will give rise to the coxopodite. In addition,
the trans-acting signals controlling the LT and DKO
enhancers are described; surprisingly, the coxopodite progenitors begin to
proliferate ~24 hours earlier than the telopodite progenitors. Together,
these findings provide a complete and high-resolution fate map of the
Drosophila appendage primordia, linking the primary domains to
specific cis-regulatory elements in Dll (McKay, 2009).
To determine how each of the cell fates in the limb primordia is
specified, genetic experiments were carried out to identify the regulators of
the LT and DKO enhancers. Consistent with LT's dependency on
wg and dpp for leg disc expression, LT is activated in the embryo in cells that receive both inputs, as monitored by anti-Wg and anti-PMad staining. To determine whether wg is required for LT activity, a
temperature-sensitive allele of wg was used to allow earlier Dll
activation. Switching the embryos to the restrictive temperature at
stage 11 resulted in the absence of LT activity, despite the presence
of Dll protein (probably derived from Dll304 activity. In addition, ectopic
activation of the wg pathway [using an activated form of armadillo (arm*)] resulted in more LT-lacZ-expressing cells (McKay, 2009).
Like wg, the dpp pathway is necessary for
LT-lacZ expression in leg discs. Paradoxically, dpp
signaling represses Dll in the embryo because dpp mutants
show an expansion in Dll304-lacZ expression. By contrast, LT-lacZ is not expressed in dpp null embryos. LT-lacZ, but not Dll protein, was also repressed by two dpp pathway repressors, Dad and brk. Conversely, stimulation of the dpp pathway [using an activated form of the Dpp receptor (TkvQD)] resulted in ectopic activation of LT ventrally (McKay, 2009).
Taken together, these data demonstrate that LT is activated by Wg
and Dpp in the embryonic limb primordia, just as it (and Dll) is in
the leg disc. Similarly, DKO activity also requires Wg and Dpp input (McKay, 2009).
Although LT is activated by wg and dpp in the
leg primordia, these signals are also present in each abdominal segment.
Consequently, there must be additional factors that restrict LT
activity to the thorax. One possibility is that LT is repressed by
the abdominal Hox factors, such as Dll304. Alternatively, LT might be regulated by Dll, itself. In Dll null embryos LT-lacZ
was initially expressed in a stripe of cells instead of a ring, but then
expression decayed. Ectopic expression of Dll resulted in weak ectopic expression of LT-lacZ in the thorax and abdomen. These data suggest that LT activity is restricted to the thorax in part because of the earlier restriction of Dll304 activity to the thorax (McKay, 2009).
The related zinc-finger transcription factors encoded by
buttonhead (btd) and Sp1 are also expressed in the
limb primordia and are also required for ventral appendage specification. In
strong btd hypomorphs, the activity of LT was still detected
but the number of cells expressing LT-lacZ was decreased and its
pattern was disrupted. LT-lacZ expression was completely eliminated in animals bearing a large deficiency that removes both btd and Sp1. By contrast,
Dll304 was activated normally in these animals (data not shown).
Importantly, LT-lacZ expression was rescued by expressing
btd in these deficiency embryos. By contrast,
expressing Dll, tkvQD, or arm* did not
rescue LT expression in these deficiency embryos. Ectopic
expression of btd resulted in weak ectopic activation of
LT-lacZ in cells of the thorax and abdomen. Strikingly, the simultaneous expression of Dll and btd resulted in robust ectopic expression of
LT-lacZ in abdominal segments in the equivalent ventrolateral
position as the thoracic limb primordia. btd and
Dll were not sufficient to activate LT in wg null
embryos (data not shown). These data indicate that the thoracic-specific expression of the LT enhancer is controlled by the combined activities of btd and/or Sp1, Dll and the wg and dpp pathways (McKay, 2009).
Although the data suggest that LT is activated by a
combination of Wg, Dpp, Btd and Dll, these activators are also present in the
precursors of the KO, which activate DKO instead of LT.
Because the KO is a sensory structure, the role of members of the
achaete-scute complex (ASC) that are expressed in these cells was tested. In embryos hemizygous for a deficiency that removes the
achaete-scute complex, LT-lacZ expression was expanded at the expense
of the Ct-expressing cells. Consistently, ectopic expression of the ASC gene
asense (ase) repressed LT and increased the number
of Ct-expressing cells. These data suggest that there is a mutual antagonism between
the progenitors of the telopodite and those of the KO. It was also found that
DKO-lacZ expression in the leg primordia was lost in Dll or
btd null embryos, consistent with the loss of KOs in these mutants. DKO activity was also lost from the limb primordia in embryos deficient for the ASC. These results indicate that DKO is activated by the same genes that promote LT expression but, in addition, requires proneural input from the ASC (McKay, 2009).
One of the most interesting findings from this work is that the temporal
control of Dll expression in the limb primordia by three
cis-regulatory elements is linked to cell-type specification. The earliest acting
element, Dll304, is active throughout the appendage primordia. At the
time Dll304 is active, the cells are multipotent and can give rise to
any part of the dorsal or ventral appendages, or KO. A few hours later,
Dll304 activity fades, and two alternative cis-regulatory elements
become active. Together, these two elements allow for the uninterrupted and
uniform expression of Dll within the appendage primordia. However,
their activation correlates with a higher degree of refinement in cell fate
potential: LT, active in only the outer ring of the appendage
primordia, is only expressed in the progenitors of the telopodite. By
contrast, DKO, active in the cells within the LT ring, is
only expressed in the progenitors of the KO. Thus, although the pattern of Dll
protein appears unchanged, the control over Dll expression has
shifted from singular control by Dll304 to dual control by
LT and DKO. Moreover, not only is there a molecular handoff
from Dll304 to LT and DKO, the two later enhancers
both require the earlier expression of Dll. Thus, the logic of
ventral primordia refinement depends on a cascade of Dll regulatory
elements in which the later ones depend on the activity of an earlier one (McKay, 2009).
The high-resolution view of the embryonic limb primordia provided in this study
allows clarification of some contradictions that currently exist in the
literature. Initial expression of Dll in the thorax overlaps entirely
with Hth-nExd (referring to nuclear Extradenticle). Subsequently, hth expression is lost from most, but not all, of the Dll-expressing cells of the leg primordia. The first reports describing these changes failed to recognize the persistent overlap
between Dll and Hth-nExd in some cells. As a result, and partly because of the analogy with the third instar leg disc, the predominant view of this fate map became that the
Dll-positive, Hth-nExd-negative cells of the embryonic primordia gave rise to
the telopodite, while the surrounding Hth-positive cells gave rise to the
coxopodite. The expression pattern of esg, a gene required for
the maintenance of diploidy, was also misinterpreted as being a marker
exclusively of proximal leg fates. Counter to these earlier studies, the current experiments unambiguously show that the Dll-positive, Hth-nExd-negative cells in the
center of the primordia give rise to the KO, the ring of Dll-positive,
Esg-positive, Hth-nExd-positive cells gives rise to the telopodite, and the
remaining Esg-positive, Dll-negative cells give rise to the coxopodite (McKay, 2009).
The spurious expression of DKO-lacZ in Dll-non-expressing
cells outside the leg primorida complicates the interpretation of several
experiments. Attempts to refine DKO activity by changing the size of
the cloned fragment proved unsuccessful. Nevertheless, the evidence supports
the idea that DKO-positive, Dll-positive cells of the leg
primordia give rise to the Keilin's organ, and not the adult appendage (McKay, 2009).
The progenitors of the coxopodite begin to proliferate at approximately 48
hours of development, consistent with previous measurements of leg imaginal
disc growth, whereas the progenitors of the telopodite do not resume
proliferating for an additional 12 to 24 hours. According to estimates of the
cell cycle time in leg discs, this difference in the onset of proliferation results in one to two additional cell divisions in the coxopodite, consistent with images of late second instar leg discs presented in this study. Why might the telopodite and coxopodite begin
proliferation at different times? One possibility is that the cells of the
coxopodite give rise to the peripodial epithelium that covers the leg imaginal
disc, and therefore require additional cell divisions relative to the
telopodite. It is also possible that the telopodite is delayed because the
neurons of the Keilin's organ serve a pathfinding role for larval-born neurons
that innervate the adult limb. Perhaps this pathfinding function requires that the KO and
telopodite remain associated with each other through the second instar.
Consistently, the leg is the only imaginal disc that has not invaginated as a
sac-like structure in newly hatched first instar larvae (McKay, 2009).
A possible explanation for the delay in the onset of telopodite
proliferation is the persistent co-expression of hth and Dll
in these cells; hth (and tsh) expression is turned off in
these cells at about the same time they begin to proliferate. Consistent with
this idea, maintaining the expression of hth throughout the primordia
blocks the proliferation of the telopodite. Also noteworthy is the finding that the genes no ocelli and elbow have been shown to mediate the ability of Wg
and Dpp to repress coxopodite fates. Together with the current findings, it is possible that the activation of these two genes in the LT-expressing progenitors is the
trigger that turns off hth and tsh in these cells (McKay, 2009).
The experiments suggest that once LT is activated, and under
normal growth conditions, there is a lineage restriction between the
telopodite and coxopodite. By contrast, previous lineage-tracing experiments
using tsh-Gal4 concluded that the progeny of proximal cells could
adopt more distal leg fates. However, tsh is still expressed in the telopodite progenitors far into the second instar, providing an explanation for these results. In contrast to this early restriction, there is no evidence for a later lineage restriction within the telopodite. For example, the progeny of a Dll-positive cell can lose
Dll expression and contribute to the dac-only domain (McKay, 2009).
Interestingly, the lineage restriction between coxopodite and telopodite is
not defined by the presence or absence of Hth-nExd or Tsh because both
progenitor populations express hth and tsh after their fates
have been specified. By contrast, when these two domains are specified, the
telopodite expresses Dll, while the coxopodite does not, suggesting
that Dll may be important for the lineage restriction. However, later in
development, some cells in the telopodite lose Dll expression and
express dac, but continue to respect the coxopodite-telopodite
boundary. Thus, either Dll expression in the telopodite is somehow
remembered or the telopodite-coxopodite boundary can be maintained by
dac, which is expressed in place of Dll immediately adjacent
to the telopodite-coxopodite boundary. Also noteworthy is the finding that
clones originating in the coxopodite can contribute to the trochanter, the
segment inbetween the proximal and distal components of the adult leg that
expresses both Dll and hth in third instar imaginal discs.
However, the progeny of such clones do not contribute to fates more distal
than the trochanter. Likewise, a clone originating in the telopodite can also
contribute to the trochanter, but will not grow more proximally into the coxa. Thus, the lineage restriction uncovered here seems to be determined by distinct combinations of
transcription factors expressed in the coxopodite and telopodite progenitors
at stage 14. The progeny of cells that express Dll, tsh and
hth can populate the telopodite or trochanter, whereas the progeny of
cells that express tsh and hth, but not Dll, can populate the coxopodite or trochanter. In light of Minute-positive results, however, the lineage restriction between coxopodite and telopodite does not satisfy the classical definition of a compartment boundary. A similar non-compartment lineage restriction has also been documented along the PD axis of the developing Drosophila wing (McKay, 2009).
The radically distinct morphologies of arthropod and tetrapod legs argue that these appendages do not share a common evolutionary origin. Yet, despite dramatic differences in morphology, it has been known for some time that transcription factors encoded by the Distalless (Dll)/Dlx gene family play a critical role in the development of both structures. This study shows that a second transcription factor family encoded by the Sp8 gene family, previously implicated in vertebrate limb development, also plays an early and fundamental role in arthropod leg development. By simultaneously removing the function of two Sp8 orthologs, buttonhead (btd) and Sp1, during Drosophila embryogenesis, adult leg development was found to be completely abolished. Remarkably, in the absence of these factors, transformations from ventral to dorsal appendage identities are observed, suggesting that adult dorsal fates become derepressed when ventral fates are eliminated. Further, Sp1 was shown to play a much more important role in ventral appendage specification than btd, and Sp1 lies genetically upstream of Dll. In addition to these selector-like gene functions, Sp1 and btd are also required during larval stages for the growth of the leg. Vertebrate Sp8 can rescue many of the functions of the Drosophila genes, arguing that these activities have been conserved, despite more than 500 million years of independent evolution. These observations suggest that an ancient Sp8/Dlx gene cassette was used in an early metazoan for primitive limb-like outgrowths and that this cassette was co-opted multiple times for appendage formation in multiple animal phyla (Estella, 2010).
Prior to this study, understanding of the roles that btd and Sp1 play in ventral appendage development in Drosophila was largely derived from ectopic expression experiments showing that btd could induce ectopic leg development when expressed in dorsal imaginal discs. In addition, based on a large deficiency that removes >50 genes, it was suggested that these genes may function upstream of Dll in ventral appendage specification. What was lacking in this previous study was the ability to specifically analyze the functions of these genes, both in embryogenesis and during adult development, using loss-of-function null alleles. Using a newly derived deficiency, together with rescue experiments, this study showed that these Zn-finger transcription factors play non-redundant roles in ventral appendage development. Moreover, for all of the readouts examined in this study (leg allocation, leg growth, proliferation, and PD axis formation) btd plays a much more minor or no role compared to Sp1. Early, Sp1, but not btd, is required to define the group of cells that will give rise to the legs and perhaps additional ventral body structures as well. Thus, Sp1 is a selector-like gene for the entire ventral appendage. Later in development, both genes are required for the proper growth of the leg, although to very different degrees. It was also shown that vertebrate Sp8 retains both the selector and growth-promoting functions, suggesting that there has been a remarkable amount of functional conservation between the vertebrate and fly genes (Estella, 2010).
During larval development, it was found that Sp1 is required for the proper growth of the entire leg, from the coxa through the tarsus. In contrast, btd plays a much more limited role in the tibia and femur. At this stage, neither gene is required for leg identity, nor are they required for the development of ventral body structures that arise from the most proximal cells in the leg imaginal disc. These 'late' phenotypes are consistent with the expression patterns of these genes in the third instar leg imaginal discs, where they appear to mark the entire presumptive leg, but not more proximal cells. This is interesting, because prior to these observations there were no markers that distinguished between the hth-expressing cells that give rise to the coxa from the hth-expressing cells that give rise to the ventral body wall. Dll, for example, is expressed in the cells that give rise to the distal tibia and tarsus, and lineage tracing with the Dll-LT element marks the entire telopodite (trochanter, femur, tibia, and tarsus). The addition of the btd and Sp1 expression patterns and mutant phenotypes to previously characterized PD genes therefore adds an important demarcation that distinguishes leg from body fates (Estella, 2010).
This analysis also reveals dramatic differences in the post-embryonic functions of btd and Sp1. Specifically, most of the growth phenotypes observed when both genes are removed can be phenocopied by knocking down only Sp1. In contrast, btdXG81 clones (or btdXA clones) have no phenotypes in the antenna, and, in the leg, result in only partial fusions between the femur and tibia. Thus, Sp1, not btd, plays an important and non-redundant function in ventral appendage development at this stage (Estella, 2010).
Selector and selector-like genes have the property that they specify an entire organ or body part. The classic example is engrailed (en) which 'selects' posterior compartment identities in Drosophila. Another example is eyeless (ey), which is both necessary and sufficient for eye development in Drosophila. In the leg, previous work highlighted the role of Dll in ventral appendage specification. In the absence of Dll, the distal portion of the leg fails to develop, while dorsal appendages remain wild type. Moreover, ectopic expression of Dll can induce distal legs to develop in dorsal positions. Taken together, these observations suggested that Dll is a selector-like gene for the distal leg (Estella, 2010).
Despite the requirement for Dll in leg development, it has been known for sometime that the ventral appendage primordia form in the absence of Dll. Moreover, homeotic transformations are not observed in the absence of Dll. Thus, Dll cannot be considered a selector-like gene for the entire ventral appendage. These observations raise the question of what factor or factors initially specify the cells that will give rise to the ventral appendage. It is proposed that Sp1 fulfills this selector-like role (Estella, 2010).
The suggestion that Sp1 is a selector-like gene for the entire ventral appendage stems in part from the observation that when the function of this gene is removed early in development, ∼10% of the animals have dramatic transformations of ventral structures to dorsal structures. In many of these cases, both wing and notum tissue were observed developing in ventral positions. Molecularly, Dll and dac expression is lost in transformed leg discs, and ectopic expression of vg and eyg, two markers for the dorsal appendages, are observed instead. The expression of Dll-304, which is traditionally been considered a marker for the ventral appendage, in Df(btd,Sp1) embryos may seem at odds with the idea that Sp1 is required for the initial specification of leg fates. However, fate-mapping studies show that Dll-304-expressing cells give rise to both the ventral (leg) and dorsal (wing and haltere) appendages. Thus, Dll-304 cannot be considered a ventral marker, and its activity in Df(btd,Sp1) embryos only confirms the establishment of appendage primordia without ventral or dorsal identity (Estella, 2010).
In sum, the striking transformations of fate seen in Df(btd,Sp1) animals suggest that Sp1 promotes ventral fates, both the entire leg and ventral body wall, and that in the absence of this gene, dorsal fates are de-repressed. This change in developmental fate is analogous to other classical homeotic transformations, for example, when the leg is transformed to antenna in the absence of Antennapedia (Antp). Note that btd null clones made at the same early time in development only result in mild growth defects, but legs are still generated. Thus, btd is not required for this function. However, because an Sp1 null allele (btd+) is not currently available, we cannot at this time be completely certain that btd plays no role in this process (Estella, 2010).
Because wing development is normally limited to T2, it was unexpected to observe leg to wing transformations in the T1 and, to a lesser extent, T3 segments. One potential explanation for this violation of antero-posterior identity is due to the timing of clone induction. Although the Hox genes are responsible for determining the segmental identities of the dorsal appendages, it may be that they are deployed at different times in the ventral and dorsal primordia in the different thoracic segments. If this is the case, then the resulting transformations may be very sensitive to the time they were generated and to their segmental origins. It is also worth noting that the wing primordia and T2 identity can be generated in the absence of Hox input. Thus, wing fates, as opposed to haltere or humeral (dorsal T1) fates, represent a Hox-free default state, which may predominate in these aberrant developmental situations (Estella, 2010).
Together with previous studies, these findings allow a presentation of a more complete view of ventral appendage specification, which is brocken down into three main phases. In the first phase, Sp1, btd, and Dll (via it's early Dll-304 enhancer) are initially activated in parallel in a ventral domain in each thoracic hemisegment of stage 11 embryos. The activation of all three genes is dependent on Wg signaling. This early, Dll-304-driven expression of Dll does not require either btd or Sp1. This initial group of cells is fated to give rise to both the entire ventral and dorsal thoracic imaginal discs, in other words, the entire adult thorax. In the second phase, which begins at stage 14, Dll-304 is no longer active and Dll is controlled by late-acting enhancers such as Dll-LT, which is activated by Wg and Dpp signaling. Interestingly, as shown in this study, these late-acting Dll enhancers also require Sp1, but not btd, thus placing Sp1 genetically upstream of Dll. At this stage, the Dll+ cells will only give rise to the leg telopodite. Sp1 is also required for telopodite formation but is carrying out at least two additional functions. One is that, unlike Dll, Sp1 is required to specify more proximal leg segments (the coxapodite). Second, the ventral to dorsal homeotic transformations described above suggest that Sp1 is also required to repress dorsal fates. Finally, in the third phase, Dll begins to autoactivate it's expression and no longer depends on Wg and Dpp inputs. At this stage, Dll also no longer requires Sp1 to be expressed. Instead of working through Dll, btd and Sp1 continue to play a critical role in leg development but now work in parallel to Dll to promote the growth of the entire leg. Thus, the specification of the ventral primordia depends on a feed-forward logic in which Sp1 activates late embryonic Dll expression followed by a phase in which both btd and Sp1 contribute to appendage growth in parallel to Dll (Estella, 2010).
Besides having a PD axis, arthropod and vertebrate appendage morphologies have little in common. Moreover, the developmental logic of limb formation in Drosophila is very different from that of vertebrate limb development. In flies, Hedgehog signaling induces two antagonistic secondary signals, Dpp and Wg, which in turn establish the PD axis by activating genes such as Dll and dac. In vertebrate limb development, Sonic hedgehog induces the activity of fibroblast growth factor-like molecules such as FGF8 in the ectoderm, which drives the proliferation of the underlying mesenchyme and the nested expression of Hox genes to create a PD axis. Despite these differences, it is striking that multiple vertebrate orthologs of both Sp1 and Dll are expressed during vertebrate limb development. In addition, orthologs of both hth and exd (Meis and pbx, respectively) are expressed in the proximal domain of the developing mouse limb. Although the existence of multiple Dll and Sp1 orthologs (Dlx1/Dlx2/Dlx5/Dlx6 and Sp8/Sp9, respectively) makes it much more challenging to assess their functions in detail, the available data demonstrate that, as in flies, both sets of genes are critical for vertebrate limb development. The current results, illustrating that vertebrate Sp8 can rescue many of the Sp1 and btd loss of function phenotypes in Drosophila, support the idea that appendage development in these two phyla represents a case of 'deep homology'. Interestingly, that orthologs of both Sp1 and Dll gene families are used in both phyla argue that, for appendage development, the functions of these transcription factors have been much more conserved than those of the signaling pathways used in limb development. The same conclusion holds for eye development where the transcription factors, more than the deployment of specific signaling pathways, have been conserved over vast evolutionary distances. These observations imply that, once established, transcription factor networks may be very stable, while the organization of signaling pathway networks may be much more plastic and easily modified to accommodate radically distinct morphologies (Estella, 2010).
The expression of the Hox gene Distal-less (Dll) directs the development of appendages in a wide variety of animals. In Drosophila, its expression is subjected to a complex developmental control. This study examined a 17kb genomic region in the Dll locus that lies downstream of the coding sequence; control elements of primary functional importance were found for the expression of Dll in the leg and in other tissues. Of particular interest is a control element, which has been called LP, which drives expression of Dll in the leg primordium from early embryonic development, and whose deletion causes severe truncation and malformation of the adult leg. This is the first Dll enhancer for which, in addition to the ability to drive expression of a reporter, a role can be demonstrated in the expression of the endogenous Dll gene and in the development of the leg. In addition, the results suggest that some enhancers, contrary to the widely accepted notion, may require a specific 5' or 3' position with respect to the transcribed region (Galindo, 2011).
The genomic region immediately downstream of the Dll transcription unit has remained virtually unexplored since the gene was first characterised at the molecular level. The only enhancer described in this region was a maxillary enhancer which requires Dfd to drive Dll expression. This enhancer was contained within the ETD6 fragment, affected in the DllB allele, but not in DllJ. Beyond the ETD6 fragment lie several kilobases of genomic DNA without any major transcripts. In contrast, the genomic region upstream of Dll had been extensively investigated in search of control regions involved in the leg expression of Dll. This has identified at least four new enhancers in the Dll downstream region than can drive expression in the embryonic and early larval leg primordia (LP), late larval leg disc primordia (LL), leg bracts (BR) and wing margin (WM). In addition, the results have helped refine the location of the maxillary enhancer (MX) (Galindo, 2011).
The known control elements for leg expression included an early embryo enhancer (304), a late embryo and early larval enhancer (215/LT), a Keilin organ enhancer (DKO) and a self-maintenance element (M) . These spread over 20 kb upstream of the Dll transcription unit, and together they seemed to account for the whole pattern of expression of Dll. This conclusion was based mostly on the pattern of expression they impose on reporter genes. The only functional information available on regulatory regions was the rescue experiments with the 312 and 313 minigenes, and these suggested that the upstream region was able to rescue the lack of Keilin's organs in a Dll null mutant. This study has revisited and extended these rescues, and it was observe that a few of the rescued individuals can develop into pharate adults displaying a severe leg phenotype, which indicates that this 5′ region is not enough to support a complete Dll gene expression pattern in leg development. This conclusion is supported by the phenotypes of the DllJ and Dll1092 regulatory mutants and, most strikingly, by the newly induced DllR28 mutant. This mutant is a relatively small deletion and its phenotype is remarkably similar to the minigene rescues, which suggests that the enhancer that it affects accounts for the most crucial part of the leg function of the 3′ region (Galindo, 2011).
The crucial regulatory element disrupted by DllR28 is the LP enhancer. The deleted region is well covered by reporter fragments 5 and 1, with extensive overlap among them, and the only leg enhancer is LP, present only in fragment 1. Therefore, it is unlikely that any other leg enhancer which is covered by the DllR28 mutation has passed unnoticed, and the LP enhancer can be mapped to a 0.8 kb interval up to some 3 kb downstream of the Dll1092 insertion site. Impairing the function of this enhancer has dramatic consequences resulting in deformities in medial leg and truncation of the distal segments. These regions derive from cells that fell within the LP-expressing territory up to first instar. It was observed that in late third instar the morphology of the DllR28 imaginal discs is abnormal, expression of Dac is extended distally and the expression of Dll is weakened both in the central domain and in the peripheral ring. Therefore, the activity of the LP enhancer is required for the early determination of leg PD fates and the subsequent efficient distal expression of Dll. The LP enhancer is not only functionally different from 304 and 215/LT, but also its timing and the regulation of its expression are also different. LP starts to work in stage 11, soon after 304 and earlier than 215/LT. It integrates positive effects from three main signalling pathways, Wg, Dpp and EGFR, and it does not absolutely require Dll for its own expression (Galindo, 2011).
LP is the only Dll enhancer described to date with any functional significance in leg development. The combination of 215/LT + M can drive expression of lacZ in a central domain in the leg disc which is coincident with the endogenous Dll, but to date no leg-specific regulatory mutation has been mapped to this region. Another major caveat against the central role attributed to the combination of the 215/LT and M enhancers comes from the fact that both this combination and 215/LT itself are dependent on Dll expression and therefore they may represent an autoactivatory input to reinforce the expression of Dll, rather than the actual trigger of Dll expression in the leg. It is possible that 215/LT contains a 'shadow' leg enhancer whose functionality would be required to reinforce and maintain the activity of the downstream leg enhancers in extreme physiological conditions or during certain developmental periods (Galindo, 2011).
An additional leg enhancer, expressed from early-mid third instar in leg imaginal discs, was found that was called LL. LL is an autoregulatory enhancer, which autonomously requires Dll. Its pattern of expression coincides with the endogenous Dll domain, and in this respect it is similar to the other autoregulatory enhancer described to date, the M enhancer. Thus, it would seem that Dll expression may require a variety of enhancers with an autoactivatory component: 215/LT, M and LL (Galindo, 2011).
