BIOLOGICAL OVERVIEW

Distal-less is the first genetic signal for limb formation to occur in the developing zygote. Limbs are defined to include antennae, labium, legs and wings. Limbs form from imaginal discs, the small sacs of epithelial cells that grow and develop during the larval stage. The signal from Distal-less affects the formation of imaginal discs, distinguishing the site of imaginal disc development from the general body wall (B. Cohen, 1993), thus setting the stage for limb development.

What signals are required for the induction of Distal-less? In the developing fly Wingless (WG) is arrayed at compartment boundaries in the dorsal to ventral stripes of each parasegment. These stripes intersect cells secreting Decapentaplegic (DPP), arrayed in a single anterior to posterior stripe. It is at the points where WG and DPP stripes intersect that leg imaginal discs are specified and Distal-less is induced (B. Cohen, 1993).

A third signaling molecule, Hedgehog, is also required for Distal-less induction. HH is secreted from the posterior compartments of imaginal discs. These three secreted signaling molecules (HH, WG and DPP) specify the distal-axis of imaginal discs. Each is required for Distal-less induction (Diaz-Benjumea, 1994).

One must be careful in applying as a generalization, the above model to the origin of all discs. The involvement of three secreted signals is readily apparent for the origin of leg discs, but wing discs do not exhibit wingless expression until the second larval instar (Couso, 1993). At this stage wg expression is detected first in the anterior compartment, and Wingless is involved in the specification of the wing primordium. A primary target of Wingless in the specification of the wing primordium is pdm-1 which has an important role is specifying the proximo-distal axis of the wing disc. The interaction between the three secreted proteins and Distal-less in the second larval instar wing disc has not been documented, but it is clear that Dll performs a secondary and late function required for the normal patterning of the wing (Ng, 1996).

Thus Distal-less induces ventral appendage development in Drosophila. It appears that Dll suppresses dorsal appendage development. Lack of Dll function causes a change in the identity of ventral appendage cells (legs and antennae) that often results in the loss of the appendage. Ectopic Dll expression in the proximal region of ventral appendages induces nonautonomous duplication of legs and antennae by the activation of wingless and decapentaplegic. Ectopic Dll expression in dorsal appendages produces transformation into corresponding ventral appendages; wings and halteres develop ectopic legs and the head-eye region develops ectopic antennae. In the wing, the exogenous Dll product induces this transformation by activating the endogenous Dll gene and repressing the wing determinant gene vestigial. It is proposed that Dll induces the development of ventral appendages and also participates in a genetic address that specifies the identity of ventral appendages and discriminates the dorsal versus the ventral appendages in the adult. However, unlike other homeotic genes, Dll expression and function is not defined by a cell lineage border (Gorfinkiel, 1997).

The combination of Wg and Dpp induces the formation of the P/D axis in the leg of Drosophila. It was originally suggested that the Wg/Dpp combination may establish an organizer at the distal tip that controlled patterning along the P/D axis and that this organizer is characterized by expression of the homeobox gene aristaless. Even if such an organizer does exist then it is shown that al is not absolutely required for its activity because removing al at the tip using a null allele does not prevent formation of the P/D axis, although it does prevent the formation of the structures normally found at the tip of the leg. However, there is an absolute requirement for Dll activity in the formation of the P/D axis in the part of the leg that is more distal than the most proximal segment (the coxa). Yet, Dll protein does not show a graded distribution -- this presents a paradox between where Dll is expressed and where its activity is required -- late in development Dll protein can be detected only in the tarsus and distal tibia, but the genetic data reveal that Dll function is also required cell autonomously in more proximal regions, the femur and all of the tibia (Campbell, 1998).