An integrated model of the regulation of Dll in the legs would be as follows: At embryonic stage 10, Dll expression is activated in the single primordium for the Keilin's organ (the vestigial larval leg), the leg and the wing imaginal disc, and is required for the formation of these three structures. This activation of 304 is achieved by Wg, while Dpp, EGFR and the Hox proteins Ubx and AbdA act as repressors; hence this mixed appendage primordium is located in the thoracic segments only and at the dorsal edge of the ventral stripe of wg expression. Slightly later, at stage 11, 304 ceases to act, the wing primordium loses Dll expression, and separates and moves away dorsally. Dll expression remains in the leg and Keilin primordia but is now driven by LP, which interprets inputs differently than 304: thus, while LP is similarly activated by Wg, it is also activated by Dpp and EGFR, which were repressors of 304. A requirement has been described for EGFR signalling in leg development between 6 and 7 h of development (stage 11) with concomitant transient activation of MAPK activation. This precise timing indicates that EGFR activates Dll through the LP enhancer. Later on, during stages 12 and 13, 215/LT becomes active and collects activatory inputs from Wg, Dpp and Dll itself to reinforce the action of LP. This mode of regulation remains during first instar, and is responsible for the specification of most of the imaginal leg (the telopodite), giving raise to trochanter, femur, tibia, and tarsus. At the first to second instar transition, the activity of LP ceases, the leg imaginal disc separates from the Keilin organ and the expression of Dll disappears from the presumptive femur and distal tibia, which acquire the expression of dac. Expression of Dll remains in the distal part of the leg (tibia and tarsus), driven by continuing Wg and Dpp signalling through 215/LT. At early third instar, the expression of Dll becomes independent of Wg and Dpp and seems to rely exclusively on autoactivatory maintenance driven by 215/LT + M and the new 3′ autoactivatory enhancer described here, LL. This self-maintained expression remains until the late pupa, when sensory-organ specific expression driven by the BR enhancer appears in the bracts of the leg bristles (Galindo, 2011).
While this model accounts for Dll regulation in Drosophila, and presumably other holometabolous insects with separate larval and imaginal leg primordia, it is likely that a very similar mechanism operates in less derived hemimetabolous insects and other arthropods, which develop their legs directly at embryogenesis. These less derived arthropods also display dynamic Dll expression showing the disappearance of Dll expression from the presumptive medial leg (femur and tibia in insects), which in Drosophila is correlated with inactivation of the activatory 3′ enhancer LP. This reduction in Dll expression does not occur in the antenna of any of these species, and this differential regulation contributes to the different pattern and morphology of these appendages (Galindo, 2011).
It was already suspected that the Dll wing margin enhancer had to lie in the downstream region, since the upstream region could not drive any expression in the wing imaginal disc. This work has shown that the minigene rescues with the 312 and 313 fragments produce pharate adults in which the wing margin has a typical Dll phenotype of lack of bristles. In consequence, there must be a wing enhancer in this downstream region. Two regions were found that can drive GFP expression in the wing margin. The first one, shared by fragments 3 and 7 may be the same as the LL enhancer, and it is active in the wing margin late in pupal development. It could be a manifestation of the self-regulatory enhancer LL in the wing margin, but in any case it is probably irrelevant since the expression of Dll is required for the determination of the wing margin bristles earlier, in late third instar. The second one, the enhancer that has been called WM is most likely the missing wing margin enhancer. WM is contained in Fr1, like LP, but probably 3′ of it since the DllR28 deletion does not affect the wing margin. WM can drive GFP expression only in a narrow line of cells at the presumptive wing margin itself, while Dll protein expression is stronger in the wing margin, but then decays gradually in the wing pouch, in what has been interpreted to be a graded response to wg. The most likely explanation is that this enhancer may need to act in conjunction with another element elsewhere in the Dll locus, most likely an autoactivatory enhancer. Thus, cells in the early wing disc close to the margin would switch Dll expression on, but as the disc grows some of these cells will find themselves further away from the margin, and outside of the functional Wg gradient. In these cells, some weaker Dll expression would still remain thanks to the self-maintenance activity of Dll. In this scenario, the gradient of Dll protein observed in the wing disc (strong levels near the margin, weaker in the blade), would be the result of the life history of the disc cells, while the pattern of Fr1-GFP would just represent a snapshot of the cells currently exposed to Wg. It would be interesting to test this possibility in the context of previous and recent re-assessments of the long-range Wg gradient hypothesis (Galindo, 2011).
Finally, a BR enhancer was found that is co-expressed with Dll-Gal4 and probably represents the driver required for the function of Dll in the leg bracts. Bracts are determined by directional EGFR signalling from the bristle. It was known that a typical phenotype in different combinations of Dll mutant alleles and in Dll− somatic clones was the lack of the bracts, which are characteristic of medial and distal leg segments. This study has shown through the small deletion in DllR28 that this phenotype maps to the downstream region of Dll, and the corresponding control region was identified in the overlap of Fr5 and Fr6 since both fragments can drive expression of reporter genes in the bracts (Galindo, 2011).
Two cautionary lessons could be obtained from these results. First, some enhancers may have specific positional requirements with respect to the coding region in order to function efficiently. In this respect, the LP, LL and WM enhancers did not work or worked much less efficiently when placed 5′ of the Gal4 transcription unit, but did drive expression of GFP when placed downstream of the transcription unit. Since the objective of the present work was not to study the positional specificity of enhancers, the results do not permit a completely watertight interpretation, but some of the alternatives can be discarded on close inspection (Galindo, 2011).
The nature of the vector backbone is unlikely to be the cause of the difference, since both use the same hsp70 minimal promoter, which is standard for many Drosophila vectors. In addition, PTGal has been used in the characterization of several regulatory regions, with at least 14 publications listed by Pubmed. Finally, this positional effect was not present in the MX or BR enhancers, both of which could drive correct expression either upstream or downstream of both reporters. In the case of MX, this fragment works in three different constructs (lacZ, Gal4 and GFP). The difference between LP, LL and WM 5′ and 3′ reporters could also be due to a specific requirement for these enhancers to be situated at a minimal distance from the promoter; this minimal spacing could be achieved more easily when situated 3′ of the transcription unit. However, examination of the distances between the LP and LL enhancers and the hsp70 promoter in fragments 1 and 7 constructs does not support this explanation either, due to the 5′ position of the enhancers within the 5 kb inserts and to the small size of the GFP coding sequence (1.2 kb). In any case, any argument based on construct distances fades if it is considered that the endogenous distance of LP to the Dll promoter is much larger at nearly 40 kb. Still, other possibilities cannot yet be discarded, such as the presence in fragments 7 and 1 of uncharacterized insulators, located 3′ to the actual LP and LL enhancers. To definitely prove this 3′ position effect, cloning of the LP and LL enhancers 5′ of the hsp70 promoter and the GFP reporter in the pH-Stinger vector would be required (Galindo, 2011).
Regardless of the precise basis of this position effect, its functional significance may reflect some constraint in the control of the transcription of Dll, or it may help to prevent the ectopic activation of genes further downstream, and therefore represent a more general safety mechanism in the control of gene expression. In a similar study, the downstream region of the wingless gene was investigated and regulatory regions for the eye, wing and ventral (leg and antenna) imaginal discs identified. Although the patterns of expression of the reporter genes closely resembled endogenous Wg, some details in their pattern and activation timing differed with respect to the endogenous protein. Small differences like these have been usually disregarded, but may stem from the fact that regulatory regions have been largely characterised in reporter constructs in which the genomic region was cloned upstream of the lacZ reporter gene, even if their native position is downstream of the coding region. These results beg further research that might challenge the prevalent view that the 5′ or 3′ positioning of enhancers is not as important as distance to the promoter, and may illuminate new models of enhancer–promoter communication (Galindo, 2011).
From these results, a second cautionary principle arises. Even if the pattern of expression of a reporter construct is similar to the endogenous gene product, one cannot necessarily conclude that the DNA region cloned in such a construct is either absolutely required or fully sufficient to control the expression of the gene. Similarly, in vitro binding assays inform as to the potential ability of DNA fragments to bind certain proteins, not of the functional outcome in vivo. Multiple enhancers, either similar or unrelated, can contribute towards the final output in both normal and extreme conditions. Therefore, expression data of reporter constructs should be complemented with functional information in order to obtain meaningful insights into the regulation of the genes under study (Galindo, 2011).
This report presents a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunoprecipitate the tagged chromatin from specific cell types. This approach was applied to Distalless (Dll), a gene required for limb development in Drosophila. The local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5' regulatory region is in close proximity to the Dll promoter. In Dll-nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type-specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and nonexpressing cell types, respectively (Agelopoulos, 2012).
In summary the local chromatin conformation at Dll varies in a
developmentally relevant manner: its 5' regulatory DNA is present
in different states depending on whether it is expressed or
repressed by abdominal Hox proteins. In contrast
to previous studies where 3D chromatin organization was compared
in very different tissues (e.g., forebrain versus limb, these experiments compared a small group of Dll-expressing cells in the thorax that are fated to give rise
to the appendages with the homologous groups of cells in the
abdomen. The fates of these two populations of cells differ
only due to the expression of Hox selector proteins. Because
long-distance interactions were observed only in the thorax, the
results suggest that abdominal Hox proteins suppress limb
development at least in part by preventing distant enhancer
elements from being brought into proximity with the Dll promoter.
It is further speculated that abdominal Hox proteins block
these long-range interactions by interfering with the binding of
GAF and other activators, perhaps by promoting the assembly
of H2Av-containing nucleosomes (Agelopoulos, 2012).
It is also noteworthy that the interactions observed in Dll-expressing
cells are not limited to communication between
individual enhancers and the promoter. Instead, the entire 5'
Dll regulatory region appears to be in a more compact state
because many of these sequences are in close proximity to
each other and to the Dll promoter. These observations suggest
that the entire 5' 12 kb region functions as a single unit, consistent
with the presence of additional Dll CRMs within this region. Thus, whereas isolated CRMs and shadow enhancers are often sufficient to drive accurate
reporter gene expression, multiple CRMs may be integrated
within larger functional regulons when in their native context (Agelopoulos, 2012).
Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules (McKay, 2013).
This paper addresses a long-standing question in developmental biology:
how does a single genome give rise to a diversity of structures?
The results indicate that the combination of transcription factors
expressed in each thoracic appendage acts upon a shared set of
enhancers to create different morphological outputs, rather than
operating on a set of enhancers that is specific to each tissue. This conclusion is based upon the surprising observation
that the open chromatin profiles of the developing appendages
are nearly identical at a given developmental stage.
Therefore, rather than each master regulator operating on a set
of enhancers that is specific to each tissue, the master regulators
instead have access to the same set of enhancers in different tissues,
which they differentially regulate. It was also found that tissues
composed of similar combinations of cell types have very similar
open chromatin profiles, suggesting that a limited number of
distinct open chromatin profiles may exist at a given stage of
development, dependent on cell-type identity (McKay, 2013).
Different tissues were dissected from developing flies to compare
their open chromatin profiles. These tissues are composed of
different cell types, each with its own gene expression profile.
Formaldehyde-assisted isolation of regulatory elements (FAIRE) data thus represent the average signal across all cells
present in a sample. However, data from embryos and imaginal
discs indicate that FAIRE is a very sensitive detector of functional
DNA regulatory elements. For example, the Dll01 enhancer is
active in 2–4 neurons of the leg imaginal disc; yet, the FAIRE
signal at Dll01 is as strong as the Dll04 enhancer, which is active
in hundreds of cells of the wing pouch. Thus, FAIRE may detect nearly all of the DNA regulatory elements that are in use among the
cells of an imaginal disc. This study does not rule out the existence of DNA regulatory elements that are not marked by open chromatin or are otherwise not
detected by FAIRE (McKay, 2013).
Despite this sensitivity, the approach of this study
does not identify which cells within the
tissue have a particular open chromatin
profile. For a given locus, it is possible that all cells in the tissue
share a single open chromatin profile or that the FAIRE signal
originates from only a subset of cells in which a given enhancer
is active. Comparisons between eye-antennal discs, larval
CNS, and thoracic discs suggest that the latter
scenario is most likely, with open chromatin profiles among cells within a tissue shared by cells with similar identities at a given developmental stage (McKay, 2013).
The observation that halteres and wings share open chromatin
profiles demonstrates that Hox proteins like Ubx can differentially
interpret the DNA sequence within the same subset of enhancers
to modify one structure into another. This is consistent
with the idea that morphological differences are largely dependent
on the precise location, duration, and magnitude of expression
of similar genes, and it is further supported by the similarity in gene
expression profiles observed between Drosophila appendages and observed between vertebrate
limbs. However, that such dramatic differences
in morphology could be achieved by using the same
subset of DNA regulatory modules in different tissues genome-wide
was not known. The current findings provide a molecular framework
to support the hypothesis that Hox factors function as
'versatile generalists,' rather than stable binary switches. The similarity in open chromatin profiles between wings and legs suggests that this framework also extends to
other classes of master regulators beyond the Hox genes. It is also noted that, like the Drosophila appendages, vertebrate
limbs are composed of similar combinations of cell types that
differ in their pattern of organization. Moreover, the Drosophila
appendage master regulators share a common evolutionary
origin with the master regulators of vertebrate limb development, suggesting that the concept of shared open chromatin profiles may also apply to human development (McKay, 2013).
The data suggest that open chromatin profiles vary both over
time for a given lineage and between cell types at a given stage
of development. Given the dramatic differences in the FAIRE landscape
observed during embryogenesis and between the CNS and
the appendage imaginal discs during larval stages, it appears as
though the alteration of the chromatin landscape is especially
important for specifying different cell types from a single genome.
After cell-type specification, open chromatin profiles in the
appendages continued to change as they proceeded toward terminal
differentiation, suggesting that stage-specific functions
require significant opening of new sites or the closing of existing
sites. These findings contrast with those investigating hormone-induced
changes in chromatin accessibility,
in which the majority of open chromatin sites did not change after
hormone treatment, including sites of de novo hormone-receptor
binding. Thus, it may be that genome-wide remodeling of chromatin
accessibility is reserved for the longer timescales and eventual
permanence of developmental processes rather than the
shorter timescales and transience of environmental responses (McKay, 2013).
Different combinations of 'master regulator' transcription factors,
often termed selector genes, are expressed in the developing
appendages. Selectors are thought to specify the identity
of distinct regions of developing animals by regulating the
expression of transcription factors, signaling pathway components,
and other genes that act as effectors of identity.
One property attributed to selectors to
explain their unique power to specify identity during development
is the ability to act as pioneer transcription factors. In such models, selectors
are the first factors to bind target genes; once bound, selectors
then create a permissive chromatin environment for other transcription
factors to bind. The finding that the same set of
enhancers are accessible for use in all three appendages, with
the exception of the enhancers that control expression of the
selector genes themselves and other primary determinants of
appendage identity, suggests that the selectors expressed in
each appendage do not absolutely control the chromatin
accessibility profile; otherwise, the haltere chromatin profile (for example) would differ from that of the wing because of the expression of Ubx (McKay, 2013).
What then determines the appendage open chromatin profiles?
Because open chromatin is likely a consequence of transcription
factor binding, two nonexclusive models are possible.
First, different combinations of transcription factors could
specify the same open chromatin profiles. In this scenario,
each appendage's selectors would bind to the same enhancers
across the genome. For example, the wing selector Vg, with its
DNA binding partner Sd, would bind the same enhancers in
the wing as Dll and Sp1 bind in the leg. In the second model, transcription
factors other than the selectors could specify the
appendage open chromatin profiles. Selector genes are a small
fraction of the total number of transcription factors expressed in
the appendages. Many of the non-selector transcription
factors are expressed at similar levels in each appendage,
and thermodynamic models would predict them to bind the
same enhancers. This model could also help to
explain how the appendage open chromatin profiles coordinately
change over developmental time despite the steady
expression of the appendage selector genes during this same
period. It is possible that stage-specific transcription factors
determine which enhancers are accessible at a given stage of
development. This would help to explain the temporal specificity
of target genes observed for selectors such as Ubx. Recent work supports the role of hormone-dependent
transcription factors in specifying the temporal identity
of target genes in the developing appendages (Mou,
2012). Further experiments, including ChIP of the selectors
from each of the appendages, will be required to determine the
extent to which either of these models is correct (McKay, 2013).
Binding of Ubx results in differential activity of enhancers
in the haltere imaginal disc relative to the wing, despite
equivalent accessibility of the enhancers in both discs, indicating
that master regulators control morphogenesis by differentially
regulating the activity of the same set of enhancers. It is likely
that functional specificity of enhancers is achieved through
multiple mechanisms. These include differential recruitment of
coactivators and corepressors, modulation of binding specificity
through interactions with cofactors, differential
utilization of binding sites within a single enhancer, or regulation of binding dynamics through an altered
chromatin context. This last mechanism
would allow for epigenetic modifications early in development
to affect subsequent gene regulatory events. For example, the
activity of Ubx enhancers in the early embryo may
control recruitment of Trithorax or Polycomb complexes to the
PREs within the Ubx locus, which then maintain Ubx in the ON
or OFF state at subsequent stages of development. Consistent with this model,
Ubx enhancers active in the early embryo are only accessible
in the 2-4 hr time point, whereas the accessibility of Ubx PREs
varies little across developmental time or between tissues at a
given developmental stage (McKay, 2013).
The current results also have implications for the evolution of morphological
diversity. Halteres and wings are considered to have a
common evolutionary origin, but the relationship between insect
wings and legs is unresolved. The observation that wings and legs share open
chromatin profiles supports the hypothesis that wings and legs
also share a common evolutionary origin in flies. Because legs
appear in the fossil record before wings, the similarity in their
open chromatin profiles suggests that the existing leg cis-regulatory
network was co-opted for use in creation of dorsal
appendages during insect evolution (McKay, 2013).
Three signaling molecules are required for Distal-less induction in leg and antennal discs. hedgehog (hh) is locally expressed in posterior compartments, under the control of engrailed. wingless (wg)
is expressed in ventral-anterior cells having been induced earlier by HH signals. decapentaplegic (dpp) is expressed in dorsal-anterior compartments, also having been induced by HH signals. All three, hh, wg and dpp are required for the induction of Distal-less and the establishment of the proximal-distal axis of the developing leg and antenna (Diaz-Benjumea, 1994).
Limb development requires the formation of a proximal-distal axis perpendicular to the main
anterior-posterior and dorsal-ventral body axes. The secreted signaling proteins Decapentaplegic and
Wingless act in a concentration-dependent manner to organize the proximal-distal axis. Discrete
domains of proximal-distal gene expression are defined by different thresholds of Decapentaplegic and
Wingless activities. distal-less is expressed in a central domain that corresponds to the presumptive tarsal segments and the distal tibia. The dachshund gene is required for development of the femur and tibia. Dac is expressed in a ring corresponding to the presumptive femur, tibia and first tarsal segment, but is absent from the more distal tarsal segments of the leg disc. Although there is little or no overlap between Dll and Dac domains at early stages, by mid third instar the combination of Dac and Dll expression defines three regions along the P-D axis. Dll and Dac are expressed in circular domains centered on the point at which the ventral Wg domain and the dorsal Dpp domain meet. Dll expression in the center of the disc depends on the combined activities of wg and dpp. Wg and Dpp act directly to induce Dll, as analysis of constitutively active Thick-veins clones has shown (Tkv is the receptor for Dpp); analysis of shaggy/zeste white 3 clones (Sgg is required for transduction of the Wingless signal) reveals that both Wg and Dpp transduction pathways are activated cell autonomously. Continuous signalling is not required to maintain Dll or Dac expression. The spatial domains of Dac and Dll expression are defined by different threshold levels of both Wg and Dpp activities. Both Dpp and Wg act to directly repress Dac in the center of the disc. Dac repression is actively maintained by Wg and Dpp signaling long after Dac and Dll have been induced and are stably expressed in the absence of further signaling. Subsequent modulation of the relative sizes of these domains by growth of the leg
is required to form the mature pattern (Lecuit, 1997).
Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of
cells that include the source of two secreted signaling molecules, Decapentaplegic and
Wingless. Wingless, but not Decapentaplegic, is responsible for the initial distal identity specification of the limb primordia. Proximal limb precursors expressing escargot encircle the Distal-less expressing distal primordium. Dll expressing cells show a dynamic cell migration in the early stage of limb formation, migrating basally during stage 12. Cells that have just started to express Dll also express thickveins. This suggests a requirement for regulated Dpp signaling at the level of receptor expression. Limb formation is restricted to the
lateral position of the embryo through exertion of negative control by Decapentaplegic
and the EGF receptor, both of which determine the global dorsoventral pattern. dpp specifies proximal cell identities. In the absence of dpp Escargot and Snail are lost. A late function
of Decapentaplegic locally determines additional cell identities in a dosage dependent
manner. Loss of Decapentaplegic activity results in a deletion of the proximal structures of
the limb, in contrast to the deletion of distal structures when decapentaplegic mutations affect the
imaginal disc. The limb
pattern elements appear to be added in a distal to proximal direction in the embryo, which is just the opposite of what is happening in the growing imaginal disc. It is proposed that Wingless and
Decapentaplegic act sequentially to initiate the proximodistal axis. This model is contrary to that of Cohen (1993) who argues that Dpp and Wingless are both required to induce the limb. Since Dll expression persists and expands dorsally in the absence of Dpp, it is clear that Dpp plays no role in inducing initial Dll expression but that the dorsoventral limit of Dll expression is defined by repression as a result of Dpp expression. Similarly, EGF-R is required to repress Dll expression in the ventral ectoderm (Goto, 1997a).
Two lines of evidence suggest that Distal-less and vestigial expression at the wing margin, as well as bristle specification, are organized by WG. First, using a temperature-sensitive mutation of wg, it is possible to remove wg activity at chosen times during wing development. Dll expression is abolished within 48 hours following a shift to the nonpermissive temperature, and vg expression is eliminated except for a thin stripe of cells straddling the D/V boundary compartment boundary. Second, ectopic expression of wg as well as ectopic activation of the Wg-signal transduction pathway, caused by eliminating the Shaggy/Zeste-white 3 activity, up-regulates the expression of Dll and Vg within the wing-blade primordium. Expression of a teathered WG protein, genetically engineered to be attached to the cell surface and thus unable to diffuse like normal WG protein, drastically alters the up-regulation of vg and Dll in surrounding cells. Normal WG upregulates Dll in wild-type cells up to 10 or more cell diameters away from the WG source, while teathered WG up-regulates these genes only in their immediate wild-type neighbors. Thus normal WG can act as a long range morphogen, exerting a graded influence on vg and Dll in surrounding cells, while teatherd WG exerts only a short-range, all-or-non influence on surrounding cells (Zecca, 1996).
Armadillo is required autonomously and continuously to mediate the response of wing cells to WG-Secreting cells located at a distance. Clones of arm mutant cells were generated in wing discs. These cells stop dividing and either die or are actively eliminated from the disc epithelium. When stained for either VG or DLL expression 36 hours after mitotic recombination is induced, none of the cells within such clones express either protein (Zecca, 1996).
Short-range interaction between dorsal and ventral (D and V) cells
establishes an organizing center at the DV compartment boundary that
controls growth and specifies cell fate along the dorsal-ventral axis of the
Drosophila wing. The secreted signaling molecule Wingless (WG) is
expressed by cells at the DV compartment boundary and has been implicated
in mediating its long-range patterning activities. Does WG acts directly at a long-range to specify cell fates in the wing? To investigate this question, mutant clones of two components of the WG transduction pathway, dishevelled and armadillo were examined. Cells mutant for dsh show reduced levels of Dll and vg expression. Cells mutant for a temperature sensitive hypomorphic allele of arm, likewise show loss of expression of Dll and vg when larvae are shifted to non-permissive temperatures. Reducing WG levels at the margin reduces both the maximum level of Dll expression near the DV boundary and the distance from the DV boundary at which Dll can be activated. An intermediate level of WG activity is not sufficient to support the specification of wing margin bristles, suggesting that WG has fallen below a critical threshold for activation of AS-C gene expression, while remaining above the respective thresholds of activation for both Dll and vg. Thus, WG acts
directly, at long range, to define the expression domains of its target genes,
Distal-less and vestigial. Expression of the Achaete-scute genes, Distal-less and
vestigial at different distances from the DV boundary is controlled by WG in a
concentration-dependent manner, with AS-C requiring the highest levels of WG. Dll, expressed in a wider range, requires the next highest level, and vg, which is expressed across the entire wing pouch, requires the lowest levels. It is proposed that WG acts as a morphogen
in patterning the D/V axis of the wing (Neumann, 1997).
Vestigial contributes Distal-less gene expression in the wing blade.
In the wild-type wing, Dll is expressed in the wing pouch in
a circle of cells that lies within an area defined by the expression of vestigial. Ectopic expression of vg elevates the levels of Dll expression within the developing wing
blade and triggers ectopic expression of Dll outside this area. This differs from the effects of ectopic expression of wg, which are always restricted to the developing wing blade
and never induce ectopic expression of Dll. Coexpression of
both, wg and vg, results in synergistic effects similar to those
described before for other targets and an increase in the area
of ectopic expression. The effects of vg are independent of wg, since they
are achieved even in apterous mutants, where wg is never expressed
along the DV boundary. These results indicate that Vestigial can indeed regulate gene
expression in the wing blade. In addition, the correlation
between levels suggests that different concentrations of
Vestigial elicit different effects and that, in some instances,
these effects seem to be independent of the levels of Wingless.
Thus Vestigial might act in concert with Wingless, and not
simply as an effector of wg, in the regulation of gene
expression. Very high levels of Vestigial expression, or long-term exposure to vestigial
expression, results in the cessation of proliferation, loss of gene expression and, eventually, in cell death. This might account for the small wings that are often visible when vg is overexpressed in the
developing wing and would correlate with the zone of non-proliferation that appears at the wing margin, where the levels of vg expression are elevated (Klein, 1999).
Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the
amount of beta-catenin/Armadillo, which in turn activates gene transcription. The Drosophila
protein D-Axin is shown to interact with Armadillo and Drosophila APC. D-Axin was identified in a yeast two-hybrid screen for proteins that bind the Armadillo repeat domain of Arm. d-axin codes for a protein of 743 amino acids. A region near its N-terminus shows similarity to the regulator of G protein signaling (RGS domain), whereas its C-terminus contains a region homologous to a conserved sequence near the N-terminus of Dishevelled. Thus D-Axin has a domain structure very similar to that of proteins of the mammalian Axin family. Unlike mammalian Axin family members, which bind to GSK-3beta, D-Axin does not bind to the homologous protein Shaggy/Zeste white3. d-axin is expressed maternally and is ubiquitously expressed during development. Embryos devoid of maternal and zygotic d-axin have completely naked ventral cuticle, lacking all denticles (Hamada, 1999).
During wing disc development, Wg signaling is induced along the dorsoventral compartment boundary in the wing imaginal disc. Arm accumulates in the cytoplasm, associates with its partner Pangolin, and activates expression of target genes such as Distal-less. Mutation of d-axin results in the
accumulation of cytoplasmic Armadillo and results in elevation of Distal-less.
Ectopic expression of d-axin inhibits Wingless signaling. Hence, D-Axin negatively regulates Wingless
signaling by down-regulating the level of Armadillo. It is speculated that the Axin family of proteins functions to establish a threshold to prevent premature signaling events caused by Wg/Wnt and to restrict areas that are capable of responding to Wg/Wnt. These results establish the importance of the Axin
family of proteins in Wnt/Wingless signaling in Drosophila (Hamada, 1999).
Dll expression in imaginal discs of leg and labial limb primordia, requires both wg and dpp, whereas expression in the antennal and labral limbs does not. Only the primordia of legs and
labial limbs are bisected into compartments by the parasegment boundary under the control of wg and en. Dll expression occurs in cells surrounding and including the lateral tips of the ventral wg stripes in the thoracic segments of cells also expressing engrailed (B. Cohen, 1993).
Rows of cells expressing wg in an array of dorsal to ventral stripes, one per segment, intersect cells expressing dpp in a long stripe running anterior to posterior. Dll is expressed in surrounding cells as well. Dll is not required for allocation of imaginal cells. Thus Dll is required in only a subset of cells in the leg disc primordium, (to distinguish the presumptive leg cells from the surrounding body wall). Perhaps leg and wing are derived from a common imaginal primordium. ( B. Cohen, 1993).
The leg primordium enhancer is 12Kb upstream of the Dll gene. The head enhancer region is adjacent to and downstream of this location. The leg enhancer is a target of bithorax-complex mediated repression (B. Cohen, 1993).
Distal-less is a downstream gene of the HOM gene
Deformed, and Distal-less function is required for the elaboration of a subset of the maxillary
epidermal identities specified by Deformed. The regulatory effect of Deformed on Distal-less is mediated by a ventral maxillary-specific enhancer located 3' of the Distal-less transcription unit. Deformed and Distal-less, both of which encode homeodomain transcription factors
that are persistently expressed in ventral maxillary cells, combinatorially specify a subsegmental
code required for a group of cells to differentiate maxillary cirri. Ectopic dfd is sufficient to activate ectopic Dll in other segments. Whether the dfd activation is direct or indirect is unknown (O'Hara, 1993).
Distal-less is a target of Ultrabithorax in parasegment 6. Ubx is shown to be down-regulated by Engrailed in
the posterior compartment of this parasegment. In the posterior compartment of parasegment 6, Dll is normally expressed in a small cluster of cells. If Ubx is expressed uniformly via a heat-shock promoter, Dll is inappropriately repressed in these posterior compartment cells. In the anterior compartment of parasegment 6, Dll is normally repressed by high levels of UBX. However, if en is expressed uniformly via a heat-shock promoter, Ubx is repressed and Dll is derepressed. Because Dll is required for the development of larval sensory structures, these results demonstrate that EN-mediated repression of Ubx in the posterior compartment is necessary for the morphology of parasegment 6 (Mann, 1994).
dwnt-5 is a putative downstream target of Distal-less. dwnt-5 may then act in the specification of the limb primordia (Eisenberg, 1992)
To regulate their target genes, the Hox proteins of
Drosophila often bind to DNA as heterodimers with the
homeodomain protein Extradenticle (Exd). For Exd to
bind DNA, it must be in the nucleus, and its nuclear
localization requires a third homeodomain protein,
Homothorax (Hth). A conserved N-terminal
domain of Hth directly binds to Exd in vitro,
and is sufficient to induce the nuclear localization of Exd
in vivo. However, mutating a key DNA binding residue in
the Hth homeodomain abolishes many of its in vivo
functions. Hth binds to DNA as part of a Hth/Hox/Exd
trimeric complex, and this complex is
essential for the activation of a natural Hox target
enhancer. Using a dominant negative form of Hth evidence is provided that similar complexes are important for
several Hox- and exd-mediated functions in vivo. These
data suggest that Hox proteins often function as part of a
multiprotein complex, composed of Hth, Hox, and Exd
proteins, bound to DNA (Ryoo, 1999).
During leg development, expression of the homeobox gene
Distal-less, which is required for ventral limb development, is mutually
antagonistic with Hth/Exd function: Dll is a repressor of
hth and Hth can also repress Dll. Hth's
ability to repress Dll requires Hth's homeodomain.
From ectopic expression assays, it is concluded that
although the Hth homeodomain is not required to induce
Exd's nuclear localization, it is necessary for many Hth
functions, including the regulation of specific target genes such
as Dll. The one known exception is that all forms of Hth,
including GFP-Hth 51A and GFP-HM, are able to interfere
with distal leg development when expressed with the Dll:Gal4
driver. This phenotype, however, is also observed
when wild-type Exd is expressed with this driver, and
therefore does not require any Hth input. The different in vivo activities of Hth and
Hth 51A indicate that Hth has functions in addition to
localizing Exd to nuclei, and that these functions require Hth
to bind DNA (Ryoo, 1999).
The proximal distal axis of the Drosophila leg is patterned by expression of a number of transcription factors in discrete domains along the
axis. The homeodomain protein Homothorax and the zinc-finger protein Teashirt are broadly coexpressed in the presumptive body wall and
proximal leg segments. Homothorax has been implicated in forming a boundary between proximal and distal segments of the leg. Evidence is presented that Teashirt is required for the formation of proximal leg segments, but Tsh has no role in boundary formation (Wu, 2000).
The leg disc consists of a single epithelial sheet in which
the presumptive distal segments are specified in the center
and the presumptive proximal segments are specified in the
periphery. Cross-sections show that proximal segments,
which express Hth and Tsh, fold back over the distal
segments, which express Dll and Dac. Hth
and Tsh expression is limited to the proximal region of
the disc through repression by the combined activities of
Wg and Dpp. Although the Hth and Tsh expression
domains overlap through much of the proximal region,
Hth expression extends more distally than Tsh. This is visible as a band of Hth expression that does not overlap Tsh in
a basal optical section. This band coincides with the outer ring of Dll
expression. The Tsh domain overlaps the proximal edge of the Dll ring
by one or two cells. Tsh expressing cells are
also found beneath the disc epithelium. Their location suggests that these may be adepithelial cells. Hth functions as a repressor to modulate Tsh expression.
More distally located hth mutant
clones lose Tsh expression. Loss of Tsh expression in hth
correlates with ectopic expression of Dachshund. hth mutant clones cause ectopic expression of Dac close to the endogenous Dac domain,
but do not do so in more proximal regions. The differential
effect on Dac expression of hth clones located at different
positions along the PD axis has been attributed to a role of
Hth as a repressor of Wg and Dpp signaling. Thus the paradoxical loss of
Tsh in more distal hth clones can be explained as an
indirect effect of Hth on Dac expression. Dac can repress
both Tsh and Hth when overexpressed. Thus the
different distal limits of the Hth and Tsh expression domains
presumably reflect a difference in their sensitivity to repression by Dac.
The observation that Tsh levels increase in proximal
hth clones suggests that Hth serves as a repressor of
Tsh. Thus Hth modulates Tsh expression levels in the proximal
leg in two ways. Hth may act directly to reduce Tsh expression levels in the proximal leg, and indirectly via repression
of Dac to define the distal limit of Tsh expression (Wu, 2000).
Reducing Tsh activity produces a phenotype quite
distinct from that of removing Hth activity. Tsh is required
for the development of trochanter and coxa but does not
appear to have a role in segment boundary formation.
Hth and its partner Extradenticle are required to prevent
fusion of coxa and trochanter with the femur. To better understand the basis for the defects
in tsh mutant legs, Hth, Dll and Dac expression
were studied in tshGAL4/tshHD1 and tshGAL4/tshGAL4
mutant discs. In wild-type discs Hth and Dac expression overlap in the proximal ring of Dll expression. Hth function in this ring is required for the affinity boundary between proximal and distal regions of the leg. This basic relationship
holds in the tshGAL4/tshHD1leg disc. Hth, Dll and Dac expression overlap, and the affinity boundary between
proximal and distal leg segments appears to be intact. The principal difference in these discs is expansion
of the Dll domain into the proximal, Hth-expressing region.
The spatial relationship between Hth and Dac is normal. Ectopic Dll expression is
not sufficient to repress Hth but does appear to reduce the size of the coxa
and trochanter and to cause problems that result in loss of
pattern elements from the remaining portions of these
segments. Even slight reductions in Tsh activity causes
loss of sensory bristles from the coxa. In contrast, small
clones of hth mutant cells are capable of differentiating
bristles (Wu, 2000).
Taken together, these observations suggest that Tsh and
Hth have distinct functions in the proximal leg. Hth limits
the proximal extent of Dac expression, and is required for
the affinity boundary between trochanter and femur. Tsh
limits the proximal extent of Dll expression and is required
for proper growth and differentiation of proximal segments, but does not appear to have a role in PD boundary
formation (Wu, 2000).
Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).
A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).
How do these three transcription factors regulate the formation of nine segments? Since the requirements for and the expression of the leg gap genes encompasses all leg segments, it is unlikely that there are additional leg gap genes yet to be identified. Rather, a collection of distinct combinatorial approaches is used to establish a segmental pattern of Serrate, Delta and fringe expression (Rauskolb, 2001).
In early third instar leg discs, there are two domains of gene expression: proximal cells express Hth and distal cells express Distal-less. Hth autonomously promotes the expression of Serrate, while Distal-less may prevent expression more distally, giving rise to a ring of expression in the coxa. Additionally, Distal-less-expressing cells may signal to the Hth-expressing cells to restrict Serrate expression to the distal edge of the Hth domain. As the leg disc grows, cells in an intermediate position, lying between the Hth and Distal-less domains, begin to express Dac. Dac, as shown in this study, is both necessary and sufficient to induce the expression of Serrate, Delta and fringe within the femur. Since they are not expressed in all Dac-expressing cells, other factors appear to be required to promote their expression in the proximal femur. The nonautonomous induction of Serrate expression by Hth suggests that this may be accomplished by a signal (X) emanating from the neighboring Hth-expressing cells. By mid third instar stages, expression of Serrate, Delta and fringe is also observed in tarsal segments 2 and 5, within cells expressing Distal-less but not Dac. Given that Distal-less is necessary and sufficient to repress their expression, Serrate, Delta and fringe expression within the tarsus appears to be induced by a mechanism that overrides the repressive effects of Distal-less. Subsequently, expression of Serrate, Delta and fringe is observed within the tibia, in cells expressing both Dac and Distal-less. Dac is necessary for expression of Serrate within the tibia, and its role here may be to overcome the repressive effects of Distal-less. It is also worth noting that the tibia ring of expression is not established at the time when cells first express both Dac and Distal-less. This may be because Dac levels may not be sufficiently high enough to overcome the repression by Distal-less. Clearly levels of Dac expression are critical because simply increasing Dac levels is sufficient to promote Serrate expression in cells already expressing endogenous levels of Dac. This observation can be explained if high levels of Dac expression in cells already expressing Dac override the function of inhibitory regulators of Serrate expression, such as Distal-less, where the expression of these genes overlap. Although late stages of leg segmentation were not investigated in this study, it has been noted that Hth, Dac and Distal-less are co-expressed in the presumptive trochanter late in leg development. It is thus hypothesized that Serrate, Delta and fringe expression is established by the combined activities of the three leg gap genes in the trochanter (Rauskolb, 2001).
Although these here have focused on the regulation of Serrate expression, it is thought that not only Serrate, but also Delta and fringe, receive primary regulatory input from the leg gap genes. Delta and fringe expression, like Serrate, is positively regulated by Dac. Moreover, Dl and fringe mutants have stronger leg segmentation phenotypes than Ser mutants, and thus Delta and fringe expression cannot simply be regulated downstream of Ser. The identification of two separate Ser enhancers, directing expression in the proximal versus distal leg, argues against Serrate being regulated downstream of Dl and fringe. Thus, the simplest model is that expression of all three genes is regulated directly by the leg gap genes. The regulation of Serrate, Delta and fringe expression in each segment appears to occur through independent and separable enhancer elements, supported by the analysis of the Ser reporter genes. This is reminiscent of what occurs during Drosophila embryonic segmentation, where separable enhancer elements direct different stripes of pair-rule gene expression (Rauskolb, 2001).
Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).
Intriguingly, the only notable variation between insect species is in the number of tarsal segments, with an unsegmented tarsus believed to be the ancestral state. Thus, the combinatorial regulation of segmentation by the leg gap genes may represent an ancient mechanism common to all insect species, a hypothesis supported by the conserved expression of Hth, Dac and Distal-less in the developing legs of many insect species (Rauskolb, 2001 and references therein).
The Iroquois complex (Iro-C) genes are expressed in the
dorsal compartment of the Drosophila eye/antenna
imaginal disc. Previous work has shown that the Iro-C
homeoproteins are essential for establishing a dorsoventral
pattern organizing center necessary for eye development.
In addition, the Iro-C products are
required for the specification of dorsal head structures. In
mosaic animals, the removal of the Iro-C transforms the
dorsal head capsule into ventral structures, namely,
ptilinum, prefrons and suborbital bristles. Moreover, the
Iro-C minus cells can give rise to an ectopic antenna and
maxillary palpus, the main derivatives of the antenna part
of the imaginal disc. These transformations are cell-autonomous,
which indicates that the descendants of a
dorsal Iro-C minus cell can give rise to essentially all the ventral
derivatives of the eye/antenna disc. These results support a
role of the Iro-C as a dorsal selector in the eye and
head capsule. Moreover, they reinforce the idea that
developmental cues inherited from the distinct embryonic
segments from which the eye/antenna disc originates play
a minimal role in the patterning of this disc (Cavodeassi, 2000).
The expression of genes required to make
antenna was examined in the Iro-C minus clones, such as the homeobox gene
Distal-less (Dll).
Using Dll-lacZ, Dll expression was found to start at the
early third instar and is confined to a small group of cells
in the centre of the antennal primordium. Later
expression of Dll encompasses all the antennal segments with
the exception of the most proximal one. In the non-overproliferating Iro-C minus clones, Dll is activated, although this only
occurs in clones located at the dorsal/posterior quadrant of
the eye disc. In the antennal primordium,
activation of Dll depends on simultaneous signaling by Dpp
and Wg. wg is expressed by most of the cells of dorsal Iro-C minus clones. Many of these clones
also express Dpp, which could be provided by either the
morphogenetic furrow or, in clones that are near the disc
margin, by a source of Dpp induced by the ectopic eye
organizer associated with the clone. Thus, the clone cells were exposed to both Dpp
and Wg, which in combination should activate Dll.
Iro-C minus cells at the dorsal/anterior region, which are also
exposed to Wg and Dpp, probably fail to
activate Dll because they are located within the domain of
expression of homothorax (hth).
The removal of the Iro-C does not appreciably modify the
expression of this gene. The presence of Hth might
impair the activation of Dll since, in the leg disc, this protein,
by driving the nuclear localization of Extradenticle (a cofactor
of many homeoproteins),
appears to reduce the sensitivity of cells to Wg and Dpp
signaling. Thus, at the dorsal eye disc,
Iro-C and hth probably cooperate to repress Dll.
Large outgrowths resulting from Iro-C minus clones encompass
hth-expressing cells in their anterior part and
display ectopic Dll expression in the posterior part. In summary,
the results suggest that, upon removal of Iro-C in dorsal cells,
the cells become exposed to Wg and Dpp, and this and the
absence of Iro-C products promote Dll activation. The
interaction of Dll with hth, a gene with an antenna-selector
function, should allow the growth
of an ectopic antenna (Cavodeassi, 2000).
The Notch signaling pathway defines an evolutionarily conserved cell-cell interaction mechanism that throughout development controls
the ability of precursor cells to respond to developmental signals. Notch signaling regulates the expression of the
master control genes eyeless, vestigial, and Distal-less, which in combination with homeotic genes induce the formation of eyes, wings,
antennae, and legs. Therefore, Notch is involved in a common regulatory pathway for the determination of the various Drosophila appendages (Kurata, 2000).
Because Distal-less in combination with
extradenticle (exd) and homothorax
(hth) specifies the antennae, Dll
expression was monitored in the eye discs that are capable of forming ectopic antennae. In wild-type larvae, Dll protein is expressed in the antennal
but not in the eye disc. In all of the tested discs in ey-GAL4 UAS-Nactey2 animals that form ectopic antennae from the
eye disc, significant Dll expression was detected ectopically. This
indicates that Notch signaling directly or indirectly
induces ectopic expression of Dll in the eye-antennal disc,
leading to the ectopic induction of antennae (Kurata, 2000).
The observation that Nact can induce
both ectopic eyes and, in a specific genetic background, antennae, led
to a consideration of the possibility that Notch signaling also
might induce the formation of other appendages in a different genetic
context. To test this hypothesis, the activation of
Notch signaling was combined with ectopic expression of
Antennapedia (Antp). The latter is known to
determine the identity of the second thoracic segment (T2),
which on the dorsal side gives rise to a pair of wings and on the
ventral side to a pair of second legs. For this purpose, transgenic
flies of the constitution ey-GAL4 UAS-Nact
UAS-Antp were generated. About 26 of the flies
escaping pupal lethality were found to have ectopic wings on the head. Almost all ectopic wing structures consisted of
dorsal and ventral wing blades bordered by bristles of the wing margin, but lacking wing veins. In contrast, in wing
structures induced by the ectopic expression of vg, the wing
margin is not formed, suggesting that Notch signaling
and Antp are acting upstream of vg. Furthermore,
about 17% of these flies show ectopic leg structures
induced by secondary transformation of the ectopic antennal tissue into
leg structures (e.g., arista into tarsus). Therefore, activation of Notch
signaling when combined with the ectopic expression of Antp
driven by ey-GAL4 is capable of inducing wing and leg structures on the head (Kurata, 2000).
In wild-type larvae, the vg gene is expressed in the wing
but not in the eye disc. By contrast, in ey-GAL4
UAS-Nact UAS-Antp animals in which ectopic
wing structures are induced in the eye disc all of the tested eye discs show significant ectopic expression of Vg protein. It therefore
appears that activation of Notch signaling in the context of
Antp expression induces vg expression in the eye
discs and that there are synergistic effects between Notch
signaling and Antp expression. Notch signaling pathway has been shown to be used to
specifically activate the boundary enhancer of the vg gene
necessary for dorso-ventral wing formation. The same enhancer also may
be used for ectopic formation of the wing, a point that has to be
investigated further. A dorso-ventral boundary also is established by
Notch in the eye disc that controls growth and polarity in the Drosophila eye.
In ey-GAL4 UAS-Nact UAS-Antp ectopic
legs also are induced on the head; this is
accompanied by Dll expression (Kurata, 2000).
In view of the above observations it is proposed that the effects of
Notch signaling on the various appendages depend on the
context provided by control genes such as ey and
Antp. In the eye primordia, Notch signaling
induces ey expression, which induces a cascade of downstream
genes leading to eye morphogenesis. In conjunction with
Antp, Notch signaling induces vg,
leading to wing formation. At low levels of ey expression,
Notch signaling induces Dll, leading to antenna
morphogenesis. In the case of the leg, Notch also induces Dll expression that, in conjunction with Antp,
leads to leg formation (Kurata, 2000).
Segmental identity is specified by the homeotic genes that are active
in a particular combination in each segment. Within a given segment,
the appendages are specified by a different set of subsidiary control
genes; the eyes are specified by ey, the wings and haltere
by vg; the legs by Dll and the antennae by
Dll in combination with extradenticle
(exd) and homothorax (hth).
The results presented here indicate that they all are regulated by Notch signaling and that they share the same cell signaling pathway, which raises the
possibility that the appendage specificity is provided by a
combinatorial interaction between Notch and the homeotic and the subsidiary control genes (Kurata, 2000).
The repression of one control gene by the expression of another seems
to be a widespread mechanism to ensure that the developmental pathways
are mutually exclusive so that the formation of intermediary cell types
is prevented. Similar to the repression of ey by
Antp, ey directly or indirectly
represses Dll. In hypomorphic ey mutants, the
activation of Notch signaling leads to ectopic expression of
Dll in the eye disc, suggesting that ey might
repress Dll in the wild-type eye disc. In dpp-GAL4
UAS-ey transheterozygous flies Ey is expressed on the ventral side
of the posterior half of the antennal discs under the control of the
dpp-enhancer, whereas Dll is not
detectable in this area. A similar mutually exclusive expression is found in the leg discs of these flies, suggesting that ey represses Dll expression (Kurata, 2000).
Based on these findings, a model is proposed to explain the difference
between the eye and antennal pathway starting from a common signaling
mechanism. Notch signaling induces the expression of both
ey and Dll. However, in the eye primordia
ey represses Dll and induces eye morphogenesis.
By contrast, in the antennal disc ey is repressed by a
repressor, resulting in Dll expression that confers antennal
(ventral appendage) specificity. Two of the possible candidates for the
repressor are the homeobox genes exd and hth,
because both exd- and
hth- mutant clones in the rostral membrane
region of the antennal disc can result in ectopic eye development,
which presumably is caused by derepression of ey.
Both exd and hth also may function in conjunction
with Dll, serving as corepressors. The present
study extends the fundamental role of Notch by indicating
that the implementation of entire developmental programs leading to
appendage formation and organogenesis may be controlled by
Notch activity (Kurata, 2000).
The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific
downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the
absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from
the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream
of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).
To test whether Osa is acting directly on Wg target genes or
regulating the expression of some other gene that interacts with the
wg pathway, attempts have been made to determine at what level in the wg pathway Osa acts. In third-instar wing discs, wg
is expressed in a narrow stripe of cells that straddles the
dorsal/ventral (DV) boundary of the wing pouch and
directs growth and patterning of the wing blade with respect to the DV
axis. Cells adjacent to the DV boundary respond to the wg signal by posttranscriptionally up-regulating cytosolic Arm. Arm then translocates to
the nucleus and binds to Pan to activate the expression of
downstream target genes such as Distal-less (Dll). When an activated form of the protein kinase Sgg, which constitutively
targets Arm for degradation (UAS-Sgg*) is
expressed in the dorsal compartment using the ap-Gal4 driver,
Arm is not up-regulated. Expression of UAS-Osa in the dorsal compartment
similarly prevents the expression of Dll on the dorsal side
of the DV boundary. However, these cells still respond to
the Wg signal by up-regulating cytosolic Arm. Therefore,
Osa represses Wg target genes without affecting the up-regulation of
Arm. This places the activity of Osa in the nucleus and argues that Osa may directly repress the expression of Wg target genes (Collins, 2000).
In Drosophila, the Hox gene Abdominal-B is required
to specify the posterior abdomen and the genitalia.
Homologs of Abdominal-B in other species are also
needed to determine the posterior part of the body. The function of Abdominal-B in the formation of Drosophila genitalia has been studied, and the absence of Abdominal-B in the genital disc of Drosophila is shown to transform
male and female genitalia into leg or, less frequently, into
antenna. These transformations are accompanied by the ectopic expression of genes such as Distal-less or dachshund, which are normally required in these appendages. The extent of wild-type and ectopic Distal-less
expression depends on the antagonistic activities of the
Abdominal-B gene (as a repressor), and of the decapentaplegic and wingless genes (as activators). Absence of Abdominal-B also changes the expression of Homothorax, a Hox gene co-factor. These results suggest that
Abdominal-B forms genitalia by modifying an underlying
positional information and repressing appendage
development. It is proposed that the genital primordia should
be subdivided into two regions, one of them competent to
be transformed into an appendage in the absence of Abdominal-B (Estrada, 2001).
Abd-B clones were induced, and they transform posterior abdominal segments into more anterior ones but are normal in the analia. Rare clones transform to distal antennae (second and/or third antennal segment and arista).
Transformations to legs or antennae are cell autonomous.
The formation of legs requires
the activity of genes such as homothorax (hth), dac and Dll, which specify the proximal, medial and distal parts of the leg,
respectively. Dll expression in wild-type discs is regulated by the combined activities of wingless and dpp in the genital
primordia, and is confined to two groups of cells in
male and female discs, the female domains being smaller and
expressing lower levels of Dll protein.
Since Abd-B is transcribed in the entire genital primordia of the
two sexes, some cells co-express Abd-B and Dll. In
the male disc, hth is not expressed in the Dll-expressing cells
and is also excluded from a large group of cells surrounding
them. Levels of antibody signal vary within the
disc, and are higher in the female repressed primordium. In
females, the hth domain of expression occupies the whole
primordium. Lower levels of Hth are detected in a region
encompassing the Dll-expressing cells, whereas higher levels
are observed in the male repressed primordium. In
both sexes, hth expression is absent from the anal primordium.
dac is expressed differently in male and female genital
primordia: in male discs, Dac protein is
detected in two broad lateral bands, while in female
discs it is found in the central region, almost coincident with
the wg-expressing region. Therefore, the expression
patterns of hth, dac and Dll differ substantially from those
observed in legs (Estrada, 2001).
It is known that expression of Dll is not required to make male
genitalia and that it has only a minor role in the formation of
the female one. To ascertain the role
of hth in the genitalia, hth minus clones were induced during the third larval period and they were examined in the adult structures. In
the female genitalia, hth minus clones cause extra growths with
additional vaginal teeth. In males, these clones show
occasionally some abnormalities in the clasper teeth. hth clones in the analia are wild type.
Possible interactions between Dll and hth in the
genital disc were sought. In these experiments, unless stated,
the results apply both to male and female genital primordia. Dll minus
clones in the Dll domain of the male disc have no hth expression. Similarly, in hth minus clones Dll is not ectopically
expressed. Dll was also expressed ectopically and
the effect on hth expression was examined. Dll-expressing cells close
to the wild-type Dll domain repress hth expression, although not
all the cells do so. By contrast, clones far from the Dll domain
do not affect hth expression (Estrada, 2001).
To characterize the transformation of genitalia into leg or antennal tissues, Abd-B minus clones were examined. Abd-B
minus clones in the genital primordia tend to segregate from
the rest of the tissue, round up and have smooth borders,
suggesting they have acquired different affinities. By
contrast, clones in the analia have indented borders and do
not segregate. Abd-B minus clones in the genital
primordium close to the normal Dll domain show ectopic,
cell-autonomous Dll expression, whereas those far
apart do not show such expression. dac is also activated cell
autonomously in many Abd-B minus clones.
As expected, Dll target genes, such as Bar, also become
activated in these clones (Estrada, 2001).
Abd-B minus clones exhibit differential effects on hth, depending on their position: those close to the Dll domain show no hth
expression, whereas those located away from the Dll domain
show a slight increase in hth signal. Clones in intermediate
positions do not significantly change hth levels.
This distribution, however, is clearer in females, since in males
there is a wide region with no hth expression. The repression of hth observed in some Abd-B minus clones may be mediated by the ectopic Dll
(Estrada, 2001).
In the genital disc, the transcription of Dll depends, as in the
leg disc, on dpp and wg signals. Abd-B
represses Dll expression. Moreover, increasing Abd-B levels
in the Dll domain suppresses Dll transcription. The antagonistic activities of dpp/wg and
Abd-B in determining the Dll distribution was analyzed. Mutations in PKA ectopically activate wg and dpp expression. PKA minus clones in the genital primordia activate Dll, although only in some places. This activation is not mediated by changes in Abd-B levels. Similarly, although Dll is derepressed in
many late Abd-B minus clones, derepression
of either dpp or wg was not observed. It is
concluded that there is an antagonism between the activation
of Dll by dpp/wg signaling and its repression by Abd-B. This
is not mediated by changes in the expression of either dpp,
wg or Abd-B (Estrada, 2001).
To characterize this antagonism further, Abd-B minus
clones that were made were also unable to transduce the dpp signal. This
signal requires the presence of the type II receptor encoded by the gene punt. In put;Abd-B double mutant clones, Dll is not activated, indicating that, in the absence of Abd-B, Dpp (and possibly Wg) are still required to activate Dll. Abd-B minus clones far from the wild-type Dll domain fail to activate Dll ectopically, suggesting that activation of Dll in the absence of Abd-B depends on the range of diffusion of Dpp and Wg, as in the leg disc and in the anal primordium (Estrada, 2001).
Dll is required for the development of legs and antennae, and
induces these structures when expressed ectopically in the wing
or eye-antennal discs. However, although Dll is also expressed in the genital primordia this expression does not lead to the formation of any of
these appendages. To test if Abd-B prevents Dll function Abd-B was eliminated in Dll-expressing cells; these cells
formed leg tissue. However, it is possible that the high
levels of Dll observed in these mutant cells account for the leg
transformation. To test this, use was made of the GAL4/UAS
system to increase Dll expression in the genital disc (dpp-GAL4/
UAS-Dll flies). Male and female genitalia of this
genotype are abnormal, but not transformed into leg.
To extend these observations, the ability of Dll
to promote Bar transcription, a gene expressed in the leg disc
and activated by Dll, was examined. Bar is not expressed
in the female genital primordium and only in a few cells within
the Dll domain in the male genital primordium;
however, Abd-B minus clones show Bar expression in both sexes. When Dll is ectopically expressed in the genital disc,
Bar expression is activated in some of the cells that express Dll. These results suggest that, in females, Dll levels
are insufficient to activate Bar when Abd-B is present, but that
increasing Dll expression or removing Abd-B activates Bar
transcription. Abd-B, therefore, prevents some Dll activity in
females. In males, although there is Bar transcription, leg tissue
is not formed, probably because Abd-B modifies or prevents
the activation of other Dll target genes. A similar case has been
reported in the wing disc: ectopic Dll activates bric a brac, a
gene downstream of Dll, both in the wing pouch and the body
wall region of the wing disc; however, legs appear in the wing,
but not in the notum (Estrada, 2001).
The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate expression of Wingless pathway nuclear targets. Wg signaling causes nuclear translocation of Armadillo, the fly ß-catenin, which then complexes with the
DNA-binding protein TCF (Pangolin To examine targets that are positively regulated by Wg in the wing, the zinc-finger protein Senseless (Sens) and the homeodomain protein Distal-less (Dll) were chosen. Sens is expressed in the proneural clusters on either side of the dorsoventral border, immediately adjacent to the Wg expression domain. Inhibition of Wg signaling with a dominant-negative TCF blocks Sens expression, demonstrating that it is a short-range target of Wg action. pygo-expressing cells outside the Wg expression domain completely lack Sens expression. The long-range target Dll is also always lost in clones overexpressing pygo. For reasons that are not clear, occasionally some expression persists just inside the clonal border (Parker, 2002).
The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). The function of these signaling pathways in this process has been investigated. Organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. It is proposed that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development (Kenyon, 2003).
This paper analyzes the role of Dpp and Notch in the regional specification of the eye-antennal disc. This study makes four observations: (1) domains of regional identity emerge in a complex pattern starting early in L2; (2) formation of eye and antenna primordia depend upon specific domains of dpp expression that emerge in early-L2 (eye) and mid-L2 (antenna); (3) neither Notch nor Dpp control the establishment of separate eye and antennal fields; (4) Notch can influence the establishment of an eye primordium through its control of proliferation in the eye field. Current models of regional specification have been evaluated based on these results and a new perspective on the emergence of regional identity in this tissue is presented (Kenyon, 2003).
It has been proposed that allocation of eye field and antennal field identity occurs in the latter half of L2 through the restriction of eye selectors, such as Ey, and antennal selectors, such as Dll, to distinct regions of the disc. However, two observations reported in this paper are not consistent with this interpretation: (1) Dll is not expressed ubiquitously at any time during disc development; (2) eye and antennal fields are clearly established by mid-L2 as evidenced by the restricted expression of Ey (eye field) and Cut (antennal field), and by distinct Dpp/Wg patterning centers within each field. These observations place the emergence of separate eye and antennal fields in the first half of L2 and not in the second half as previously proposed. Moreover, onset of Eya occurs in early-L2 and so is expressed by mid-L2. The beginning of eye primordium formation in early-L2, prior to the appearance of distinct fields, indicates that regional specification within this disc does not follow a two-step mechanism (i.e., establishment of separate fields followed by induction of organ primordia) but occurs in a more complex pattern. Further analysis of the transcription factors and signaling molecules active in the late-L1 and early-L2 disc is necessary to better understand how the establishment of eye field identity relates to eye primordium formation and the emergence of an antennal field (Kenyon, 2003).
Analyses of hypomorphic dpp alleles and tissue mutant for Mad implicate Dpp in the control of eya and Dll expression during late larval development. The onset of Eya and Dll expression correlate with specific changes in dpp expression during normal development. Using temperature shift experiments, it has also been established that Dpp signaling in L2 is required for the proper induction of both Eya and Dll in their respective fields. Gain-of-function analyses show that Dpp is also sufficient to induce Eya expression within the eye field and Dll expression within the antennal field. Clearly, though, Dpp must function in the context of selector factors such as Ey in order to produce two independent primordia within the eye-antennal epithelium. In the presence of Ey, Dpp signaling induces Eya expression as opposed to Dll. The absence of Ey in the antennal field at the time of Dpp signaling is of crucial importance to ensure the proper induction of Dll and subsequent formation of an antenna primordium. Indeed, as described in this study, the restriction of Ey to the eye field precedes the emergence of dpp and Dll in the antennal field during normal development (Kenyon, 2003).
In conclusion, Dpp, unlike Notch, functions as an inducer of tissue identity during specification of the eye-antennal disc, and the spatial and temporal aspect of organ primordia formation is controlled at the level of dpp transcription (Kenyon, 2003).
The related genes buttonhead (btd) and Drosophila
Sp1 (the Drosophila homolog of the human SP1 gene)
encode zinc-finger transcription factors known to play a developmental role in
the formation of the Drosophila head segments and the mechanosensory
larval organs. A novel function of btd and Sp1 is reported:
they induce the formation and are required for the growth of the ventral
imaginal discs. They act as activators of the headcase (hdc)
and Distal-less (Dll) genes, which allocate the cells of the
disc primordia. The requirement for btd and Sp1 persists
during the development of ventral discs: inactivation by RNA interference
results in a strong reduction of the size of legs and antennae. Ectopic
expression of btd in the dorsal imaginal discs (eyes, wings and
halteres) results in the formation of the corresponding ventral structures
(antennae and legs). However, these structures are not patterned by the
morphogenetic signals present in the dorsal discs; the cells expressing
btd generate their own signalling system, including the establishment
of a sharp boundary of engrailed expression, and the local activation
of the wingless and decapentaplegic genes. Thus, the Btd
product has the capacity to induce the activity of the entire genetic network
necessary for ventral imaginal discs development. It is proposed that this
property is a reflection of the initial function of the btd/Sp1 genes
that consists of establishing the fate of the ventral disc primordia and
determining their pattern and growth (Estella, 2003).
In a search for genes with restricted expression in the adult cuticle,
the MD808 Gal4 line was found to direct expression in the ventral derivatives
of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and
analia no clear expression was discerned. It was also noticed that the
insertion was located in the first chromosome and associated with a lethal
mutation. The mutant larvae showed a head phenotype resembling that described
for mutants at the btd gene: loss of antennal organ and the ventral
arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that
reported for btd, suggesting that the Gal4 insertion was
located at this gene. In addition, the imaginal expression of MD808 and of
btd was largely coincident (Estella, 2003).
Further to the genetic analysis and the expression data, DNA
fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the
btd gene. The related gene Sp1 is
immediately adjacent. It is likely that btd and
Sp1 have originated by a tandem duplication of a primordial
btd-like gene (Estella, 2003).
In early
embryos btd is expressed in the head region, but by the extended germ
band stage the expression domain has expanded to the ventral region of
cephalic, thoracic and abdominal segments. During germ band retraction most of
the abdominal and thoracic expression is lost, except in derivatives of the
peripheral nervous system and the primordia of the imaginal discs.
Sp1 is not expressed in early embryos, but from stage 11 onwards it
shows the same pattern as btd (Estella, 2003).
Special attention was paid to the btd/Sp1 expression domain in the thoracic
imaginal discs primordia, as it may suggest a novel function related to
imaginal development. Double labelling with Dll and btd probes indicates that btd precedes Dll expression, but by stage 12 the two genes are co-expressed in a group of thoracic cells. However, the Dll domain is
smaller and is included within the btd/Sp1 domain: there are cells expressing
btd that do not show Dll activity, although all the cells
expressing Dll express btd (Estella, 2003).
The ventral disc primordia include not only cells expressing Dll
but also other cells containing expression of escargot (esg)
and hdc, markers of the diploid cells that form the imaginal
primordia. In late embryonic stages, esg is expressed in a
ring domain surrounding the Dll-expressing cells and
hdc is expressed in a similar pattern. Double label experiments were carried out with btd, hdc and esg probes; the expression of the two latter genes overlaps with that of btd (and with Sp1) in the thoracic disc primordia (Estella, 2003).
The overlap of the btd and of esg domains indicates that
btd is also expressed in the hth domain, which is coincident with
that of esg. As the hth/esg domain marks the precursor cells of the
proximal region of the adult leg the embryonic
expression data indicate that btd and Sp1 are active in the
entire primordia of the ventral adult structures, including the distal and the
proximal parts (Estella, 2003).
In the mature antennal disc, btd expression is
restricted mostly to the region corresponding to the second antennal segment,
where it co-localizes with both Dll and hth. In the leg disc
btd also overlaps in part with Dll and with hth. The latter result is significant, for the expression of Dll and hth define two
major genetic domains, which are kept apart by antagonistic interactions. The
fact that btd is expressed in the two domains suggests that its
regulation and function is independent from the interactions between the two
domains. This observation is consistent with the results obtained in embryos and suggests that the btd domain includes the precursors of the whole ventral thoracic region from the beginning of development (Estella, 2003).
This work demonstrates a novel and also redundant function of btd
and Sp1: they are responsible for the formation of the ventral
imaginal discs by activating the genetic network necessary for their
development. Furthermore, Btd protein retains the capacity of
inducing the entire ventral genetic network during the larval period. It is
proposed that the activation of btd/Sp1 is the crucial event in the
determination of the ventral structures of the adult fly (Estella, 2003).
This argument is based on the finding that btd and Sp1
appear to mediate all events connected with the formation of the ventral
discs. The discussion deals with the leg disc, but there is evidence that
antennal primordium also requires btd. Moreover, the genital primordium
is lacking in Df(1)C52 embryos, suggesting that this disc is also under the same control. Most of the experiments concern the function of btd but given the expression and functional similarities between the two genes, it is assumed that Sp1 fulfils the same or a very similar role.
Therefore, btd/Sp1 will be considered to carry out a single function (Estella, 2003).
One crucial feature is that btd is an upstream activator of
Dll and hdc, which are considered developmental markers of
disc primordia: (1) btd expression precedes that of Dll and of hdc; (2) the btd expression domain includes those of Dll and hdc; (3) in btd mutants, Dll and hdc activity is much
reduced, and completely absent in Df(1)C52 embryos;
(4) ectopic btd function induces ectopic activation of Dll
and hdc (Estella, 2003).
The role of btd in embryogenesis can be illustrated in the light
of the models of Dll regulation. Dll is activated by wg and its expression modulated by the
EGF spitz and by dpp, whereas it is repressed in the
abdominal segments by the BX-C genes. The current
experiments suggest that Dll regulation is mediated by btd:
in wg mutants there is no btd expression and hence neither
Dll nor hdc activity. In dpp mutant embryos,
btd expands to the dorsal region resembling the effect on
Dll. In Ubx- embryos there is an additional
group of cells in the first abdominal segment expressing btd; the
same cells that also express Dll in those embryos. The interpretation
of the role of btd mediating Dll regulation by Ubx
is complicated by previous observations showing direct repression of Dll by the Ubx protein. It is possible that Ubx regulates Dll both directly and by controlling btd activity (Estella, 2003).
It is proposed that the localization of btd/Sp1 activity to a group of
ventral cells is a major event in the specification of adult structures.
btd and Sp1 are activated in response to spatial cues from
Wg, Dpp, EGF and BX-C, and in turn their function induces the activity of the
genes necessary for ventral imaginal development (Estella, 2003).
This hypothesis is strongly supported by the results obtained inducing
ectopic btd activity in the dorsal discs; just the presence of the
Btd product alone is sufficient to bring about ventral disc development. In
the wing and the haltere discs, Btd induces a transformation into leg, whereas
in the eye it induces antennal development. This indicates that it specifies
ventral disc identity jointly with other factors, e.g., the Hox genes, possibly
through the activation of subsidiary genes such as Dll, known to
contribute to ventral appendage identity in combination with Hox genes (Estella, 2003).
The requirement for btd and Sp1 activity appears to be
restricted only to the ventral discs, even during the early phases of the
thoracic disc primordia. In this context it is worth considering the
observation that in Df(1)C52 embryos there is
esg expression in the wing and haltere disc primordia, even though it
is absent in the leg discs. Thus, the wing and haltere discs are formed in the
absence of btd and Sp1. Because in these embryos there is an
almost complete lack of Dll expression, this observation raises the
question of the origin of the dorsal thoracic discs, which are currently
considered to derive from the original ventral primordium, formed by cells
expressing Dll. Although some of the original group of ventral cells may
contribute to the dorsal disc primordia, the data suggest that there may be
cells recruited to form the dorsal discs that do not originate in the initial
ventral primordium. Accordingly, it is worth considering that in the absence
of Dll activity the leg and wing discs are formed, although the leg only differentiates proximal disc derivatives. Thus, the activity of Dll cannot be considered a reliable marker for imaginal discs (Estella, 2003).
RNA interference experiments also indicate that both btd and
Sp1 are required for the growth of the antennal and leg discs. When
the two gene functions are reduced simultaneously, leg segments fuse and there
is an overall reduction in the size of antennae and legs. The reduction of
growth affects the proximal and distal regions of the appendage, and assigns a role to the expression observed in the imaginal discs. The two genes are able to
perform this function on their own, for the inactivation of only one is not
sufficient to impair growth. This conclusion is also supported by the
observation that mutant btd clones do not have any effect; they
still possess Sp1 activity, which is sufficient for normal
development. At this point the mechanism by which btd/Sp1 may affect growth is not known (Estella, 2003).
One particularly significant result about the mode of action of
btd comes from the analysis of the ectopic leg patterns observed with ectopic btb expression in the wing and halteres. The clones of cells ectopically expressing btd tend to recapitulate
the complete development of leg and antennal discs. For example, the whole
genetic network necessary to make a leg appears to be activated. btd
induces the activity of hth, dac and Dll, the domains of
which account for the entire disc. Furthermore, hth, dac and Dll are
activated in a spatially discriminated manner. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different
signal thresholds. In one clone, for example hth is expressed only in the peripheral region, resembling the normal expression in the leg disc; in another clone the discriminate expressions of dac and Dll define three distinct regions. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds, but the hth domain is independent from Wg and Dpp (Estella, 2003).
Pax6 genes encode transcription factors with two DNA-binding
domains that are highly conserved during evolution. In Drosophila,
two Pax6 genes function in a pathway in which twin of
eyeless (toy) directly regulates eyeless (ey),
which is necessary for initiating the eye developmental pathway. To
investigate the gene duplication of Pax6 that occurred in
holometabolous insects like Drosophila and silkworm, different truncated forms of toy and small eyes (sey) were used, and their capacity to induce ectopic eye development was tested in an ey-independent manner. Even though the Paired domains of Toy and Sey have DNA-binding properties that differ from those of the Paired domain of Ey, they all are capable of inducing ectopic eye development in an ey mutant background. One of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, only the homeodomain (HD) of Ey is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of Toy and Ey, and significant evolutionary divergence between the two Drosophila Pax6 genes (Punzo, 2004).
The existence of two Pax6 genes in Drosophila raises the
question of whether they have a redundant function or whether they have
diverged to control different sets of target genes. A recent characterization
of new alleles of ey and toy mutants by Kronham (2002) suggests a functional divergence, with a
partial redundancy remaining. Epistasis studies show that toy lies
upstream of ey, because ectopic toy is capable of inducing
ectopic ey but not vice versa.
Additionally, toy cannot induce ectopic eyes in an
ey2 mutant background whereas ey can. The
regulation of ey by toy is due to a direct binding of the
Toy-PD to the ey-enhancer, which is located in the second intron of
the ey gene. The Ey-PD contains a glycine at position 14, whereas the
Toy-PD has an asparagine at that same position. This difference allows Toy to
regulate ey through the ey-enhancer, whereas Ey cannot
regulate itself. Complementary experiments showed that ectopic expression of
a HD-deleted version of the Ey protein did not induce the endogenous
full-length gene, and therefore confirmed the lack of an auto-regulatory
feedback loop for ey. Endogenous ey can only be induced by
misexpression of the three downstream genes eya, so and dac,
or by toy. Interestingly, the mouse Pax6 gene sey
also has an asparagine at position 14 of the PD and has, therefore, the same
DNA-binding properties as toy. Moreover,
sey and toy have multiple stretches of conserved amino acids
in their C termini, whereas the C terminus of ey diverged. Thus,
ectopic sey is able to induce ectopic ey but it does not
induce ectopic toy. This suggests that the auto-regulatory feedback loop found in the vertebrate Pax6 gene evolved into a hetero-regulatory
interaction in Drosophila with toy regulating ey
expression. Overall these data indicate that not only the PDs, but also
the cis-regulatory sequences of the two Drosophila Pax6 genes, have
diverged to control different sets of target genes. This hypothesis is further
supported by the fact that only toy is expressed in the ocelli
territory of the eye disc but both toy and ey regulate the
eye-specific enhancer of the so gene, by binding partly to the same
and partly to different binding sites, and by discriminating between eye and
ocelli development during larval stages (Punzo, 2004 and references therein).
The PD and the HD are the most conserved regions within the Pax6 proteins,
indicating evolutionary constraints imposed to maintain specific binding to
target genes. Therefore, an investigation was carried out to what extent the PD of Tot and Sey, which diverged in their DNA binding properties from the PD of Ey, were able to induce the eye developmental pathway independently of ey. Moreover,
whether only the HD of ey was able to downregulate
Dll expression was investigated, and
whether this function is required during endogenous eye development to specify
the eye territory. These hypotheses were tested by generating deletion
constructs of sey similar to those described for ey and
toy, as well as Ey-Toy chimeric proteins, and ectopic
eye formation was scored. Furthermore, the ey null mutant
eyJ5.71 was rescued by transferring the genomic region of the ey gene onto the third chromosome. This allowed an analysis of mutant ey clones in a wild-type background. Both sey and
toy were found to be able to activate eye development in an
ey-independent manner, and one of the main differences between
toy and ey, besides their DNA-binding properties, was found to lie in
their C-terminal region, and therefore mainly in their transactivation
potential. This suggests that most of the differences reside in their capacity
to interact with different sets of proteins. Only the HD of
ey is able to downregulate Dll expression in an ectopic situation,
and this downregulation is required during endogenous eye
development (Punzo, 2004).
eyDeltaPD can downregulate Dll expression at the transcriptional level in an ectopic situation leading to leg truncation, whereas
toyDeltaPD and seyDeltaPD do not, even
though all three HDs have the same amino acids at positions conferring
DNA-binding specificity. These functional differences between Ey and Toy most
likely reside in the CT of Ey, which differs significantly from that of Toy
and SEy. Although previous findings have shown that DNA binding of the HD is
required for the downregulation of Dll, the C
terminus of Ey appears to confer the functional specificity of the
Dll repression (Punzo, 2004).
Several lines of evidence point to the fact that the induction of
Dll is not directly controlled by ey but rather by a
secondary late event of postmitotic differentiation. (1) In
ey2 mutants Dll is normally not expressed. Only
in very rare cases do those mutants show a transdifferentiation from eye to
antenna. (2) Over expression of P35 in ey2
mutants does not lead to Dll induction until the third larval stage when
differentiation sets in. Those Dll-expressing cells reside at the
posterior tip of the eye disc, where differentiation starts with the onset of
MF movement. (3) Rescuing the ey2 mutant by
eyDeltaHD leads to normal eye development and not to uniform
up-regulation of Dll. Only in rare cases was Dll found to be
expressed in those eye discs and in even fewer cases showed antenna like
outgrowth. These results are in line with the clonal analysis, where only a
small percentage of clones show induction of Dll, but no clone
displays an adult eye phenotype. Thus, only rarely might the size of
Dll-expressing clones be big enough the lead to a
transdifferentiation. Additionally, the ability of toy to function
redundantly to ey may account for those observations. (4) The
co-expression experiment of the various ey constructs with P35 in the
ey2 mutant strongly suggests that only the repression of
Dll is ey dependent, not the induction, since P35 in
conjunction with eyDeltaPD, which does not initiate eye
development, completely abolishes antenna duplication. Antenna duplications
are observed only in those rare cases where P35, in conjuction with
eyDeltaHD, does not rescue eye development and thus fails
to instruct the cells to enter the eye developmental pathway (Punzo, 2004).
Taken together, these findings suggest that expressing a PD-containing Pax6
protein is sufficient to prevent Dll activation. By contrast, the
Ey-HD clearly confers downregulation of Dll. A more profound study
with double mutant clones of ey and toy, preventing the
presence of any Pax6-PD containing protein, may be more conclusive. The
downregulation of Dll by ey may be direct or indirect, but
the activation is ey independent. Other studies have
shown that dpp is required for the activation of Dll in the
antenna primodium. This may explain why in the absence of ey, Dll is
activated only in cells located in or behind the MF that fail to differentiate
to photoreceptors, cells that have already seen dpp and reside
therefore normally in the posterior part of the eye disc or within the range
of dpp signaling (Punzo, 2004).
Dll repression is shown to be required in the normal eye
disc to prevent antennal development and to install the eye development
program. The failure to repress Dll in the eye primordia leads to a
transdetermination from eye to antennal structures, and the formation of an
additional antenna in the eye field. The downregulation of Dll in the
eye region of the eye-antennal discs depends on the Ey-HD and the Ey-CT,
whereas the Ey-PD (and the PD of toy) are required to install the eye
development program, mainly by activation of the subordinate target genes (Punzo, 2004).
The results strongly suggest that the functional differences between
ey and toy are not only due to their different DNA-binding
specificities and changes in the cis-regulatory sequences of their PDs, but
also to interactions with different co-factors through their C termini. Recent
studies have shown that the transcriptional activator Pax5 is converted
into a repressor by interaction with the groucho protein through its
C terminus and its octapeptide. Similarly, the Ey-CT, which differs strongly from that of Toy, is likely to interact with a different set of co-factors to confer
specific activation or repression of target genes. This hypothesis is
supported by the analysis of the CT. Only the Ey-CT, and not that of Toy, is
capable of inducing ectopic eyes on the antenna, and only the Ey-HD with an
Ey-CT is able to confer DLL repression, which is required for normal eye
development. Thus, these experiments provide new insights into the evolutionary
divergence of the two Pax6 genes in Drosophila, and their role in eye
and head development (Punzo, 2004).
While testing the functions of deletion mutants in the Hox protein Ultrabithorax (Ubx), it was found that the embryonic repression function of Ubx on Distal-less transcription in limb primordia is highly concentration dependent. The steep sigmoidal relationship between in vivo Ubx concentration and Distal-less repression is dependent on the Ubx YPWM motif. This suggests that Ubx cooperatively assembles a multi-protein repression complex on Distal-less regulatory DNA with the YPWM motif as a key protein-protein interface in this complex. Deletion mutants also provide evidence for a transcriptional activation domain in the N-terminal 19 amino acids of Ubx. This proposed activation domain contains a variant of the SSYF motif that is found at the N termini of many Hox proteins, and is conserved in the activation domain of another Hox protein, Sex combs reduced. These results suggest that the N-terminal region containing the SSYF motif has been conserved in many Hox proteins for its role in transcriptional activation (Tour, 2005).
The current results indicate that at least for its limb and Dll repression
functions, Ubx contributes to a cooperative on/off switch over a small
concentration range. When Dll repression is plotted as a function of
Ubx concentration, the best-fit curve has a Hill slope of 4.9±2.2.
These results suggest a highly cooperative assembly of a multiprotein
repression complex containing Ubx on Dll regulatory DNA. Although the
repression dose-response curves cannot be extrapolated into the number of
cooperative protein-protein interactions within a repression complex, they are
a surprisingly good fit to a model in which the Ubx-mediated repression of a Dll limb enhancer requires at least five clustered DNA sites that cooperatively bind two molecules of Ubx, Extradenticle (Exd) and Homothorax, while the fifth site binds the Sloppy paired 1 protein. The
high sensitivity of Ubx phenotypes to concentration may explain why previous
experiments using ectopic expression of Ubx have come to different
conclusions, and illustrates why the validity of conclusions from ectopic
expression studies should be interpreted with caution, unless great care is
taken to achieve near-normal physiological levels (Tour, 2005).
Why is the Ubx repressive effect on Dll so concentration
sensitive? It is instructive to look at other biological systems with similar
concentration-dependent transcriptional switches. For example, the steep
concentration dependence of the lambda transcriptional repressor allows
prophages in E. coli cells to switch, at crucial levels of cellular
distress, from one stable state to another, lysogenic to lytic. For
Ubx, one likely reason for the highly concentration-dependent effects on
Dll expression and limb development is to ensure that all the cells
in a limb field are stably programmed to adopt either the limb state, or body
wall fate. At least in extant Drosophila, a mosaic appendage that
developed from a mixed field of limb and body wall cells would presumably be
little benefit to the animal that carried it, and thus selected against during
evolution (Tour, 2005).
Tests of mutant Hox proteins in Drosophila and in mice have
demonstrated the importance of the YPWM motif for Hox function in vivo,
although both loss- and gain-of-function phenotypes were observed. In vitro, the YPWM region has been shown to mediate Hox
interactions with the PBC family of homeodomain proteins. The
PBC proteins (Exd protein in Drosophila, Pbx proteins in mammals)
bind cooperatively with Hox proteins on composite DNA sites, and are important
co-factors in the regulation of many Hox target genes (Tour, 2005).
A Ubx protein with a YAAA substitution for YPWM
exhibits reduced cooperative binding with Exd on a consensus composite
Ubx-Exd DNA-binding site. Reduced affinity between UbxDeltaYPWM and Exd
might compromise the assembly of the entire repression complex, resulting in an inefficient transcriptional repression of
Dll in the anterior segmental compartments (Tour, 2005).
The in vivo results are also consistent with models in which the YPWM
region contributes in other ways to repression cooperativity. For example, the
YPWM region appears to influence Hox activation and repression functions in a
manner that is independent of its role in enhancing the affinity of Hox/PBC
protein complexes for binding sites. In vitro, Ubx is also known to bind cooperatively to DNA in
homomeric complexes, and the YPWM motif might be required for the formation of
such complexes on Dll regulatory sequences (Tour, 2005).
No single deletion abolishes the Ubx repression function, although some
regions are required for robust repression. Hox protein repression function
appears to be quite complex. Embryonic tests of the deletion mutants, suggest that Ubx contains multiple regions that additively
contribute to repression. In addition, other results suggest that the homeodomain also contributes directly to transcriptional repression
function in a manner that is independent of its DNA-binding function (Tour, 2005).
The deletion of the Ubx YPWM region has little detectable effect on the
transcriptional activation of the dpp and tsh genes. Because
exd genetic function is required for normal levels of dpp
and tsh activation in Ubx-expressing cells, this result
is difficult to reconcile with a simple model in which the YPWM motif is
required for Exd recruitment to activation target sites in dpp and
tsh enhancers. However, it is consistent with studies that tested the
effect of YPWM mutations on the activation abilities of the Labial and Abd-A
Hox proteins in embryos. A YPWM to AAAA mutant of Labial is a more potent activator
than wild-type Labial protein of a sequence derived from the Hoxb1
autoregulatory region, whereas a YPWM-to-AAAA mutant of Abd-A converted this
protein from a repressor into an activator of dpp transcription. In
addition, this YPWM mutation has no effect on the activation function of Abd-A
on wingless. The ability of Labial and Abd-A YPWM mutants to retain
their transactivation functions is correlated with their ability to bind Exd
in vitro in a YPWM-independent fashion. The
YPWM-independent interactions between Hox proteins and Exd can be mediated by
Hox homeodomains and the C-terminal regions (Tour, 2005).
Since the Ubx-responsive elements from dpp and tsh loci
possess a mixture of Ubx monomer and Ubx-Exd heterodimer-binding sites,
possible reasons for the ability of the Ubx YMPM deletion mutant to activate
these downstream target genes are: (1) Hox activation of target genes often
involves a mixture of Exd-dependent and Exd-independent functions; (2)
removal of the YPWM motif does not completely abolish Exd-Ubx binding
interactions, and (3) the YPWM apparently serves other functions besides
binding Exd in the context of developing embryos (Tour, 2005).
The genitalia of Drosophila derive from the genital disc and
require the activity of the Abdominal-B (Abd-B) Hox gene.
This gene encodes two different proteins, Abd-B M and Abd-B R. The embryonic genital disc, like the larval genital disc, is formed by
cells from the eighth (A8), ninth (A9) and tenth (A10) abdominal segments,
which most likely express the Abd-B M, Abd-B R and Caudal products,
respectively. Abd-B m is needed for the development of A8 derivatives
such as the external and internal female genitalia, the latter also requiring
abdominal-A (abd-A), whereas Abd-B r shapes male
genitalia (A9 in males). Although Abd-B r represses Abd-B m
in the embryo, in at least part of the male A9 such regulation does not occur.
In the male A9, some Abd-B m–r– or
Abd-B r– clones activate Distal-less and
transform part of the genitalia into leg or antenna. In the female A8, many
Abd-B m–r– mutant clones produce
similar effects, and also downregulate or eliminate abdominal-A
expression. By contrast, although Abd-B m is the main or only
Abd-B transcript present in the female A8, Abd-B
m– clones induced in this primordium do not alter
Distal-less or abd-A expression, and transform the A8
segment into the A4. The relationship between Abd-B and
abd-A in the female genital disc is opposite that of the embryonic
epidermis, and contravenes the rule that posteriorly expressed Hox genes
downregulate more anterior ones (Foronda, 2006).
Abd-B is not only expressed, but also required in the embryonic
genital primordium. In the absence of Abd-B m, the number of
hdc-expressing cells in the disc is reduced, most likely because
these cells adopt now a more anterior fate, as occurs in the cuticle. When Abd-B r is absent, the genital primordium lacks some cells and is disorganized, and when both Abd-B products are absent, the primordium is reduced to a few, dispersed cells, some of which express Dll ectopically, suggesting a transformation into a leg primordium (Foronda, 2006).
The A8, A9 and A10 primordia of the mature genital discs bear anterior and
posterior compartments, with expression of en and wg in each
of these three primordia. Curiously, although three primordia in the
embryonic disc can be defined, based on the expression of Abd-B m, Abd-B r and cad, neither en nor wg is expressed in the three
separate domains at this stage. This may suggest, as was also recently proposed, that new bands of en and wg expression may be formed later in
development, in precise concordance with the three primordia defined
by the Abd-B m, Abd-B r and cad genes. It is noted
that late en expression is also characteristic of the antennal
primordium of the eye-antennal disc (Foronda, 2006).
abd-A is expressed in the whole internal female
genitalia except for the parovaria, and this is consistent with experiments
indicating that parovaria derive from the female A9 segment.
abd-A has been shown to be required for gonad development, and in the
abd-Aiab-3/Df mutant, combinations ovaries are also absent.
However, the defects observed in the female internal genitalia are not
simply due to an indirect effect of the lack of gonads, since iab-4
mutations prevent the formation of the ovaries but do not alter internal
genitalia formation (Foronda, 2006).
The results indicate that Abd-B m is required for the development
of female external and internal genitalia, both derived from the female A8.
The internal genitalia of Abd-B-Gal4LDN/UAS-lacZ
females (driving expression only where Abd-B m levels are high)
were stained with X-gal except in two structures, the oviducts and
parovaria. The absence of oviduct staining in Abd-B-Gal
4LDN/UAS-lacZ females is probably due to the
particular expression driven by this reporter, and does not imply an absence
of Abd-B m transcription in these organs, for two reasons: (1)
Abd-B m transcripts are present in the whole A8 segment of the female
genital disc, and (2) oviduct development is affected in Abd-B m
mutant females. Parovaria, by contrast, are not stained in Abd-B-Gal
4LDN/UAS-lacZ or abd-A-lacZ females, and
this agrees with their A9 provenance. This is supported by the observation that in some Abd-B m mutant females parovaria are the only structures that remain in the internal female genitalia (Foronda, 2006).
Abd-B M seems to be the main or only Abd-B product present in the
female A8, so it was expected that elimination in this segment of just Abd-B M
or of all Abd-B proteins would give similar results. This is not so. Some
Abd-B– clones transform part of the female genitalia
into leg or antenna, whereas Abd-B m mutant clones convert the eighth tergite, and probably the female genitalia, into an anterior abdominal segment. The differences between Abd-B m– and AbdB– clones in the A8 of the female genital disc reveal the existence of unsuspected regulatory interactions between the abd-A and Abd-B genes: whereas Abd-B m– clones do not affect abd-A, in AbdB– clones abd-A expression is
eliminated. This is a surprising result, because it is contrary to what is
observed in the embryo, where Abd-B represses abd-A (Foronda, 2006).
Abd-B m– clones induced in the female A8 do not
alter abd-A expression but do not change Abd-B expression
levels either. This is observed with mutations that do not make Abd-B M
protein, so the Abd-B protein detected is not the Abd-B M product.
Surprisingly, although some Abd-B r expression is detected in the
female A8, uniform Abd-B r expression is not seen throughout this
primordium and Abd-B r transcripts seem not to be derepressed in
Abd-BM5 mutant clones. No explanation is available for this
conundrum. Perhaps the probe used, although it includes sequences complementary to all of the Abd-B r cDNA sequences that have been published, does not efficiently detect all of the non-Abd-B m transcripts (Foronda, 2006).
The differences in regulatory and functional interactions among gene
products in the embryo and the genital discs are not limited to those of
Abd-B and abd-A that have been discussed above. Three other possibilities should be considered. (1) There
may be changes in phenotypic suppression: the transformation of the eighth
tergite to the fourth one in Abd-B m– clones is due
to abd-A. Because in these clones Abd-B protein is still present, this suggests that abd-A may phenotypically suppress Abd-B, differently from what is generally observed in the embryo. (2) Abd-B r represses Abd-B m in the embryo, but some Abd-B r– clones do not activate Abd-B m in the male disc. (3) abd-A represses Dll in the embryo, but not in the female genital disc, and ectopic Dll can repress abd-A instead. abd-A does not repress Dll in the leg discs either, and this resembles Ubx function, which represses Dll only early in development. By contrast, Abd-B represses Dll in the embryo, in the larval genital disc, and in the leg disc when ectopically expressed (Foronda, 2006).
Abd-B r expression is restricted to the A9 segment in male genital
discs, but shows expression in the A9 and in some cells of the A8 in female
genital discs. In spite of this, Abd-B r– clones in
the external female genitalia (A8) are phenotypically wild type. In the male
A9, some Abd-B r mutant clones eliminate Abd-B, activate
Dll and transform part of the genitalia into distal leg or antenna.
This is similar to the result obtained in some
Abd-B– clones, and it implies that Abd-B m
is not derepressed in these mutant clones. However, Abd-B m is
perhaps derepressed in those Abd-B r mutant clones where
Abd-B signal remains (Foronda, 2006).
Although Abd-B r– clones affect, almost
exclusively, male genitalia development, Abd-B r hemizygous or
trans-heterozygous flies lack genitalia and analia in both sexes. This
probably reflects the absence of proper interactions between the different
primordia needed for the growth of the genital disc. In
Abd-B r mutant females, the internal genitalia are abnormal, and in
some of these females, an absence of parovaria and the presence of
three or four spermathecae is observed. This phenotype is consistent with a
segment-autonomous transformation of A9 derivatives (parovaria) into A8
structures (spermathecae), similar to the embryonic cuticular transformation
of A9 into A8 observed in Abd-B r mutations. A transformation of parovaria into spermathecae has been described in Polycomblike mutants, and may also indicate a transformation of A9 to A8 (Foronda, 2006).
These results illustrate that there are quite different Hox cross-regulatory
interactions in the embryo and in the genital disc. The effects in the
genital discs contradict the general rule that genes transcribed more
posteriorly suppress or downregulate the expression of more anterior ones. This
rule has, nevertheless, some exceptions in genes of the Antennapedia complex.
Further, differences in Hox cross-regulation between the embryo and imaginal
discs are not unprecedented: the proboscipedia (pb) Hox gene
is positively regulated by Sex combs reduced in the embryo, but
pb activates Sex combs reduced in the labial imaginal disc (Foronda, 2006).
It has been proposed that the primordia of female and male genitalia could
be subdivided into an 'appendage-like' and a 'trunk-like' region). These two regions of the female A8 can now be defined more
precisely. The 'appendage-like' region would be that expressing abd-A
and low levels of Abd-B, and corresponds approximately to the
presumptive internal female genitalia. This domain is
roughly coincident with the region of expression of a reporter insertion in
buttonhead, the gene that defines ventral appendage development, and
this is also, approximately, the domain where Abd-B–
clones may activate Dll. If this subdivision is correct,
the 'appendage' specification defined by buttonhead would be
repressed in the wild type by Abd-B, which both limits Dll
expression to a few cells of the A8 primordium and prevents Dll
function. Abd-B– clones in this region eliminate abd-A expression and promote leg or antenna development. This subdivision may also apply to the male disc, the penis apparatus presumptive region being the main 'appendage' domain. Similar to what is described in this study, the labial disc possesses a large 'appendage' region that is revealed by Dll derepression in pb mutations. This characteristic, and the changes in Hox gene cross-regulation between the embryo and the imaginal disc, are two features shared by pb/labial disc and Abd-B/genital disc (Foronda, 2006).
Development of organ-specific size and shape demands tight coordination
between tissue growth and cell-cell adhesion. Dynamic regulation of cell
adhesion proteins thus plays an important role during organogenesis. In
Drosophila, the homophilic cell adhesion protein DE-Cadherin
regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study
shows that along the proximodistal (PD) axis of the developing wing epithelium,
apical cell shapes and expression of DE-Cad are graded in response to
Wingless, a morphogen secreted from the dorsoventral (DV) organizer in
distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft)
tumor suppressor, by contrast, represses DE-Cad expression. In
genetic tests, ft behaves as a suppressor of Wg signaling.
Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg
signaling, is negatively correlated with the activity of Ft. Moreover, unlike
that of Wg, signaling by Ft negatively regulates the expression of Distalless
(Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg
signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert
opposing regulation to coordinate cell-cell adhesion and patterning along the
PD axis of Drosophila wing (Jaiswal, 2006).
In both loss- and gain-of-function assays,
this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the
distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained
to be the direct targets of Wg, all available evidence so far
suggests that these targets positively respond to Wg signaling.
These results also show that Ft and Wg signaling intersect and control distal
wing growth and pattern, presumably through their opposing regulation of a
common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling,
Dpp signaling also regulates Q-vg-lacZ; however,
its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by
its intersection with Wg signaling (Jaiswal, 2006).
The results show that Ft negatively regulates Wg signaling. Loss or gain of
Ft induces a telltale sign of perturbations in Wg signaling, namely, changes
in the cellular pool of ß-catenin/Arm,
consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to
its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also
noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).
One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).
Drosophila wing growth is under dynamic spatial and temporal
regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact
cell proliferation in their characteristic ways and activate distinct sets of
PD markers. Although at a very high threshold, Wg signaling inhibits cell
proliferation, at a modest threshold it has been shown to stimulate growth. It is
noted that loss of Ft fails to activate Wg targets that demand a high threshold
of Wg signaling, e.g., Ac, which is required for wing margin
specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ,
which responds to a high threshold of Wg signaling, is also not upregulated by
loss of Ft. Dll responds to a higher threshold of Wg
signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).
Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and has been proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibits several mutant phenotypes such as the transformation of antennae to mesothoracic legs. This study examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, is ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation are also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, is also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes are enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are also PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis (Kim, 2008).
Many PcG genes act as zygotic as well as maternal effect genes during whole Drosophila development, but it is not well known when and how they function. Pho is known to work with its redundant DNA-binding protein, Phol and recruits other PcG complexes by binding its binding sites on PREs. pho functions as a maternal effect gene. Its maternal effect mutant embryos show several segment defects and weak homeotic transformation. When pho functions as a zygotic gene, its zygotic mutant adults show homeotic transformation of antennae and legs. In accord to these results, pho functions in identification of ventral appendage were investigated (Kim, 2008).
Mutations in a few PcG genes result in the transformation of antennae to legs. Mutation in esc induces the ectopic expression of Antp and Ubx in the antennal disc, thus transforming antennae to legs. This indicates that esc represses Antp and Ubx expression in the antennal disc during antennal development. Therefore, the possibility was investigated that pho mutation, like esc mutation, would affect the expression of the selector genes that determine the identity of antenna or leg. In the wild type antennal disc, Antp is not expressed, but hth is expressed in almost all cells except for the presumptive arista, allowing for the development of antenna. However, in the leg disc, Antp is expressed and restricts hth expression to the proximal cells, which permits leg development (Kim, 2008).
Antp is ectopically expressed in the antennal disc of the pho mutant, and its expression subsequently but partially represses hth expression in the presumptive a2 or a3. Moreover, in the pho mutant, dac, which is expressed in the presumptive a3 of wild type antennal discs, is overexpressed in the presumptive a2 or a3 where hth expression is reduced. Ectopic expression of Antp in the presumptive a2 represses hth expression, which subsequently results in the transformation from antenna to leg. Ectopic Antp expression in the presumptive a1 permits expression of hth. In addition, when dac is ectopically expressed in a3 using the UAS/GAL4 system, leg-like bristles are newly formed in a3, indicating transformation of a3 to femur. However, the antennal disc of pho mutant shows that hth expression does not completely disappear in all regions of the presumptive a2 and a3 where Antp is ectopically expressed. These indicate that a pho single mutation partially affects expression of Antp, which leads to the incomplete repression of hth. Moreover, as the increased dosage of PcG mutants causes stronger mutant phenotypes than each single mutant, double mutation of pho and Psc strongly affects the expression of Antp, which leads to the complete repression of hth. Therefore, these results indicate that a pho mutation results in the ectopic expression of Antp, which directly represses hth expression in antennal disc and indirectly regulates dac expression through hth expression, which consequently transforms antennae to legs (Kim, 2008).
In the wing imaginal disc, Polycomb (Pc) and Suppressor of zeste (Su(z)) regulate the expression of teashirt (tsh), which specifies the proximal domain with hth. The polyhomeotic (ph) gene regulates the expression of en and the hedgehog (hh) signaling pathway in the wing imaginal disc. Pc also regulates eye specification genes such as tsh and eyeless (ey). PcG genes have recently been found to regulate organ specification genes in addition to homeotic genes, segmentation genes and cell cycle genes (Kim, 2008).
Therefore, it was proposed that pho might regulate the expression of organ specification genes for several reasons. First, Dll is ectopically expressed in the proximal region of the posterior compartment in the antennal disc of the pho mutant. Additionally, Dll is ectopically expressed in the more proximal region of the leg disc in the pho mutant, while dac is ectopically expressed in both the proximal and distal regions. These ectopic expressions do not antagonize each other in their normal region of expression, and result in duplication of distal tibia. Finally, en expression extends to the anterior compartment of both the antennal and leg discs of the pho mutant (Kim, 2008).
According to these reasons the following is proposed; first, pho regulates the expression of Antp in the antennal disc, which in turn might activate Dll. It has been shown that Dll is activated in AntpNS discs, which is similar in younger and older pho discs. Second, pho regulates the expression of en, which affects the expression of Dll. As a gene determining the A/P axis during antenna and leg development, en affects expression of wg and dpp, which determine the D/V axis via Hh signaling. Wg and Dpp act as morphogens, restricting the expression domain of hth, dac and Dll. This study has demonstrated that en is misexpressed in the anterior compartment in the antennal and leg discs of the pho mutant, which leads to misexpression of wg in the anterior-dorsal compartment. Although it has been shown that in the pho zygotic mutant embryos en is hardly derepressed, the current study showed that it is depressed in the pho zygotic mutant adults, suggesting that pho is involved in regulation of en expression and indirect regulation of Dll expression. Finally, pho might directly regulate expression of Dll, because recent studies using X-ChIP analysis have shown that PcG proteins bind PREs of appendage genes including Dll and hth. Hence, pho may directly or indirectly maintain the expression of Antp and en and regulates P/D patterning genes during ventral appendage formation (Kim, 2008).
Pho and Phol are the only PcG proteins that have a zinc finger domain. A mutation in pho results in weaker phenotypes than other PcG mutations despite the functioning of Pho as a DNA-binding protein. Therefore, Pho may interact with other corepressors and repress the homeotic selector genes. In fact, Pho binds to PRE, which is facilitated by GAGA. PRE-bound Pho and Phol directly recruit PRC2, which leads to the anchoring of PRC1. Pho interacts with PRC1 as well as with the BRM complex. Pho has recently been used to construct a novel complex, called the Pho-repressive complex (PhoRC), which has selective methyl-lysine-binding activity. It is currently known that pho interacts with two other PcG genes, Pc and Pcl, in vivo (Kim, 2008 and references therein).
Pho binds to approximately 100 sites on the polytene chromosome and colocalizes with PSC in about 65% of these binding sites. PSC is a component of PRC1 and inhibits chromatin remodeling. In the third instar larvae, PSC is found in the nuclei in all regions of all imaginal discs. Therefore, it is possible that pho and Psc interact with each other during the adult structure formation from the imaginal discs. pho and Psc interact in ventral appendage formation. While the Psc heterozygote was normal, it enhanced the adult mutant phenotypes exhibited by the pho homozygous mutant. Antp is more widely expressed in the antennal disc of the double mutant of pho and Psc than in that of the pho single mutant, while Psc mutant clones induced by FRT/FLP system showed normal expression of Antp, which indicated that Psc does not directly act by itself in regulating expression of Antp, but it certainly interacts with pho (Kim, 2008 and references therein).
hth is expressed in the distal region regardless of Antp expression so that dac was expressed not only in presumptive a3 but also in other segments, which results in the formation of a new P/D axis. According to recent study showing that hth may have a PRE, these results suggest that pho and Psc might interact to maintain hth expression during antennal development. Moreover, Dll expression in the antennal disc might be repressed by an unknown factor that was affected by the double mutation of pho and Psc, suggesting that the factor might be regulated by pho interaction with Psc during antennal development. In addition, legs of the double mutant had fused segments and weakly jointed tarsi, which may be because extension of Hh signal lead to the abnormal expression of the P/D patterning genes. In sum, pho functions as a regulator of selector genes for the identification of ventral appendages and axis formation by interaction with Psc during postembryogenesis (Kim, 2008).
In addition, Pho interacts with Phol in ventral appendage formation. Adults of double mutants showed more severe defects in appendage formation than those of single mutant. The stronger ectopic expression of Antp in the antennal disc of phol; pho double mutant seems to be one of reasons for severe defects. While Antp is not expressed in phol mutant clones of the wild type antennal discs, it is more strongly ectopically expressed in phol mutant clones of the pho mutant antennal discs than in their surrounding phol/+; pho/pho cells, indicating that Phol may not regulate the expression of Antp alone, but it may do that by interaction with Pho, suggesting that this may lead to recruit PRC1 including PSC to PRE sites of Antp and other appendage genes (Kim, 2008).
Polycomb group (PcG) proteins form conserved regulatory complexes that modify chromatin to repress transcription. This study reports genome-wide binding profiles of PhoRC, the Drosophila PcG protein complex containing the DNA-binding factor Pho/dYY1 and Scm-related gene containing four mbt domains (dSfmbt). PhoRC constitutively occupies short Polycomb response elements (PREs) of a large set of developmental regulator genes in both embryos and larvae. The majority of these PREs are co-occupied by the PcG complexes PRC1 and PRC2. Analysis of PcG mutants shows that the PcG system represses genes required for anteroposterior, dorsoventral, and proximodistal patterning of imaginal discs and that it also represses cell cycle regulator genes. Many of these genes are regulated in a dynamic manner, and the results suggest that the PcG system restricts signaling-mediated activation of target genes to appropriate cells. Analysis of cell cycle regulators indicates that the PcG system also dynamically modulates the expression levels of certain genes, providing a possible explanation for the tumor phenotype of PcG mutants (Oktaba, 2008).
It was asked whether the chromosomal intervals identified by Pho or dSfmbt ChIP-chip are enriched for particular DNA sequence motifs. To this end, de novo sequence motif discovery was performed on the PhoE-, PhoL-, dSfmbtL-bound regions (superscript E refers to embryos and superscript L to larvae) and on the 196 core-PhoRC regions. Several 8-mers, based on a GCCAT core were significantly overrepresented in the Pho datasets and were used as the basis to reconstruct a position sequence-specific matrix (PSSM). This sequence, GC/AC/GGCCATT/CTT, closely matches the motif previously identified in vitro as an optimal Pho-binding site using electro mobility shift assays. However, in vivo binding data suggest a more extensive Pho-binding motif containing an additional Guanine nucleotide at the -3 position and a Thymine pair at positions +7 and +8. This extended Pho-binding motif is enriched in all data sets. However, it is noteworthy that no Pho-binding site was identified as an overrepresented motif in the subset of dSfmbtL regions not bound by Pho (i.e., the 328 dSfmbtL regions). The lack of Pho enrichment by ChIP and the absence of detectable Pho binding sites in this fraction of the dSfmbtL dataset indicate that dSfmbt is targeted to these regions independently of Pho (Oktaba, 2008).
Global identification of genomic locations to which PcG protein complexes bind and unraveling how expression of target genes is regulated by these complexes is important to understand how the PcG system controls transcription of the genome. The following main conclusions can be drawn from the work presented in this study: (1) PhoRC is sharply localized at discrete PRE sequences, many of which are co-occupied by PRC1 and PRC2. (2) 196 PREs were identified in the Drosophila genome where PhoRC is constitutively bound in embryos and larvae. In general, these PREs are located within ±1 kb from the closest gene transcription start site and the majority of target genes contain only one PRE. (3) Sequence analyses of identified PREs allowed definition of an extended Pho-binding motif that is part of the signature of PhoRC-bound PREs. (4) Functional analyses in Drosophila reveal that PcG proteins repress transcription of several key developmental regulators. In particular, the PcG system is required for maintaining the subdivision of segment primordia into anteroposterior, dorsoventral and proximodistal compartments by repressing the genes en, ap, pnr, tsh, and Dll. (5) As discussed in detail below, these analyses suggest that extracellular signaling can selectively induce transcription of previously silent PcG target genes, even though PcG protein complexes are bound at their PREs. (6) Among the PcG target genes are also cell cycle regulators such as Rbf, E2F, Dp, and CycB, and evidence is providedthat the PcG system regulates expression levels of the CycB gene in Drosophila (Oktaba, 2008).
Genome-wide binding profiling and ChIP analyses at selected target genes revealed an extensive overlap between PhoRC-, PRC1-, and PRC2-bound regions. These results, together with previous reports that Pho interacts with and is required for targeting of PRC1 and/or PRC2 at HOX gene PREs, suggest that PhoRC is needed for PRC1 and PRC2 binding at many PcG target genes. It should also be recalled that in larvae, PhoRC, PRC1, and PRC2 are all constitutively bound to PREs of the HOX gene Ubx, both in cells where Ubx is repressed and in cells where it is active. The observation that the same PRE sites are occupied in both tissue culture cells and in developing Drosophila thus further implies that PhoRC, PRC1, and PRC2 may be constitutively bound to a large fraction if not most of their target genes (Oktaba, 2008).
What are the sequences that make up a PRE? Using an algorithm based on binding site motifs for Pho, GAF/Trl, and Zeste proteins, 167 PREs have been predicted across the Drosophila genome. This study detected PhoRC binding at 26 of these predicted PREs (15%). Intriguingly, this study found a significant overlap between PhoRC-bound regions and regions bound by GAF/Trl but only a limited overlap with Zeste-bound regions. GAF/Trl mutants do not show HOX misexpression phenotypes, making its role at HOX gene PREs somewhat enigmatic. However, it is possible that GAF/Trl is required for PRC1 and/or PRC2 targeting to PREs of non-HOX genes. Finally, it is important to keep in mind that PRC1 and PRC2 are also bound at genomic regions where PhoRC has not been detected, suggesting that they are targeted there by other factors (Oktaba, 2008).
The most obvious phenotype of PcG mutants in Drosophila are homeotic transformations, caused by the global misexpression of multiple HOX genes. In this study, loss of repression of several non-HOX target genes was monitored and it was shown that these genes are indeed also misexpressed in PcG mutants. In particular, it was found that the PcG system represses key regulator genes required for the subdivision of appendages into anteroposterior, dorsoventral, and proximodistal compartments. This suggests that the PcG system is responsible for maintaining many more cell fate decisions than may be immediately evident from the phenotype. In this context, it is important to note that at some target genes, control by PcG proteins is masked by other regulatory interactions and can only be revealed in the absence of those regulatory inputs. The downregulation of Dll in PcG mutant clones by HOX proteins represents a prime example for such a masking effect; regulation of Dll by the PcG system could only be revealed in cells lacking both PcG and HOX gene functions (Oktaba, 2008).
Finally, it was found that some target genes are only strongly misexpressed in certain PcG mutants but not in others, even though PhoRC, PRC1, and PRC2 are all cobound at these genes. This implies that in those cases not all PcG protein complexes or complex components are required for repression. In the most extreme scenario, recruitment of all three complexes reflects the default state that even occurs at genes that require i.e., only H2A ubiquitylation by PRC1 but not H3-K27 methylation by PRC2 for repression (Oktaba, 2008).
The finding that PcG protein complexes are constitutively bound at PREs of target genes in both embryonic and imaginal disc cells has implications for understanding regulation by the PcG system. In particular, target genes such as ap, Dll, or pnr are not active in the wing disc primordium in embryos, remain silent as the primordium grows during the early larval stages and become transcriptionally active only at later larval stages. The factors responsible for activating ap transcription during the second larval instar are not known, but induction of Dll expression in the wing blade primordium during the third larval instar occurs in response to wg signaling, and expression of pnr in the notum primordium during the second larval instar is activated by dpp signaling. The most straightforward explanation of these observations is that these signaling pathways are able to switch on expression of these target genes even though PcG complexes are bound at their PREs and even though their chromatin bears the Polycomb-repressive H3-K27me3 mark in embryos. In wild-type wing discs, Wg-signaling would thus be able to overcome PcG repression at the Dll gene in wing pouch cells but not in the notum and hinge region where Wg protein is also present. Similarly, in wild-type animals, Dpp-signaling activates pnr expression only in the notum and not along the whole length of the anteroposterior compartment boundary where Dpp protein is expressed. One possible explanation for this selective activation in discrete parts of the disc would be a requirement for signaling pathways to act in a combinatorial manner with other (unknown) factors to relieve PcG repression. Consistent with this, removal of PcG function often results in misexpression of target genes only in specific regions of the disc, and these regions receive the same signals that are also responsible for activation of these target genes in their normal wild-type expression domain. For example, misexpression of pnr mainly occurs in PcG mutant clones in the wing blade and thus in cells that receive Dpp, the signal that also activates pnr expression in its wild-type expression domain in the notum. This raises the intriguing possibility that an important function of the PcG system may be to spatially restrict activation of target genes in response to more widely distributed extracellular signals (Oktaba, 2008).
Previous ChIP-chip studies showed that PcG protein complexes are bound to a large number of developmental regulators in mammalian embryonic stem cells. The majority of the genes bound by PcG proteins in stem cells are orthologs of PcG target genes identified in flies, including the family orthologs of en, ap, pnr, Dll, eve, and Doc whose regulation was analyzed in this study. The finding that these genes are regulated by the PcG system during Drosophila development implies that the mammalian PcG system may also regulate the orthologous genes in differentiating cells and tissues, beyond the known regulation in stem cells (Oktaba, 2008).
This study identified cell cycle regulator genes as PcG targets, and evidence is provided that the PcG system directly regulates CycB expression. Control of cell cycle regulators by the PcG system may provide a molecular explanation for the tumor phenotype observed in proliferating imaginal disc cells lacking the PRC1 components Psc-Su(z)2 or ph. The observation that the PcG system controls transcription of genes whose expression is modulated during the cell cycle suggests that the PcG system is also used to regulate target genes more dynamically than previously thought. In mammalian cells, knockout of the Psc homolog bmi-1 results in cellular senescence via loss of p16/INK4A transcriptional repression, a cyclin D regulator without any obvious ortholog in the Drosophila genome. It therefore appears that the PcG system has a conserved role in regulating expression of genes involved in body patterning but that it evolved in different ways to control cell growth and proliferation in mammals and flies (Oktaba, 2008).
Wnts and bone morphogenetic proteins (BMPs) are signaling elements that are crucial for a variety of events in animal development. In Drosophila, Wingless (Wg, a Wnt ligand) and Decapentaplegic (Dpp, a BMP homolog) are thought to function through distinct signal transduction pathways and independently direct the patterning of the wing. However, recent studies suggest that Mothers against Dpp (Mad), the key transducer of Dpp signaling, might serve as a node for the crosstalk between these two pathways, and both positive and negative roles of Mad in Wg signaling have been suggested. This study describes a novel molecular mechanism by which Dpp signaling suppresses Wg outputs. Brinker (Brk), a transcriptional repressor that is downregulated by Dpp, directly represses naked cuticle (nkd), which encodes a feedback inhibitor of Wg signaling, in vitro and in vivo. Through genetic studies, this study demonstrates that Brk is required for Wg target gene expression in fly wing imaginal discs and that loss or gain of brk during wing development mimics loss or gain of Wg signaling, respectively. Finally, it was shown that Dpp positively regulates the expression of nkd and negatively regulates the Wg target gene Distal-less (Dll). These data support a model in which different signaling pathways interact via a negative-feedback mechanism. Such a mechanism might explain how organs coordinate inputs from multiple signaling cues (Yang, 2013).
This study has shown that Brk directly represses nkd expression. The direct repression of nkd by Brk is underscored by three observations. First, a Brk site was identified in the intronic region of nkd, which Brk physically occupies in vitro. Second, ChIP analysis shows that Brk binds a DNA region near this Brk site in embryos in a manner inversely related to Wg activity. Third, reporter analysis in Kc cells indicates that Brk represses Arm-dependent activation of an intronic WRE containing this Brk site, but only when the Brk site is intact. In addition, genetic analyses has shown that the repression of nkd by Brk is functionally significant. In the developing wing, it was found that the loss of brk de-represses nkd and downregulates Wg target proteins, such as Dll and Sens. Conversely, ectopic brk inhibits nkd expression and markedly enhances Dll expression. Furthermore, removal of nkd prevents the loss of Dll in brk clones whereas co-expression of nkd abolishes the expanded Dll caused by ectopic brk. In adult wing, the loss and gain of brk phenotypically resembles the loss and gain of Wg signaling, respectively. Consistent with a repressive role of Dpp cascade on brk, it was found that ectopic Dpp signaling enhances nkd and inhibits Wg signaling). These results support a model in which Dpp signaling increases the expression of Nkd, a Wg inhibitor, by the downregulation of Brk, and thereby inhibits the Wg outputs. In another words, nkd might fall into a class of Dpp targets, which are de-repressed upon the activation of Dpp signaling. This study has thus uncovered a previously unsuspected molecular mechanism underlying the interaction between Wg and Dpp signaling pathways in Drosophila wing development (Yang, 2013).
Until recently, little has been known about the cross-interaction between Wg and Dpp signaling in Drosophila wings, in spite of the fact that the fly wing has served as an excellent model system for the dissection of the molecular basis of these signaling transduction pathways. This is in contrast to Drosophila leg imaginal discs, in which mutual repression between Wg and Dpp signaling has long been suspected. However, several studies have indicated that manipulation of Dpp signaling levels in the wing sometimes leads to phenotypes resembling those caused by loss or gain of Wg activity. Notably, ectopic Dpp signaling increases notches in the wing, which is characteristic of reduced Wg signaling. However, the underlying mechanism for this effect of Dpp is not clear. Recently, independent research groups have suggested that Mad, the key effector of Dpp signaling, might play a role in the regulation of Wg target gene expression in fly wings. The molecular basis for their findings has mainly been the physical interaction between Mad and TCF, similar to the findings in mammals, in which several Smad proteins interact with members of the lymphoid enhancer binding factor 1/TCF family of DNA-binding HMG box transcription factor. It remains to be determined whether the role of Mad is direct or indirect because the reporter assays in these studies were performed with TOPFlash or similar constructs in mammalian cell culture, which might not always accurately represent the complicated situation of the in vivo regulation of Wg target genes. Furthermore, manipulation of Mad expression in wing discs influences Dll expression in different directions. Although these intriguing discrepancies can be explained by the physical interaction between Mad and TCF, the current model offers an alternative interpretation based on the negative regulation of nkd by Brk, which might suggest an indirect role of Mad in Wg signaling. For example, the current model could provide an explanation for the previous finding that ectopic Dpp signaling, caused by Mad, Medea, TkvQD, etc., results in notched wings (Yang, 2013).
The role of Brk in Wg signaling has been previously documented in Drosophila. It has been suggested that brk is able to antagonize Wg signaling based on the activity of a midgut-specific Ubx reporter gene in which physical interactions among Brk, Teashirt and CtBP have been described. In leg discs, Wg signaling may directly repress Dpp morphogen expression via an Arm-TCF-Brk complex, offering a direct model for the cross-talk between Wg and Dpp. However, the current studies have indicated a positive role for Brk in Wg signaling through an indirect action. In addition to the repression of Dpp targets, the roles of Brk in Wg signaling described in these different models exemplify the pleiotropic actions of brk throughout development and might provide the molecular basis for tissue-specific consequences of developmental signaling pathways (Yang, 2013).
nkd was first identified as a Drosophila segment-polarity gene, mutation of which gives rise to major deficits in fly embryonic development. Its expression appears to be universally induced by Wg in fly embryos and larval imaginal discs. It is interesting that although the loss of nkd in embryos has an effect similar to gain of wg, decreased nkd function in fly wings shows little impact. However, none of the nkd alleles used in these studies has been well characterized at the molecular level. Given the complexity of nkd transcriptional regulation, it could be that these mutant forms of nkd still possess residual function in the wing. Alternately, overexpression of nkd blocks ectopic Wg signaling in the eyes and generates PCP phenotypes in the wing through a direct interaction with Dsh. Consistent with these observations, this study found that loss of brk can cause a dramatic increase of nkd expression in certain areas of the wing imaginal disc, leading to wing notches and PCP defects. The current findings suggest that nkd may indeed play roles, at a certain level, in both canonical and noncanonical Wg signaling in fly wings. However, a closer examination of nkd function in fly wings is needed (Yang, 2013).
In conclusion, this study found that Brk influences Wg signaling by directly repressing nkd expression and could serve as a node for cross-talk between the Wg and Dpp signaling pathways. Wnt-BMP cross-interactions have been implicated in many developmental and disease processes). For example, a Wnt-BMP feedback circuit mechanism is important for inter-tissue signaling dynamics in tooth organogenesis in mouse. The findings may therefore add new insights into cell differentiation and human cancer (Yang, 2013).
The appendages of arthropods and vertebrates are not homologous structures, although the underlying genetic mechanisms that pattern them are highly conserved. Members of the Sp family of transcription factors are expressed in the developing limbs and their function is required for limb growth in both insects and chordates. Despite the fundamental and conserved role that these transcription factors play during appendage development, their target genes and the mechanisms in which they participate to control limb growth are mostly unknown. This study analyzed the individual contributions of two Drosophila Sp members, buttonhead (btd) and Sp1, during leg development. Sp1 plays a more prominent role controlling leg growth than btd. A regulatory function of Sp1 in Notch signaling was identified, and a genome wide transcriptome analysis was performed to identify other potential Sp1 target genes contributing to leg growth. The data suggest a mechanism by which the Sp factors control appendage growth through the Notch signaling (Cordoba, 2016).
Understanding the molecular mechanisms that control the specification and acquisition of the characteristic size and shape of organs is a fundamental question in biology. Of particular interest is the development of the appendages of vertebrates and arthropods, i.e., non-homologous structures that share a similar underlying genetic program to build them, a similarity that has been referred to as 'deep homology.' Some of the conserved genes include the Dll/Dlx genes, Hth/Meis and the family of Sp transcription factors. The Sp family is characterized by the presence of three highly conserved Cys2-His2-type zinc fingers and the presence of the Buttonhead (BTD) box just N-terminal of the zinc fingers (Cordoba, 2016).
Members of the Sp family have important functions during limb outgrowth in a range of species from beetles to mice. In vertebrates, Sp6, Sp8 and Sp9 are expressed in the limb bud and are necessary for Fgf8 expression and, therefore, for apical ectodermal ridge (AER) maintenance. Moreover, Sp6/Sp8 phenotypes have been related to the split-hand/foot malformation phenotype (SHFM) and, in the most severe cases, to amelia (the complete loss of the limb) (Cordoba, 2016).
In Drosophila, two members of this family, buttonhead (btd) and Sp1, are located next to each other on the chromosome and share similar expression patterns throughout development. Recently, another member of the family, Spps (Sp1-like factor for pairing sensitive-silencing) has been identified with no apparent specific function in appendage development. The phenotypic analysis of a btd loss-of-function allele and of a deletion that removes both btd and Sp1 led to the proposal that these genes have partially redundant roles during appendage development. However, the lack of a mutant for Sp1 has prevented the analysis of the specific contribution of this gene during development (Cordoba, 2016).
In Drosophila, leg development is initiated in the early embryo by the expression of the homeobox gene Distal-less (Dll) in a group of cells in each thoracic segment. Later on, Dll expression depends on the activity of the Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways, which, together with btd and Sp1, restrict Dll expression to the presumptive leg territory. Therefore, the early elimination of btd and Sp1 completely abolishes leg formation and, in some cases, causes a leg-to-wing homeotic transformation (Estella, 2010). As the leg imaginal disc grows, a proximo-distal (PD) axis is formed by the differential expression of three leg gap genes, Dll, dachshund (dac) and homothorax (hth), which divides the leg into distal, medial and proximal domains, respectively. Once these genes have been activated, their expression is maintained, in part through an autoregulatory mechanism, and no longer relies on Wg and Dpp. Meanwhile, the distal domain of the leg is further subdivided along the PD axis by the activity of the epidermal growth factor receptor (EGFR) signaling pathway through the activation of secondary PD targets such as aristaless (al), BarH1 (B-H1) or bric-a-brac (bab). During these stages, btd and Sp1 control the growth of the leg but are no longer required for Dll expression (Estella, 2010). How btd and Sp1 contribute to the shape and size of the leg and the identity of their downstream effector targets is unknown (Cordoba, 2016).
One important consequence of the PD territorial specification is the generation of developmental borders that activate organizing molecules to control the growth and pattern of the appendage. In the leg, PD subdivision is necessary to localize the expression of the Notch ligands Delta (Dl) and Serrate (Ser), which in turn activate the Notch pathway in concentric rings at the borders between presumptive leg segments. However, it is still unknown how Notch controls leg growth and how the localization of its ligands is regulated.
The present study generated a specific Sp1 null mutant, which, in combination with the btd mutant and a deletion that removes both btd and Sp1, allow analysis of the individual contributions of these genes to leg development. This study finds that Sp1 plays a fundamental role during patterning and growth of the leg disc, and that this function is not compensated by btd. The growth-promoting function of Sp1 depends in part on the regulation of the expression of Ser and, therefore, on Notch activity. In addition, other candidate targets of Sp1 affecting leg growth and morphogenesis were identified. Intriguingly, some of these Sp1 potential downstream targets are ecdysone-responding genes. These results highlight a mechanism by which btd and Sp1 control the size and shape of the leg, in part through regulation of the Notch pathway (Cordoba, 2016).
The two Sp family members in Drosophila, Sp1 and btd, display a similar spatial and temporal expression pattern during embryonic and imaginal development. Previous work suggested that btd and Sp1 have partially redundant functions during development. However, the lack of an Sp1 mutant has prevented the detailed analysis of the individual contributions of each gene. This study has generated an Sp1 null mutant that allowed elucidation unambiguously of the individual contributions of each of these genes to leg development (Cordoba, 2016).
Appendage formation starts in early embryos by the activation of Dll (through its early enhancer, Dll-304), btd and Sp1 by Wg, and their expression is repressed posteriorly by the abdominal Hox genes. Some hours later, there is a molecular switch from the early Dll enhancer (Dll-304) to the late enhancer (Dll-LT) to keep Dll expression throughout the embryo-larvae transition restricted to the cells that will form the leg. At this developmental stage, Sp1 and btd play redundant roles in Dll activation, as only the elimination of both genes suppresses Dll expression and Dll-LT activity in the leg primordia. Once Dll expression is activated in the leg disc by the combined action of Wg, Dpp and Btd/Sp1, its expression is maintained in part through an autoregulatory mechanism. At this time point, during second instar, btd and Sp1 are co-opted to control the growth of the leg. The leg phenotype of Sp1 and btd single mutants demonstrates the divergent contributions of each gene to leg growth. Removing btd from the entire leg only slightly affects the growth of proximo-medial segments, whereas loss of Sp1 causes dramatic growth defects along the entire leg. The different phenotypes of Sp1 and btd mutant legs could be a consequence of their distinct expression pattern along the leg PD axis, with btd being expressed more proximally than Sp1 (Cordoba, 2016).
The growth defects observed in Sp1 mutant legs are not due to gross defects in the localization of the different transcription factors that subdivide the leg along the PD axis, nor to defects in the expression of the EGFR ligand vn. By contrast, the results suggest a role for Sp1 in the regulation of the Notch ligand Ser. Notch pathway activation is necessary for the formation of the joints and the growth of the leg, and defects in these two processes were observed in Sp1 mutant legs. Moreover, the results demonstrate that Sp1 is necessary and sufficient for Ser expression at least in the distal domain of the leg and is therefore required for the correct activation of the Notch pathway. These results are consistent with the proposed role of Sp8 in allometric growth of the limbs in the beetle where the number of Ser-expressing rings is reduced in Sp8 knockdown animals (Cordoba, 2016).
The regulation of Ser expression is controlled by multiple CREs that direct its transcription in different developmental territories. Interestingly, although the wing and leg are morphologically different appendages and express a diverse combination of master regulators (e.g. Sp1 selects for leg identity whereas Vg determines wing fate), the same set of enhancers are accessible in both appendages, with the exception of the ones that control the expression of the master regulators themselves. These results imply that appendage-specific master regulators differentially interact with the same enhancers to generate a specific expression pattern in each appendage. The current analysis of Ser CREs identified a specific sequence that is active in the wing and in the leg. In the leg, this CRE reproduced Ser expression in the fourth tarsal segment and require the combined inputs of Sp1 and Ap. It is proposed that Sp1, in coordination with the other leg PD transcription factors, interacts with different Ser CREs to activate Ser expression in concentric rings in the leg. Meanwhile, given the same set of Ser CREs in the wing, the presence of a different combination of transcription factors regulate Ser expression in the characteristic 'wing pattern' (Cordoba, 2016).
Transcriptome analysis identified additional candidate Sp1 target genes that contribute to control the size and shape of the leg. Appendage elongation depends on the steroid hormone ecdysone through several of its effectors, such as Sb. Sb, as well as other genes related to the ecdysone pathway, were misregulated in Sp1 mutant discs. The characteristic change in cell shape that normally occurs during leg eversion does not happen correctly in these mutants. Other genes identified in this study are the Notch pathway targets dys and Poxn, which are both required for the correct development of the tarsal joints. dys and Poxn downregulation is consistent with Sp1 regulation of the Notch ligand Ser. Interestingly, the upregulation of the antenna-specific gene danr in Sp1 mutants might explain the partial transformation of the distal leg to antennal-like structures observed when two copies of Sp1 and one of btd are mutated. Interestingly, btd and Sp1 are only expressed in the antenna disc in a single ring corresponding to the second antennal segment whereas in the leg both genes are more broadly expressed. Consistent with this, misexpression of Sp1 in the antenna transforms the distal domain to leg-like structures, suggesting that different levels or expression domains of Sp1 helps distinguish between these two homologous appendages (Cordoba, 2016).
A considerable group of Hsp-related genes were downregulated in Sp1 mutant legs. Although their contribution to Drosophila leg development is unknown, downregulation of DnaJ-1, the Drosophila ortholog of the human HSP40, affects joint development and leg size, suggesting a potential role of these genes during leg morphogenesis (Cordoba, 2016).
An ancient common mechanism for the formation of outgrowths from the body wall has been suggested. Members of the Sp family are expressed and required for appendage growth in a range of species from Tribolium to mice. Consistent with the current results, knockdown of Sp8/Sp9 in the milkweed bug or the beetle generated dwarfed legs with fused segments that maintain the correct PD positional values. As is the case for Drosophila Sp1 mutants, mouse Sp8-deficient embryos develop with truncated limbs. By contrast, loss of function of Sp6 results in milder phenotypes of limb syndactyly. A progressive reduction of the dose of Sp6 and Sp8 lead to increased severity of limb phenotypes from syndactyly to amelia, suggesting that these genes play partially redundant roles. This phenotypic analysis of Sp1 and btd are consistent with this model, in which Sp1 plays the predominant role in appendage growth and the complete elimination of btd and Sp1 together abolish leg formation. Therefore, Drosophila Sp1 mutants are phenotypically equivalent to vertebrate Sp8 mutants. In vertebrate Sp8 mutant limbs, Fgf8 expression is not maintained and a functional AER fails to form. In Drosophila, FGF signaling does not seem to be involved in appendage development. Nevertheless, another receptor tyrosine kinase, EGFR, is activated at the tip of the leg and act as an organizer to regulate the PD patterning of the tarsus. The current results suggest that Sp1 acts in parallel with the EGFR pathway, as the ligand vn and EGFR target genes maintain their PD positional information in Sp1 mutant legs. However, a potential relationship between Sp1 and the EGFR pathway in later stages of leg development cannot be ruled out (Cordoba, 2016).
The results suggest that the Notch ligand Ser is a target of Sp1, and mediates in part the growth-promoting function of Sp1. Interestingly, members of the Notch pathway in vertebrates, including the Ser ortholog jagged 2 and notch 1 are expressed in the AER and regulate the size of the limb. It would be interesting to investigate further the possible relationship between Sp transcription factors and the Notch pathway in vertebrates, and test whether the functional relationship described in this work is also maintained throughout evolution (Cordoba, 2016).
In both the antenna and leg, spineless expression is shown to depend on Distal-less (Dll), a master regulator of ventral appendage formation The Dll gene is required for the development of all leg segments distal to the coxa. To test whether spineless lies downstream of Dll in limb
development, spineless expression was examined in a weak Dll loss-of-function mutant, DllPK. This allele survives to the pharate adult stage when heterozygous with Dll null alleles such as DllB, and causes the deletion of distal limb structures. spineless expression is almost completely eliminated in the tarsus, antenna, and maxillary palp of DllPK/DllB heterozygotes. Thus, spineless lies downstream of Dll in all three of these appendages. In the antenna, spineless expression is reduced in animals that carry only one dose of Dll+. This presumably accounts for the weak transformation of distal antenna to leg seen in most Dll mutant heterozygotes (Duncan, 1998).
To monitor Dll expression in relation to spineless, a monoclonal antibody was isolated against Dll protein. Dll is expressed uniformly in the central portions of the leg and antennal imaginal discs. In the early third instar, when spineless is first expressed in the leg, the outer edge of the spineless tarsal ring coincides precisely with the proximal limit of Dll expression. As the leg disc grows, the boundary of Dll expression expands beyond the spineless tarsal ring, so that a proximal zone of cells that express Dll, but not spineless, is created. In the antenna, Dll expression extends more proximally than spineless at all stages examined (Duncan, 1998).
Unlike the leg, Dll null clones can be recovered anywhere in the
wing even when they are generated early in development. The primary effect of
these clones is on the differentiation of the wing margin. The
characteristic hairs or bristles found at the margin are deleted
or rudimentary in Dll null clones located at the margin.
The effect of
these clones is found to be autonomous so that, for example, a clone
situated only on the ventral side of the margin will not affect
the adjacent margin bristles or hairs in the dorsal region. In the mature wing disc, Dll is expressed in the wing pouch
in a graded fashion centered on the wing margin and appears
to be downstream of wg in this appendage. Its function in the wing is
quite distinct from that in the leg. It is not required for
growth and axis formation in the wing, because a wing disc
in which Dll has been almost completely removed by clones
is morphologically normal. Additionally, it does not
appear to affect cell adhesion in the wing because Dll clones
generated early in development can be recovered anywhere in
the adult wing. However, these clones do have distinct
phenotypes, the most striking being an autonomous deletion
of the bristles and hairs normally found at the wing margin. The margin is characterized by the expression of a
number of genes including wg, cut and achaete: wg
and cut are expressed normally in Dll clones in the wing; this is not surprising because wg appears to be
upstream of Dll and the phenotype of wg and cut mutations in
the wing is more severe than Dll. However, ac
expression at the margin is absent in cells lacking Dll, showing that Dll is required for the normal differentiation
of the wing margin (Campbell, 1998).
The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, the specific functions of genes required for antenna development were examined. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle with Distal-less is required to establish antenna fate. This study tests which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. Antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. It is concluded that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. Phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna are reported. dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. It is therefore proposed that there are significant genetic similarities between the auditory organs of humans and flies (Dong, 2002).
As with Dll and hth loss-of-function mutants, loss of spineless (ss) also results in antenna to leg transformations. The genetic relationship among these genes was investigated. The expression of both Dll and hth appears relatively normal in the ss null antennal disc. It is therefore concluded that ss is not required for either the activation or the maintenance of Dll or hth expression in the antenna. It has been reported that Dll is required for the antennal expression of ss. To test whether Hth is also required to activate antennal ss expression, the effect of ectopic hth was examined. Ectopic Hth where Dll is expressed, for example in the wing pouch and leg disc, can activate ss expression. Conversely, loss of hth in the antenna results in loss of ss expression. Taken together, these results indicate that ss functions downstream of both Dll and hth in the antenna (Dong, 2002).
There are only a few genes expressed in either the antenna or the leg but not in both. Among these are sal and salr, which are identically expressed in a ring pattern in presumptive a2, but are detected at low levels only in leg imaginal disc cells that contribute to the body wall and not to the leg itself (Dong, 2002).
In contrast, there are other genes expressed in both antenna and leg precursors that have distinct patterns in the two appendages. Among these are dac, ato, ct and ss. The domain of dac expression in the antenna (a3) is much smaller than in the leg where it is expressed in multiple segments. The function of dac in antennal development has not been described previously (Dong, 2002).
The bHLH transcription factor encoding gene, ato, is expressed in a ring in presumptive a2, but restricted to small spots in the dorsal leg disc. ato is required for the formation of most chordotonal organs in the fly. In the antenna, ato is required for formation of Johnston's organ (JO), a complex sense organ composed of a large number of chordotonal organs that is used for sensing acoustic vibrations transmitted from the arista through a3 (Dong, 2002).
cut, which is required for differentiation of external sensory (ES) class neurons, is expressed throughout the presumptive proximal antenna (a1 and a2) and head capsule but is expressed in small clusters of cells throughout the leg disc (Dong, 2002).
ss is expressed in a circular pattern in the antenna covering the presumptive a2 through the arista. In the leg disc, ss is transiently expressed in a ring pattern in the presumptive tarsal region and subsequently becomes restricted to leg bristle precursors. Consistent with the ss expression domain, cuticular defects in ss null mutants can be found from a2 through the arista. These include the elongation of a2, loss of olfactory sensilla from a3, and transformation of a4, a5, and arista to tarsal segments (Dong, 2002).
The large differences in the expression patterns of these genes between the antenna and the leg begs the question of whether these differences are due to differential regulation by antenna-determining genes such as Dll and hth. To test whether Dll or hth are responsible for the antenna-specific expression patterns of these genes, the effects on their patterns were examined in Dll and hth loss-of-function mutants. Whether Dll and hth are regulating their antenna-specific targets via ss was tested by examining their expression in ss null antennal discs (Dong, 2002).
In contrast to the leg, in the antenna dac expression is restricted primarily to a single segment (a3). Trace levels of Dac can be detected in areas of the antennal disc immediately distal and proximal to a3. Because no antennal phenotypes have been reported for loss-of-function dac mutants, it is unclear whether dac plays a role in patterning this appendage. In transheterozygous dac null mutants, a fusion of the a5 segment with the arista occurs, accompanied by a reduction in the width of the a5 segment. This fusion phenotype is similar to what is observed in dac hypomorphic and null legs. However, unlike the leg phenotype, no obvious reductions in length or loss of segments is found in the dac mutant antenna. In addition, this antennal phenotype is observed in dac null animals but not in strong hypomorphic combinations such as daclacZ/dac4. Therefore, high levels of Dac are probably not necessary for dac function in the antenna (Dong, 2002).
If Dac levels are elevated in the antenna, expression of Dll and hth is repressed and medial leg structures are induced. Therefore if Dac levels are too high, antenna development is compromised. Because bab mutants exhibit phenotypes similar to those of dac, and dac regulates bab expression in the antenna, it is likely that antennal dac function is mediated via its regulation of bab (Dong, 2002).
The antennal dac expression domain expands in Dll hypomorphs and in hth null clones. This expansion of dac expression in Dll and hth mutant antennae resembles the leg pattern of dac expression. In contrast, in the ss null antenna, there appears to be neither expansion nor reduction of dac expression. The only detectable difference in the ss null antennal disc is overgrowth in the central (distal) area such that the ring of dac expression has a larger radius. This correlates with the transformation phenotype of the ss null arista into a tarsus, which is a larger structure. Since the expression of dac relative to other genes appears normal in ss null antennae, ss is not thought to regulate dac (Dong, 2002).
The expression of ato is required for the formation of the JO. The JO is a structure unique to the antenna and is required to sense sound vibrations transmitted from the arista. ato function is generally associated with neuronal differentiation, so it is interesting that cuticular defects are associated with ato null antennae. It may be that formation of the JO is required for the normal morphology of the a2/a3 joint. The circular outline of the a2/a3 joint is lost in hth and Dll loss-of-function mutants, but is present in ss null mutants. Consistent with this, the antennal expression of ato is lost in hth null clones and in Dll hypomorphs, but persists in the ss null antenna discs. Thus although ss null mutants exhibit cuticular defects in a2 and a3, the a2/a3 joint to which the JO is attached is present. It is noted that the Dll hypomorphic combination used, DllGAL4/Dll3, does not lead to loss of a2. Thus the absence of ato expression in these antennae is not due to death of the cells that would normally express it (Dong, 2002).
sal and salr have similar sequences and are identically expressed in the antennal imaginal disc in presumptive a2. However, functions for sal and salr in the antenna have not yet been described. To investigate whether sal and/or salr are required for normal antenna development, clones null either for sal alone or for both sal and salr in the adult head were examined. Clones null for only sal in the antenna have no obvious cuticular phenotypes. However Df(2L)32FP-5 clones, which are null for both sal and salr, exhibit cuticular defects in the antenna. This supports the view that sal and salr have some redundant functions. The areas affected in the mutants are correlated with their expression domains in the antennal disc (Dong, 2002).
a2 normally forms a cup, in which a3 sits and must rotate along the PD axis, to transmit sound vibrations from the arista. An overall reduction in a2 is observed in salFCK–25/Df(2L)32FP-5 transheterozygous null antennae. In addition, a2 appears to be fused to a3 and a portion of the stalk that connects a3 to a2 is exposed. The circular outline of the a2/a3 joint, to which the chordotonal organs of the JO attach, is defective in Df(2L)32FP-5 clones and lost in salFCK–25/Df(2L)32FP-5 mutant antennae. Furthermore, a3 is unable to rotate in a2. The same antenna phenotypes are observed in salFCK–25 homozygous flies. However, these phenotypes are not observed in sal null clones generated using a sal16 FRT40A chromosome or in salFCK–25/sal16 transheterozygous antennae, that do not express sal but do express salr in the antenna. Together, the loss of the a2/a3 joint and the loss of the freedom of rotation of a3 in a2 indicate that sal/salr null antennae are defective in hearing and implicate both sal and salr in normal development of the auditory organ (Dong, 2002).
Since ato is expressed within a subset of the sal/salr domain and is activated later than sal and salr in the antenna, tests were performed to see whether Dll and hth activate ato via sal/salr. No detectable reduction of ato expression is found in a2 either in Df(2L)32FP-5 clones or in salFCK–25/Df(2L)32FP-5 transheterozygous animals. This allelic combination lacks detectable sal and salr expression in the antenna, but retains sal and salr expression in the eye. The normal expression of ato in the antennae of these mutants suggests that the activation of ato expression by Dll and hth is independent of sal/salr. Antennal sal/salr expression is also unaffected in ato null imaginal discs. Therefore, sal/salr and ato are required in parallel for development of antennae that are functional in audition (Dong, 2002).
Dll and hth are required for the expression of sal in the antenna. sal expression does not appear to be affected in ss null antenna. The fact that Ss is not required for the expression of either ato or sal in a2 is consistent with the observation that the a2/a3 joint is still present in the ss null antenna (Dong, 2002).
Expression of the homeodomain transcription factor encoded by ct almost completely fills the hth expression domain of the third instar antennal disc. In contrast, the ct and hth expression patterns in the leg disc are distinct from one another. This makes ct a strong antenna-specific candidate target for Hth. The antennal expression of ct is lost in hth null clones indicating that ct is indeed downstream of hth. To test whether the a2 expression of ct also requires Dll, ct expression was examined in Dll mutants. ct expression is not reduced in Dll null clones or in Dll hypomorphs. Therefore, although Dll and hth are both required for antennal fate, cut is an antenna-specific target of Hth activation that is independent of Dll. As with other antenna-specific targets of Dll and Hth, ct expression is also not lost in ss null antenna (Dong, 2002).
This study serves to initiate an understanding of the different roles that these homeotic genes are playing in antenna specification. During imaginal disc development, the expression of Dll and ss is found from a2, a3, a4, a5 and arista. Expression of hth is dynamic and retracts from the distal-most segments by late third instar, but hth is expressed and cell-autonomously required throughout the antenna from a1 through to the arista (Dong, 2002).
The Dll mutant phenotypes indicate that Dll is required both for the distal limb development and for antenna fate. Dll hypomorphs exhibit distal limb deletions as well as antenna to leg transformation. The transformation phenotypes of hypomorphic Dll antennae can be observed from a2 through to the arista. In these mutants, hth expression is not lost or detectably reduced. Thus medial leg structures can develop in the presence of Hth. This suggests that although loss of hth expression from the distal and medial leg, via Antennapedia-mediated repression, occurs during normal leg development, loss of Hth is not essential for leg differentiation. It also suggests that the requirement for Antennapedia in normal leg development is not only to regulate hth (Dong, 2002).
Since ss is not required to activate antenna-specific expression of genes such as sal/salr and ato that are involved in antenna differentiation, the question arises as to what ss does do in the antenna. ss represses tarsus and tarsal claw organ formation in the antenna. Since loss of ss also leads to loss of olfactory sensillae on a3, ss probably potentiates the formation of these sensillae, either cooperating with or mediating Dll and hth activities in a3. Similarly, since ectopic expression of ss elsewhere in the body can lead to the formation of ectopic aristae, ss may also cooperate with or mediate Dll and hth activities in arista differentiation (Dong, 2002).
sal and salr, like ato, are required for normal auditory functions. Since both Dll and hth are required for the antennal expression of ato and sal, Dll and hth mutant antennae are also hearing defective. In contrast, ss null antennae exhibit normal expression of both ato and sal and normal morphology of the a2/a3 joint, leading to the idea that ss mutants are likely to be functional in audition (Dong, 2002).
Homeotic genes, Dll and hth, regulate multiple targets during antennal development. These targets function in specifying antenna structures and/or in repressing leg development. For example, the ss mutant phenotype suggests that it represses leg tarsal differentiation. But ss is also required for the formation of olfactory sensory sensilla normally found in a3. Although Dll and hth repress distal leg development via activation of ss, their repression of medial leg development appears to be, at least in part, independent of ss. Instead, this is achieved via their regulation of the medial leg gene, dac, to a narrower domain of expression with lower levels in the antenna as compared to the leg. sal/salr and ato are required for proper differentiation of a2. However, no transformation phenotypes are associated with the sal/salr and ato null antenna. This indicates that while sal/salr and ato are required to make particular antenna-specific structures, they do not appear to repress leg fates. Therefore homeotic genes such as Dll and hth repress the elaboration of other tissue fates in addition to activating genes required for the differentiation of particular tissues (Dong, 2002).
In third instar imaginal discs, coexpression of Dll and Hth activates sal/salr and ato in a2 where they, in turn, are needed for JO development. The expression of ato is required for the formation of the JO and the a2/a3 joint to which it is attached. Although sal and salr are not required for the expression of ato, the a2/a3 joint is lost in the sal/salr null antenna. It is expected that this leads to improper formation of the JO, although it is also possible that defects in a2/a3 joint formation preclude JO differentiation. In addition, because sal is not lost in ato null antennae, it is concluded that sal/salr and ato are required in Drosophila parallel for proper formation of the JO. Furthermore, in the sal/salr null antenna, a3 cannot freely rotate within a2. This rotation is necessary for transmission of sound vibrations from the arista to the JO. Taken together, these findings implicate sal/salr in Drosophila audition. Interestingly, mutations associated with the human homolog of sal, SALL1 cause the human autosomal dominant developmental disorder, Townes-Brocks Syndrome (TBS). Auditory defects are also associated with the human genetic disorder, Split Hand/Split Foot Malformation (SHFM), and the SHFM1 locus is linked to the Dll homologs, DLX5 and DLX6. The sensorineural hearing defects associated with the Distal-less and spalt genes in both Drosophila and Homo sapiens, in conjunction with a recent finding that atonal functions in mouse as well as fly audition, leads to the proposal that insect and vertebrate hearing share a common evolutionary origin. Further developmental genetic dissection of the Drosophila auditory system should therefore provide additional insights into human ear development and suggest that Drosophila could provide a useful model system for studying both TBS and SHFM (Dong, 2002).
Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. The distal antenna (dan) and distal antenna-related (danr) genes encode novel 'pipsqueak' motif nuclear proteins that probably function as DNA binding proteins serving as sequence-specific transcription factors but may serve instead as more general
chromatin modification factors. dan and danr are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore it is suggested that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less The overlap between Hth and Dll has been proposed to define antennal
identity, because co-expression of the two proteins in ectopic locations can
induce formation of ectopic arista structures in other discs. To ask
whether Hth and Dll have a role in defining the non-overlapping expression
domains of Cut and Dan/Danr, clones of cells were examined lacking hth
or Dll activity in the antenna. Dan expression is lost in cells
mutant for hthc1 in the region where the two expression
domains overlap. This
suggests that Hth activity is required for Dan expression. Likewise, clones of
cells lacking Dll activity have lost Dan expression in the distal region
of the disc. Many Dll mutant clones were found adjacent to the edge of the
Dan domain,
suggesting that loss of Dan may cause these clones to sort out proximally.
Thus both Dll and Hth are required for Dan expression (Emerald, 2003).
Ectopic expression of Hth in the leg disc under dppGal4
control, induces Dan expression in distal, Dll-expressing cells. It is known that Hth-expressing cells sort out from the distal region of the disc.
This is also visible in GFP labeled cells in this study. Nonetheless,
dppGal4 directed expression of Hth induces Dan expression
in distal cells. This raises the possibility of a non-autonomous effect of Hth
expression leading to sustained Dan expression. Ectopic expression of Dll in
the leg disc under dppGal4 control, induces Dan expression
in proximal, Hth-expressing cells. In this case, ectopic Dan was limited to cells expressing the Gal4 driver (Emerald, 2003).
These observations indicate that the regulatory relationship between Hth,
Dll and Dan (Danr) is complex. Dll and Hth are each required for Dan
expression. However, it is clear that Dan is not expressed in every cell in
which Hth and Dll are co-expressed in the antenna. All Dan-expressing distal
antenna cells express Dll but not all express Hth. These observations point to a
non-autonomous effect of Hth on Dan expression, which may explain how Hth can
be required for sustained expression of Dan in distal cells where Hth is not
expressed (Emerald, 2003).
The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc (see Dan and Danr). Hernandez and Fernandez are the names of twin brothers in Tintin comic-books. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).
To test whether hern and fer are sufficient to induce eye or antennal development, they were expressed ectopically using the GAL4/UAS system. When either the hern or the fer genes are misexpressed in the leg discs with dpp-GAL4 or Dll-GAL4 (EM212) drivers, distal legs are transformed to aristae. These transformations are accompanied by the ectopic expression of hth, sal, and ss, three genes expressed in the antennal primordium but not in the distal region of mature wild-type leg disc. Clones expressing either the hern or the fer genes in the leg or wing disc have smooth borders and frequently activate the sal and hth genes cell-autonomously. In dpp-GAL4/UAS-fer or ptc-GAL4/UAS-hern leg (or wing) discs, the expression of ss is also activated. Curiously, although ss is downstream of hth in the antenna and leg, ectopic ss in the leg disc can also activate hth in a few cells (Suzanne, 2003).
The hth or ss genes, together with Dll, are sufficient to develop ectopic distal antennae when expressed in different regions of the adult. The hern or fer genes are also able to elicit this transformation in the leg and they activate hth and ss. Conversely, when high levels of the Hth or Ss products are induced in the leg discs, ectopic expression of the hern and fer genes is found. To study the interactions between these genes in normal development, the relationship between Dll, hth, ss, and hern/fer in the antennal primordium was examined. A reduction of Hth activity using a dominant negative form of hth (UAS-EN-HTH1-430) results in a decreased activity of the MD634 and AC116 GAL4 lines, which reveal hern and fer expression, respectively. Similarly, in antennal discs of a Dll strong hypomorph or a ss null mutation, the expression of hern and fer disappears. These results suggests that hth, Dll, and ss are required to maintain hern and fer expression in the antenna. By contrast, high levels of hern or fer may reduce hth expression. In dpp-GAL4/UAS-fer or dpp-GAL4/UAS-hern larvae, the expression of hth (and sal) in the third antennal segment is eliminated or strongly reduced dorsally (where levels of hern and fer are high) and does not change or is ectopically activated ventrally (where levels of hern and fer are low). Similarly, fer-expressing clones are able to downregulate hth expression in the antennal primordium. These results suggest that levels of hern and fer expression may be important for a normal antennal development (Suzanne, 2003).
The differentiation of legs or antennae depends on the activity of the hth and Antp genes. The ss gene, however, is also able to transform distal leg (and also maxillary palp and rostral membrane) into distal antenna, and the absence of ss, like that of hth, transforms antenna into leg. Although ss seems to be downstream of Dll and hth in antenna specification, ectopic ss can activate hth in some cells of the leg disc. Similarly, misexpression of ss in the rostral membrane induces Dll expression. It seems, therefore, that ss can trigger an antennal genetic program when misexpressed in certain places (Suzanne, 2003).
The fer and hern genes are both required and sufficient to make part of the distal antenna. Four different genes, hth, ss, hern, and fer, are able to form distal antenna, together with Dll, when ectopically expressed. Their mutual regulation seems to differ when misexpressed in the leg disc or when normally expressed in the antennal primordium. In the leg disc, hern or fer activates hth and ss and, reciprocally, hth and ss induce hern and fer expression. Moreover, even ss can promote hth transcription, although just in a few cells. Taken together, these results suggest that the four genes can form distal antenna by activating each other's transcription when ectopically expressed (Suzanne, 2003).
In the third antennal segment, Dll, hth, and ss are required to activate hern/fer expression. Since ss is downstream of Dll and hth in the antenna, the activation of hern/fer by Dll and hth could be mediated by ss. It is noted, however, that the levels of hern and fer may modulate hth expression. Moderately increased levels of fer can activate hth in dpp-GAL4/UAS-fer discs but, when the levels of hern or fer in the antenna are highly increased, the transcription of hth is prevented. These results suggest that the total amount of hern and fer expression may be regulated in the antennal primordium. Accordingly, in clones mutant for danr (hern), the expression of dan (fer) is upregulated. Also supporting the conclusion that levels of hern and fer have to be regulated, it was found that, in ey-GAL4/UAS-hern or ey-GAL4/UAS-fer flies, where levels of either hern or fer are highly increased in the eye–antennal disc, both the eye and the antenna disappear (Suzanne, 2003).
The hern and fer genes can form ectopic aristae and eye tissue, but only in a limited number of regions of the adult cuticle. This is similar to what happens with other genes making ectopic antennae (hth, ss) or eye (eye-specification genes). This is due to the particular developmental context of the region where the genes are ectopically activated (Suzanne, 2003).
DNA replication-related element factor (DREF) is required for expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Over-expression of DREF in the eye imaginal disc induces ectopic DNA synthesis, apoptosis and inhibition of photoreceptor cell specification, and results in rough eye phenotype in adults. In the present study, half dose reduction of the Distal-less (Dll) gene enhanced the DREF-induced rough eye phenotype, suggesting that Dll negatively regulates DREF activity in eye imaginal disc cells. Biochemical analyses revealed the N-terminal (30aa to 124aa) and C-terminal (190aa to 327aa) regions of Dll interact with the DNA binding domain (16aa to 125aa) of DREF, although it is not clear yet whether the interaction is direct or indirect. Electrophoretic mobility shift assays showed that Dll thereby inhibits DNA binding. The repression of this DREF-function by a homeodomain protein like Dll may contribute to the differentiation-coupled repression of cell proliferation during development (Hayashi, 2006).
The transformation of antenna to leg is a classical model for understanding segmental fate decisions in Drosophila. The spineless (ss) gene encodes a bHLH-PAS transcription factor that plays a key role in specifying the identity of distal antennal segments. This report identifies the antennal disc enhancer of ss and then uses enhancer-lacZ reporters to work out how ss antennal expression is regulated. The antennal determinants Distal-less (Dll) and homothorax (hth) are key activators of the antennal enhancer. Dll is required continuously and, when present at elevated levels, can activate the enhancer in regions devoid of hth expression. In contrast, homothorax (hth) is required only transiently both for activation of the enhancer and for specification of the aristal portion of the antenna. The antennal enhancer is repressed by cut, which determines its proximal limit of expression, and by ectopic Antennapedia (Antp). Repression by Antp is not mediated by hth, suggesting that ss may be a direct target of Antp. ss+ is not a purely passive target of its regulators: ss+ partially represses hth in the third antennal segment and lies upstream of Dll in the development of the maxillary palp primordia (Emmons, 2007; full text of article).
This study used lacZ reporters to identify the enhancers responsible for most aspects of ss expression during embryonic and imaginal development. Antennal expression is driven by two large fragments from the ss 5' region, B6.9 and EX8.2. Both of these fragments drive expression in the antennal segment of the embryo and in the distal portion of the pupal antenna. B6.9 is also expressed in the antennal disc through most or all larval development. Dissection of B6.9 allowed localization of the larval antennal enhancer to a fragment of 522 bp. The B6.9 and 522 reporters were used as a proxy for ss expression in experiments to determine the effects of potential upstream regulators of ss. This strategy has its strengths and weaknesses, but has been made necessary by an inability to generate antisera against Ss. A major strength of the approach is that it was possible to assess the effects of regulators on individual enhancers. It is likely that monitoring endogenous ss expression would give results that are less clear cut since both the antennal and tarsal enhancers of ss are active within the antenna. A potential weakness is that the reporters may not faithfully reproduce the normal expression of ss. However, as far as is possible to tell, the antennal reporters reproduce ss expression very well. The expression of B6.9 and EX8.2 in the embryonic antennal segment and the pupal antenna corresponds very closely to that of endogenous ss. Expression of B6.9 and 522 in the larval antennal disc appears very similar or identical to that of ss+, and the transient requirement for hth+ in the activation of these reporters corresponds well to the transient requirement for hth+ in aristal specification. The tarsal enhancer P732 likely also reproduces the spatial pattern of ss+ expression as its tarsal expression domain corresponds well to the region deleted in ss− mutants (Emmons, 2007).
The results of this dissection of the B6.9 fragment were surprising. Removal of the left-hand 2 kb of B6.9 to produce S4.9 resulted in the loss of antennal specificity; S4.9 reporters are expressed in both antennal and leg discs. The E2.0 subfragment of S4.9 shows a similar expression pattern, and expression of this fragment in both leg and antennal discs is independent of Hth, but requires Dll continuously. On further subdivision of the E2.0 fragment, it was found that antennal and leg expression are separable; the 522 fragment is largely specific for the antenna, whereas the 531 fragment drives expression primarily in leg discs. To summarize, antennal specificity is present in B6.9, lost in S4.9 and E2.0 and regained in 522. How can sense be made of this? The region deleted from B6.9 to produce S4.9 clearly plays an important role in enforcing antennal specificity. Since this region contains a PRE, one might suspect that it functions in larval stages to maintain repression of the enhancer outside of the antennal segment. However, that the E2.0 fragment has lost the requirement for Hth in both the antenna and leg (S4.9 has not been tested) suggests that the PRE-containing region might function in both locations. One possibility is that this region represses the enhancer in both antennal and leg discs. In the antenna, this repression can be overcome by the combined action of Hth and Dll, while in the leg Dll alone is not sufficient for activation. When the PRE-containing region is deleted, repression is absent or reduced, so that Dll can activate the enhancer without assistance from Hth, and expression is seen in both antennal and leg discs. Why then is antennal specificity restored in the 522 subfragment? Perhaps this fragment is lacking a subset of Dll interaction sites so that it can no longer be activated by Dll alone, but requires combined activation by Hth and Dll. Although this model is consistent with many of the results, it does not provide a ready explanation for the leg specificity of the 531 fragment (Emmons, 2007).
In addition to activation by combined Hth and Dll, the ss antennal disc enhancer is repressed by Cut and by ectopic Antp. Each of these regulators will be discussed separately. It was found that hth+ is required only transiently for activation of the B6.9 reporter. hth− clones induced in the embryo or first instar lose expression of B6.9 autonomously in both A3 and the aristal primordia. However, some time in the second of early third instar. Regulatory instar expression of B6.9 becomes independent of hth. Consistent with this transient requirement, it is shown that hth+ is required only early in larval development for specification of the arista. hth− clones induced in the first and second instars show a transformation of the entire antenna to a leg-like appendage. However, clones induced after this time show normal aristal development. These temporal requirements are reflected in the expression pattern of hth: hth is expressed throughout the antennal primordium early in development, but in the second or early third instar is repressed in the central domain, which will produce the arista (Emmons, 2007).
The stable activation of B6.9 by Hth suggests that this fragment contains a 'cellular memory module'. The presence of a PRE within B6.9 is consistent with this idea. The ss locus binds Polycomb protein in salivary gland chromosomes and was recently shown to contain PREs by chromatin immunoprecipitation. In the latter work, ss PREs were localized to within the E1.6 subfragment of B6.9 as well as the EX8.2 fragment, both of which showed pairing dependent suppression in this work. PREs are generally thought of as functioning to stably repress genes. However, PREs can also be associated with activating elements to form memory modules that mediate stable activation. It seems likely that B6.9 contains such a module that responds to Hth. Like a memory module from the hedgehog gene, activity of the ss module is set sometime around the second instar. Surprisingly, it was found that activation of the 522 reporter by Hth can also be persistent, although not as stable as for B6.9. The 522 fragment does not appear to contain a PRE, suggesting that Hth may directly recruit factors to the 522 element that cause semi-stable transcriptional activation (Emmons, 2007).
ss is not a completely passive target of hth; ss partially represses hth in antennal discs, which causes hth to be expressed at a lower level in A3 than in A2. This repression appears to be important for normal development as ectopic expression of Hth can delete A3. Moreover, clones ectopically expressing Hth are largely blocked from entering A3 from the proximal (A2) side, suggesting that the different levels of Hth present in A2 and A3 cause a difference in cell affinities between these segments. Hth-expressing clones are similarly restricted to the two most proximal segments in leg discs, although here there is no endogenous expression of hth more distally (Emmons, 2007).
In contrast to hth, Dll is required continuously for expression of both B6.9 and 522 as Dll− clones induced even very late in development lose expression of these reporters. This continuous requirement for Dll indicates that stable activation of the B6.9 memory module by Hth does not by itself commit the reporter to expression; rather, activation by Hth appears to render B6.9 open to interaction with Dll and perhaps other positive factors (Emmons, 2007).
Three lines of evidence suggest that Dll is the primary activator of the ss antennal enhancer. (1) It was found that expression of B6.9 and 522 is sensitive to the dosage of Dll+. Expression of both reporters is reduced in animals carrying only one dose of Dll+, and for 522, expression is enhanced in clones having extra doses of Dll+. This dose sensitivity suggests that ss is a direct target of Dll. (2) It was found that expression of both reporters is often induced within clones expressing ectopic Dll, even in the apparent absence of Hth expression. Such activation is seen in clones in the distal leg, wing and elsewhere. (3) It was found that the embryonic antennal enhancer carried by B6.9 is absolutely dependent upon Dll+, but independent of hth. Taken together, these observations suggest that Dll is a primary activator of the ss antennal enhancers. Hth may provide antennal specificity by boosting the level of activation by Dll in the antennal disc (Emmons, 2007).
Surprisingly, it was found that the regulatory relationship between ss and Dll is reversed in the maxillary palp. Here, ss is expressed prior to Dll and is required for the normal initiation of Dll expression. Although some Dll expression ultimately takes place in the palp primordium in ss− animals, this expression is weak and occurs in only a few cells. It has not been worked out how ss is activated in the palp. However, it seems likely that dpp plays a role as the 531 subfragment of B6.9 drives expression in a stripe in the region of the palp that roughly coincides with a stripe of dpp expression. The positioning of ss upstream of Dll in the palp may explain why the region ventral to the antenna is so sensitive to ectopic expression of Ss. Strong activation of Dll here by ectopic Ss combined with endogenous expression of hth might be expected to cause frequent induction of ectopic antennae, as is observed. Since ss is normally expressed in the palp, why should earlier ectopic Ss cause the palp primordium to develop as antenna? It seems likely that timing is key, but level of Ss expression could also be important (Emmons, 2007).
The reciprocal regulatory roles of ss and Dll in the antenna and palp suggest a particularly close relationship between these genes. This relationship is reinforced by the finding that ss is required for the development of bracts in the femur, as is Dll (Emmons, 2007).
The finding that Dll and Hth are both activators of the ss antennal reporters is consistent with the proposal that antennal identity is defined by the combined activity of these regulators. However, the results indicate that this model is an oversimplification. Examination of clones expressing Dll, Hth, or both proteins together revealed little correlation between activation of the B6.9 and 522 antennal reporters and combined expression of Dll and Hth. Strikingly, Dll-expressing clones often activate the reporters ectopically without any apparent concomitant expression of Hth, and clones expressing both proteins usually do not activate the reporters. These experiments also reveal strong context dependence. Examples include the leg, where Dll-expressing clones can activate the reporters distally, but not proximally (where endogenous hth expression occurs) and the wing disc, where clones expressing Dll or both Dll and Hth activate the reporters in the wing pouch, but not at all in the notum. The level of expression of both proteins also appears to be key as high levels of Dll can activate the reporters in the leg in the absence of Hth and elevated levels of Hth can repress expression in the normal antennal domain. Previous results have shown that antennal structures can be induced by ectopic expression of Dll in the wing hinge region or proximal leg (which express hth endogenously) or by combined expression of Dll and Hth elsewhere. While this is true, the results indicate highly variable effects in such ectopic expression experiments and fail to detect the strongly synergistic activation of antennal identity by combined Hth and Dll implied by the model. The results indicate that Dll is the primary activator of the ss antennal reporters, that Hth serves to promote this activity and that activation by Dll and Hth is highly context-dependent (Emmons, 2007).
Consistent with direct control of the antennal reporters by Dll and Hth, two highly conserved regions within the 522 fragment contain apparent binding sites for Dll, Hth, and the Hth dimerization partner Extradenticle. The functional importance of these binding sites is currently being tested (Emmons, 2007).
This study has show that the proximal boundary of B6.9 and 522 expression is defined by repression by cut. This repression likely explains why ectopic Cut causes a transformation of arista to tarsus. cut has been shown to define the proximal expression limit of distal antenna (dan) and distal antenna related (danr); since ss lies upstream of these genes , it seems very likely that their regulation by cut is indirect. The mechanism of action of Cut is not well understood, since only one direct target has been characterized in Drosophila (Emmons, 2007).
Ectopic expression of Antp in the antenna represses the B6.9 and 522 reporters. This finding was expected, since it is well known that expression of Antp or other Hox genes in the antenna causes a transformation to leg. The conventional view is that this transformation results from the repression of hth by ectopic Hox proteins. Repression of hth early in development would be expected to lead secondarily to loss of ss expression and loss of distal antennal identity. However, it was found that clones expressing Antp repress the B6.9 and 522 reporters even when these clones are induced very late in development, long after the requirement for activation by hth has passed. Late repression of the antennal reporters by Antp must therefore occur independently of hth and could be direct. One possibility, currently being tested, is that Antp might compete with Dll for binding to the 522 enhancer. Late repression of the ss antennal enhancer by Antp is consistent with the effects of Antp-expressing clones on antennal identity: such clones induced in the mid to late third instar cause transformations of distal antenna to leg (Emmons, 2007).
Clones induced late that ectopically express Antp in a sustained fashion were examined. In contrast, previous work studied the effects of pulses of Antp expression induced by one-hour heat shocks in a heat shock/Antp line. It had been found that transformations of arista to tarsus were induced by such pulses only when they were administered at the end of the second instar. Why do pulses of Antp at this time cause a stable, heritable transformation of the distal antenna? The current results suggest an explanation. The period sensitive to Antp pulses coincides roughly with when the ss antennal enhancer becomes independent of hth. This correlation suggests that pulses of Antp in the second instar cause heritable transformations by interfering with the stable activation of ss by Hth. Recently, it has been reported that ectopic Antp does not repress hth in the antenna early in larval development. This observation suggests that Antp might act directly on the ss antennal enhancer to prevent its stable activation by Hth (Emmons, 2007).
The regulation of ss by ectopic Antp suggests that Antp may normally play a significant role in repressing ss antennal enhancer activity in the legs. Although this idea has not been tested directly, it seems unlikely that Antp is primarily responsible for keeping the ss antennal enhancers inactive in the leg. Antp null clones do cause activation of the ss target gene dan in leg discs, implying ectopic activation of ss. However, this activation occurs only proximally, with the distal leg appearing to develop independently of Antp. Expression of Antp in the proximal leg may account for why Dll-expressing clones fail to activate B6.9 or 522 in this location. Ectopic activation of the ss antennal enhancers in the leg primordia of the embryo is not seen in an Antp null mutant (Emmons, 2007).
These studies suggest that antennal structures are specified in a combinatorial fashion by Hth, Dll, Ss and probably other factors. In A3, all three proteins are required for normal antennal identity. In ss− antennae, hth continues to be expressed in A3 (although at elevated levels), as does Dll. Despite this continued expression of hth and Dll, A3 develops without antennal characteristics and produces only naked cuticle. Thus, Hth and Dll are unable to specify A3 characters in the absence of Ss. Conversely, assuming that ss is stably activated in the antenna by Hth, as is B6.9, then hth− clones induced late would show persistent expression of both ss and Dll in A3. Such clones are transformed to leg, implying that Ss and Dll have no ability to direct A3 identity in the absence of Hth. Taken together, these observations suggest that Hth, Dll and Ss must act together to specify A3 identity. This requirement for combined action accounts for why ectopic expression of Ss does not induce A3 tissue in the medial leg, since hth is not normally expressed here. The view of combinatorial control suggests that many A3-specific target enhancers might be identifiable in genome searches as regions that contain clustered binding sites for Hth, Dll and Ss; tests of this prediction will be presented elsewhere (Emmons, 2007).
In contrast to A3, the aristal primordium appears to be specified by ss and Dll acting together in the absence of hth expression. hth is expressed in the aristal region early in development, where it functions to establish ss expression, but it is soon repressed here. Therefore, for most of development, the arista is specified by Ss and Dll acting without input from Hth. Consistent with this picture, the arista adopts leg identity in ss null mutants, and ectopic expression of ss causes the distal tip of the leg to develop as arista (Emmons, 2007).
In ss− mutants, the distal antenna is terminated by a single tarsal segment (the fifth). In contrast, in ss mutants that lack only antennal enhancer activity (e.g. the breakpoint mutations ssD114.3 and ssD114.7, the distal antenna develops with a near complete set of tarsal segments. This difference likely reflects the activity of the tarsal enhancer in the antenna. In support of this view, the ss tarsal enhancer drives expression in the segmented base of the arista, a region known as the basal cylinder. This region transforms to tarsal segments 2-4 in Antp-induced transformations of antenna to leg. However, the question arises as to why normal antennal expression of ss causes the proximal arista to develop as basal cylinder, whereas ss expression driven by the tarsal enhancer alone causes this same region to develop as tarsal segments. Likely, the key difference is that expression driven by the tarsal enhancer is transient, whereas expression driven by the antennal enhancer is sustained. Perhaps transient expression of ss allows growth and subsegmentation to produce a full set of tarsal segments, whereas sustained expression inhibits growth, producing the basal cylinder. Consistent with this idea, sustained expression of ss driven by the GAL4 method can cause deletion of tarsi in the legs. The levels of expression driven by the tarsal and antennal enhancers may also be important as flies having only one dose of ss show a partial transformation of the basal cylinder to tarsus. The ss tarsal enhancer drives weak expression in A3 as well as in the basal cylinder, likely accounting for the presence of some specialization of A3 in ss mutants lacking the antennal enhancers (Emmons, 2007).
The view that antennal identity is specified by the combined action of Hth, Dll and Ss contradicts the now prevalent view that antennal identity is determined solely by hth. The major evidence supporting the latter view is that early hth− clones transform the entire antenna to leg, and ectopic expression of Hth can induce ectopic antennal structures in the anal plates. Moreover, Dll shows little antennal specificity, being expressed in the distal portions of all of the ventral appendages, and ss expression in the antenna is dependent upon hth+. Should hth be viewed as the antennal 'selector' gene? hth does not seem to be a selector in the same sense as the Hox genes; it is expressed very broadly in the embryo and in other imaginal discs and plays no role in activating ss in the antennal segment of the embryo. Moreover, the ability of ectopic Hth to induce antennal structures is very limited: transformations of anal plate to distal antenna have been reported following ectopic expression of Hth or Meis1, a mammalian homolog. However, others have been unable to reproduce this effect by ectopic expression of Hth, matching the results of this study. That anal plates are susceptible to transformation at all is likely due to the fact that Dll and ss are coexpressed here in normal development. A further dissimilarity is that hth acts only as an establishment regulator of ss in the antennal disc, unlike the continuous requirements usually seen for the Hox genes. Ultimately, assessment of the importance of hth will depend on whether its function in the antenna is conserved. The expression pattern of hth in the antenna does appear to be conserved in the milkweed bug Oncopeltus. However, localization of nuclear Exd (a proxy for Hth expression) indicates that Hth is not differentially expressed in the antenna and leg of the cricket. Expression of hth in the crustacean Porcellio also appears to be identical in the second antenna and the legs. Characterization of hth, Dll and ss expression and function in additional arthropods will be required to assess properly the importance of these genes in antennal specification (Emmons, 2007).
It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, a 62bp enhancer was isolated from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. It is concluded that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring (Duncan, 2010).
This report examines the regulation of an enhancer from the antennal gene ss that drives expression specifically in the third antennal segment (A3). The work provides the first look at how the homeodomain proteins Dll, Hth, and Exd function in the antenna to activate antennal target genes. These proteins form a trimeric Dll/Hth/Exd complex on the enhancer, suggesting that Dll acts much like a Hox protein in antennal specification. This work also reveals how the Hox protein Antp functions in the leg to repress antennal development. The conventional view has been that the primary function of Antp is to repress hth in the distal leg, which then prevents the activation of all downstream antennal genes. However, this study found that Antp represses the ss A3 enhancer directly. This repression is essential within a proximal ring in the leg that coexpresses the antennal gene activators Dll, Hth, and Exd. Antp competes with Dll for binding to the enhancer, and this competition is part of a molecular switch that allows the ss A3 element to be activated in the antenna, but represses its activation in the leg. The results suggest that repression of antenna-specific genes in the proximal ring is the sole function of Antp in the leg imaginal disc (Duncan, 2010).
At 62 bp, the ss A3 enhancer (called D4) is one of the smallest enhancers to be identified in Drosophila, and yet it is quite strong; only a single copy is required to drive robust expression of lacZ reporters. The enhancer is also very specific, driving expression in A3 and nowhere else in imaginal discs. It has been proposed that antennal identity in Drosophila is determined by the combined action of Dll, Hth, and Exd. Consistent with this proposal, all three of these factors were found to be required for D4 expression. Although these activators are coexpressed in both A2 and A3, D4/lacZ expression is restricted to A3 by Cut, which represses the enhancer in A2. Like ss itself, D4/lacZ is also repressed by ectopically expressed Antp (Duncan, 2010).
A previous report (Emmons, 2007) showed that the antennal expression pattern of ss is reproduced by lacZ reporters containing a 522 bp fragment from the ss 5' region. This fragment contains five conserved (41%-90% identity) domains, each of which was deleted and tested for effect on expression in vivo. Expression in the arista and the third antennal segment (A3) prove to be under separate control; expression in the arista requires domains 1, 3 and 5, whereas expression in A3 is lost only when domain 4 is deleted. Moreover, reporters containing domain 4 alone show expression in A3 and nowhere else in imaginal discs. Thus, domain 4 is both necessary and sufficient for A3-specific expression. Domain 4 (D4) is 62 bp in length and is highly conserved, being invariant at 50/62 base pairs in the 12 Drosophila species sequenced (Duncan, 2010).
Surprisingly, Dll, Hth, Exd, Cut, and Antp all act directly upon D4. The activators Hth and Exd bind with strong cooperativity to directly adjacent sites. Their joint binding site matches the optimum site for in vitro binding of the mammalian homologs of Hth and Exd (Meis and Prep), consistent with the robust activity of the enhancer in vivo. Mutation of either of these sites abolishes activity of the enhancer. The coactivator Dll binds three sites in D4; one of these sites (Dlla) is required for almost all activity of the enhancer. Dll shows strong cooperativity with Hth and Exd for binding to D4, indicating that Dll interacts physically with these proteins. This interaction requires DNA binding, as Dll protein containing a missense change that blocks DNA binding (a change of asn51 to ala in the homeodomain) shows no ability to associate with D4-bound Hth and Exd. A curious feature of the cooperativity seen in the binding studies is that although Hth and Exd increase the affinity of Dll for D4, Dll appears to have little effect on the affinity of Hth and Exd for the enhancer. Since Hth and Exd already bind cooperatively with one another, it may be that additional cooperative interactions with Dll have little effect. Alternatively, it may be that Hth and Exd interact with Dll only after binding DNA. If so, Hth and Exd would be expected to increase Dll binding to D4, but Dll would have little effect on the binding of Hth and Exd, as observed. Interactions between Dll and Hth in the absence of DNA have been reported in immunoprecipitation experiments. However, this study was unable to repeat these observations. Moreover, the finding that the asn51 mutant of Dll fails to associate with D4-bound Hth and Exd argues strongly against such interactions (Duncan, 2010).
The repressor Cut also acts directly upon D4. Binding of Cut requires two sites, one overlapping Dlla and the other overlapping the joint Hth/Exd site. These binding sites suggest that D4 is controlled by Cut in much the same way that a structurally similar Abdominal-A (Abd-A) regulated enhancer from the rhomboid gene is controlled by the repressor Senseless (Sens). In the rhomboid enhancer, adjacent Hth and Exd sites are also present, and these create a binding site for Sens. Activity of the rhomboid enhancer is controlled by a competition between binding of the Sens repressor and binding of the activators Abd-A, Hth, and Exd. It seems likely that D4 is controlled similarly, with the repressor Cut competing for binding with the activators Dll, Hth, and Exd. It will be of interest to determine whether enhancers similar to D4 are used more widely to control Cut targets involved in its role as an external sense organ determinant (Duncan, 2010).
A key finding in this work is that Antp represses D4 by direct interaction. Antp binds a single site in D4, which overlaps or is identical to the Dlla binding site. Like Dll, Antp binds cooperatively with Hth and Exd. Using purified proteins, it was showm that binding of Dll and Antp to the Dlla site is mutually exclusive. This indicates that Antp represses the enhancer at least in part by competing with Dll for binding. Similar competition may occur at other enhancers; when Antp expression is driven artificially in the distal leg, variable deletions of the tarsal segments occur. These defects might arise because Antp competes with Dll for binding to its target genes in the distal leg. In most other contexts examined, Antp is an activator of transcription; why it fails to activate D4 is not clear. The similar behavior of Dll and Antp in binding to D4 supports the idea that Dll behaves like a Hox protein in activating D4 (Duncan, 2010).
Although the initial focus of this study was on the antenna, the finding that Antp interacts directly with D4 led to an examination of D4 regulation in the leg, where Antp is normally expressed. In second leg imaginal discs, Antp is required only in a proximal ring of cells that coexpresses Dll and Hth. This ring appears in the early third instar, and is of uncertain function. Large Antp− clones in T2 leg discs that do not enter this ring appear to develop completely normally, regardless of whether they are located distal or proximal to the ring. However, clones that overlap the ring show activation of D4/lacZ within the ring cells. Importantly, such clones have no effect on the expression of Dll or Hth within the ring. By examining Antp− clones of increasing age the following sequence of events is inferred. First, D4/lacZ is activated in cells of the ring that are included within Antp− clones. Second, many such clones begin expressing the antennal markers Ss and Cut, indicating a transformation to antenna, and round up as if they have lost affinity for neighboring cells. Third, such clones appear to extend and move distally in the disc (Duncan, 2010).
The events described for Antp− clones in the leg make sense of several previously enigmatic observations. It has been noted that many Antp− clones in the leg do not transform to antenna and appear to develop normally. The finding that only clones that overlap the proximal ring undergo transformation accounts for this observation. Antp− clones that do contain transformations usually show apparent nonautonomy in that not all cells in the clone are transformed to antenna. The current results account for this observation as well, since within an Antp− leg clone only those cells located in the proximal ring undergo transformation to antenna; cells located elsewhere in the clone retain normal leg identity. Most importantly, these observations provide an explanation for why ss is controlled directly by Antp. Antp− clones have no effect on hth or Dll expression in the proximal ring. Therefore, Antp must function in the ring at the target gene level to repress antennal genes that would otherwise be activated by combined Hth and Dll (and Exd). Since several such targets are known, it seems likely that several, perhaps many, antennal genes in addition to ss are repressed directly by Antp (Duncan, 2010).
Transformed Antp− clones in the leg often show ectopic hth expression in distal locations. If hth is not directly controlled by Antp in the leg, as this study suggests, then why is hth ectopically expressed within such clones? A likely explanation is that downstream antennal genes that have become activated in such clones feed back to activate hth. This interpretation is strongly supported by the finding that ectopic expression of the antennal genes ss, dan, or danr in the distal leg causes ectopic activation of hth. Thus, the distal expression of hth seen in Antp− leg clones is likely a consequence rather than a cause of the transformation to antenna. Whether repression of hth in the antenna by ectopic Antp is also indirect is not clear. Dll is also expressed ectopically in transformed Antp− leg clones, suggesting that it is also subject to feedback activation by downstream antennal genes (Duncan, 2010).
The function of the proximal Dll- and Hth-expressing ring in the proximal leg is not well understood. The ring is highly conserved among the insects, and may serve as a boundary between the proximal and distal portions of the legs. In the context of this work, a striking feature of the ring is that it contains a microcosm of gene expression domains corresponding to the three major antennal segments. Thus, proceeding from proximal to distal through the ring, cells express hth alone, hth + Dll, and hth + Dll + strong dachshund. These expression combinations are characteristic of the A1, A2, and A3 antennal segments, respectively. Looked at in this way, the ring would appear to resemble a repressed antennal primordium within the leg (Duncan, 2010).
It has been known for almost thirty years that Antp is required in the leg to repress antennal identity. However, an understanding of how this repression occurs has been lacking. The current results indicate that Antp functions within the proximal ring to directly repress antennal genes that would otherwise be activated by combined expression of Dll, Hth, and Exd. This appears to be the only function of Antp in the leg, at least during the third instar larval stage. The results are entirely consistent with the idea that second leg is the 'ground state' ventral appendage (the limb type that develops in the absence of identity specification) and that the role of Antp in the leg is to preserve this ground state by repressing the activation of 'head-determining' genes (Duncan, 2010).
Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. This study shows that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of NotchOFF or NotchON neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their NotchON progeny systematically die through Reaper activity, whereas later, their NotchOFF progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. It is concluded that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype (Bertet, 2014).
Although apoptosis is a common feature of neurogenesis in both vertebrates and Drosophila, the mechanisms controlling this process are still poorly understood. For instance, several studies in Drosophila have shown that, depending on the context, Notch can either induce neurons to die or allow them to survive during binary cell fate decisions. This is the case in the antennal lobes where Notch induces apoptosis in the antero-dorsal projecting neurons lineage (adpn), whereas it promotes survival in the ventral projecting neurons lineage (vPN). In both of these cases, the entire lineage makes the same decision whether the NotchON or NotchOFF cells survive or die. This suggests that, in this system, Notch integrates spatial signals to specify neuronal survival or apoptosis (Bertet, 2014).
This study shows that, during tOPC neurogenesis, neuronal survival is determined by the interplay between Notch and temporal patterning of progenitors. Indeed, within the same lineage, Notch signaling leads to two different fates: it first induces neurons to die, whereas later, it allows them to survive. This switch is due to the sequential expression of three highly conserved transcription factors-Dll/Dlx, Ey/Pax-6, and Slp/Fkh-in neural progenitors. These three factors have distinct functions, with Dll promoting survival of NotchOFF neurons, Ey inducing apoptosis of NotchOFF neurons, and Slp promoting survival of NotchON neurons. These data suggest that Ey induces death of NotchOFF neurons by activating the proapoptotic factor hid. Thus, Dll probably antagonizes Ey activity by preventing Ey from activating hid. The data also suggest that Notch signaling induces neuronal death by activating the proapoptotic gene rpr. Thus, Slp might promote survival of NotchON neurons by directly repressing rpr expression or by preventing Notch from activating it. In both cases, the interplay between Notch and Slp modifies the default fate of NotchON neurons, allowing them to survive. Further investigations will test these hypotheses and determine how Dll, Ey, Slp, and Notch differentially activate/repress hid and rpr (Bertet, 2014).
Although the tOPC and the main OPC have related temporal sequences, their neurogenesis is very different. This difference is in part due to the fact that newly specified tOPC neuroblasts express Dll, which controls neuronal survival, instead of Hth. Why do tOPC neuroblasts express Dll? The tOPC, which is defined by Wg expression in the neuroepithelium, is flanked by a region expressing Dpp. Previous studies have shown that high levels of Wg and Dpp activate Dll expression in the distal cells of the Drosophila leg disc. Wg and Dpp could therefore also activate Dll in the neuroepithelium and at the beginning of the temporal series in tOPC progenitors. Another difference between the main OPC and tOPC neurogenesis is that Ey and Slp have completely different functions in these regions. Indeed, unlike in the main OPC, Ey and Slp control the survival of tOPC neurons. This suggests that autonomous and/or nonautonomous signals interact with these temporal factors and modify their function in the tOPC (Bertet, 2014).
Finally, tOPC neuroblasts produce neurons for three different neuropils of the adult visual system, the medulla, the lobula, and the lobula plate. This ability could be due to the particular location of this region in the larval optic lobes. Indeed, the tOPC is very close to the two larval structures giving rise to the lobula and lobula plate neuropils-Dll-expressing neuroblasts are located next to the lobula plug, whereas D-expressing neuroblasts are close to the IPC. Interestingly, Dll and D neuroblasts specifically produce lobula plate neurons. This raises the possibility that these neuroblasts and/or the neurons produced by these neuroblasts receive signals from the lobula plug and the IPC, which instruct them to specifically produce lobula plate neurons. These nonautonomous signals could also modify the function of Ey and Slp in the tOPC (Bertet, 2014).
In summary, this study demonstrates that temporal patterning of progenitors, a well-conserved mechanism from Drosophila to vertebrates, generates neural cell diversity by controlling multiple aspects of neurogenesis, including neuronal identity, Notch-mediated cell survival decisions, and the mode of intermediate precursor division. In the tOPC temporal series, some factors control two of these aspects (Ey), whereas others have a specialized function (Dll, Slp, and D). This suggests that temporal patterning does not consist of a unique series of transcription factors controlling all aspects of neurogenesis but instead consists of multiple superimposed series, each with distinct functions (Bertet, 2014).
This study characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE many additional leg enhancers were identified, suggesting that this logic is broadly used by many genes during Drosophila limb development (Newcomb, 2018).
The EGFR signaling pathway is widely used in animal development, and is frequently a target in human disease and developmental abnormalities. Yet despite its importance in animal biology, many questions remain about how this pathway functions. Among these questions is whether secreted ligands that activate this pathway can induce distinct cell fates in a concentration-dependent manner. This study tests this idea by specifically eliminating a single source of EGFR ligands from the center of the Drosophila leg imaginal disc, which fate maps to the distal-most region of the adult leg. One plausible scenario is that this single source of secreted EGFR ligands, which is referred to as the EOC, activates distinct gene expression responses at different distances from this source. Alternatively, eliminating ligands secreted from the EOC might only affect gene expression locally, close to or within the EOC. Taken together, the current data are most consistent with the second scenario. This conclusion is largely supported by the observations that CRM deletions that eliminate vn and rho expression from the EOC have mild developmental consequences, both in the L3 leg imaginal discs and adult legs. These phenotypes are significantly weaker than those generated when the entire EGFR pathway is compromised using a temperature sensitive allele of the EGFR receptor. The difference between these two phenotypes is most likely explained by removing only a single source of EGFR ligands in the enhancer deletion experiments versus affecting EGFR signaling throughout the leg disc in the Egfrtsla experiments. This explanation is further supported by the observation that there are indeed additional CRMs, some of which were defined in this study, that drive EGFR ligand production in more medial ring-like patterns during the L3 stage (Newcomb, 2018).
One possible caveat to these conclusions is that there are a total of seven rho-like protease genes in the Drosophila genome that could, in principle, play a role in distal leg development. This study focused on rho and roughoid ru, based on previous results showing that triple rho ru vn clones generate severe leg truncations that phenocopy strong Egfrtsla truncations. In addition, it is noted that if other rho family proteases were active in the EOC, leg truncations and patterning defects would not be expected in the leg discs of the rhorhoE-Df vnvnE-Df double mutant, because those proteases should be able to produce active Spi. These observations suggest that the remaining five rho-like protease genes play a minor (or no) role in leg development. However, this conclusion will ultimately benefit from further genetic and expression analysis of these additional rho-like genes (Newcomb, 2018).
An additional previous observation that contrasts with the suggestion that EOC activity has only a limited role in specifying distal leg fates is the partial rescue of the PD axis when only a small number of distal leg cells were wild type in legs containing large rho ru vn clones. However, it is noted that even in these 'rescued' legs, medial defects in PD patterning were apparent. It is also noteworthy that in these earlier experiments, only adult legs were examined. When the same experiment was repeated, but L3 discs were analyzed, it was found that rho ru vn clones generated phenotypes that were very similar to those produced by the double vnE rhoE enhancer deletions. Taken together, these observations suggest that timing must be considered in the interpretation of these experiments. When assayed at the late L3 stage, both enhancer deletion and rho ru vn clone experiments argue that EOC activity is limited to specifying only the most distal fates, marked by the expression of al and C15. Starting in mid L3, and perhaps continuing into pupal development, there are additional sources of EGFR ligands that, when compromised, can affect adult leg morphology. Nevertheless, at least at the L3 stage, these data suggest that EGFR ligands produced from the EOC have a limited and local role in specifying distal leg fates (Newcomb, 2018).
Integration of inputs from signaling pathways and organ selector genes at CRMs in order to execute distinct developmental programs is a recurrent theme during animal development. This study identified two leg EGFR ligand CRMs that integrate the inputs from the Wg and Dpp signaling pathways and the leg selector genes Dll and/or Sp1 in a manner that is very similar to a previously characterized leg enhancer DllLT. In addition, when the same regulatory logic was applied to the whole genome, a battery of leg enhancer elements was identified. Interestingly, each of these enhancers drives expression in a specific manner with slightly different timing despite the fact that many of the inputs are shared. It is conceivable that the different expression patterns directed by these enhancers are in part a consequence of additional inputs and/or the difference in the TF binding site grammar. In support of this idea, vnE and rhoE differ in the number of binding sites for many inputs and vnE requires Sp1 while rhoE does not. Both of these differences may contribute to the earlier onset of vnE expression compared to rhoE. The remaining enhancer elements identified in this study direct a plethora of PD-biased leg expression patterns -- ranging from ubiquitous, to central and 'ring' patterns (see Genome-wide analysis of combinatorial inputs of Dll, Sp1, Wg, and Dpp in leg discs), which likely integrate inputs in addition to the ones described here. Future studies of these CRMs would help reveal the complex network of regulation that orchestrates leg development in the fruit fly. Such detailed understanding of the cis-regulatory architecture of fly leg development would likely give insights into organogenesis and evolution in other animals as well (Newcomb, 2018).
The EGFR signaling pathway has tremendous oncogenic potential and understanding the various mechanisms regulating its activation is not only interesting from the point of view of animal development but also has important practical implications. While the core components of the EGFR pathway have been thoroughly studied because of their potent tumorigenic capability in humans, little is known about the transcriptional regulation of EGFR ligands that bind the receptor and activate the pathway. The reiterative use of EGFR signaling in many developmental processes implies that different cis-regulatory elements are likely utilized by each EGFR ligand in different organs and tissues in order to correctly read the diverse cues in any specific developmental context. It is conceivable that genomic variation in EGFR pathway CRMs might lead to a predisposition to different types of EGFR-dependent tumors in humans, since such CRMs may respond to potent growth-promoting signaling pathways, such as Wnt and BMP (Newcomb, 2018).
This study has characterized in detail two Drosophila EGFR CRMs, vnE and rhoE, and showed how they integrate the cues from two transcription factors, Dll and Sp1, and two signaling pathways, Wg and Dpp, in order to execute a leg patterning developmental program. Analogous EGFR CRMs are likely to exist in mammals, especially because complex interactions between BMP, Wnt, Shh, multiple Dlx paralogs and other factors, are implicated in the induction of FGF signaling in mammalian limb development. Consistent with this idea, specific single nucleotide polymorphisms (SNPs) in humans in non-coding loci of genes encoding EGFR ligands have been shown to be associated with different types of cancer. Such loci may be enhancer elements analogous to vnE and rhoE. It is also noted that the regulatory logic uncovered in this study is likely to be relevant to many CRMs and genes that share spatial and temporal expression programs. Exploiting this regulatory logic in other systems might streamline the identification of enhancer elements that will aid in the discovery of mechanisms that are relevant to EGFR-related human disease and developmental birth defects (Newcomb, 2018).
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