Thus, studies of Dll expression in developing legs have suggested that the protein is restricted to the presumptive tarsus and distal tibia. However, mosaic analysis indicates a requirement for Dll gene function in the presumptive femur and proximal tibia early in development. If Dll is expressed in the more proximal parts of the leg early but then lost from this region, this would account for these observations. To show that Dll is expressed in more proximal regions earlier in development, a cell line was generated in which Dll expression switches on expression of lacZ and provides an inherited marker for Dll expression. Analysis of third instar leg discs from this line reveals that clones expressing beta-gal can be detected anywhere in the leg disc (i.e. including regions outside of the central Dll protein-positive domain), but at much higher frequency in the center of the disc, suggesting Dll is expressed early in development in all the leg disc cells. As an alternative approach the phenomenon of perdurance of protein products (in this case green fluorescent protein) was utilized to investigate whether evidence of earlier Dll transcriptional activity could be detected in the more proximal regions of the leg in which Dll transcription and Dll protein are absent later in development. In late third instar leg discs, the domain of high level GFP expression is generally coincident with Dll protein revealed by antibody staining, but much weaker staining can also be detected in the presumptive proximal tibia/femur. At earlier stages the level of GFP expression outside the Dll protein domain is much stronger, suggesting that the weak staining later (and the stronger staining earlier) is due to perdurance of GFP. Weak lacZ expression can also be detected in the presumptive proximal tibia/femur and again is likely due to perdurance. By the time leg eversion occurs, the low level expression of GFP is generally no longer evident in the femur and proximal tibia. However, in the adult, GFP expression is clearly expressed in most of the cells in the proximal tibia and at high levels in single cells associated with each bristle in the femur. There is a variable amount of expression elsewhere in the femur. Dll is required in the distal regions of the leg for the expression of tarsal-specific genes including al and bric-a-brac. Dll mutant cells in the leg sort out from wild-type cells suggesting one function of Dll here is to control adhesive properties of cells (Campbell, 1998).

The results of this detailed clonal analysis with a Dll null allele can be summarized as follows and are in general agreement with previous studies. Before about the early third instar there is an autonomous requirement for Dll in cells of the leg more distal than the coxa, apart from the dorsal femur, but later in development this requirement is limited to a distal domain corresponding to the tarsus and distal tibia. This domain corresponds to the region where Dll protein can be detected at these stages with antibodies. Dll is expressed in all cells of the presumptive leg, but does not distinguish between expression in the embryo or later during larval life. It should be noted that at late larval stages the domain showing high levels of Dll expression corresponds to the region where Dll function is required, so it is not unreasonable to suggest that similar high levels of expression will correspond to the regions where Dll function is required at earlier stages. If the above view is correct, the leg may be loosely divided into three regions along the P/D axis: (1) proximal - no Dll expression; (2) intermediate - Dll expressed and required early, but not later, and (3) distal - continuous Dll expression and requirement. This would then provide a clear explanation for the paradox raised by the observation that the apparent sites of Dll expression does not explain all the mutant phenotypes. Additionally, it is possible that one source of positional information along the P/D axis is the length of time a cell expresses Dll. Two exceptions to this simple hypothesis are the femur and the trochanter. (1) There is a differential requirement for Dll in the dorsal and the ventral femur (i.e. at the same P/D level) and at present there is no explanation for this, although it is possible that it is related to the situation in the embryo where dorsal Dpp appears to act as a repressor rather than activator of Dll expression. (2) The trochanter, which is proximal, appears to require Dll activity even late in development. This late requirement may correspond to the proximal ring of Dll expression in the leg discs. It should also be noted that expression in this domain appears to be independent of wg and dpp. An additional peculiarity is that although Dll mutant tissue can differentiate normally sized bristles, it fails to form bracts at the base of these bristles even when the clones are generated late in development in proximal locations. This probably reveals a later function for Dll during pupal development because Dll expression extends more proximally during pupal life, and in the adult Dll appears to be expressed in a support cell of each bristle. Not all of these develop bracts, but Dll may be required in these cells to allow the development of this structure (Campbell, 1998).

If the model suggesting cell fate along the P/D axis as specified by Wg and Dpp directly proves to be correct, then one static, simplified version of this model could hold that different cell fates are established above strict concentration thresholds of Wg/Dpp, which in turn would correspond to precise distances from the sources of these molecules. However, one possible problem with this simplified model is growth: as the imaginal disc grows in size, the distance of any one cell from the source will vary so that for such a strict model to produce precise patterning, all cell fates may have to be established simultaneously. Relevant data suggest this is not the case. To account for growth, it has been proposed that different target genes may require Wg and Dpp for different periods of time before expression becomes independent of these signals. Following growth, this could result in overlapping domains of target genes, and these domains could be established at different times in development. However, an alternative way of viewing this model is to propose that different cell fates are established not simply on the basis of how much Wg and Dpp they receive but by how long they receive it. Early in development most of the presumptive leg cells will receive a specific level of Wg and Dpp, but as the disc increases in size, the presumptive proximal cells at the edge of the disc will begin to receive less Wg and Dpp, as they become situated further and further from the sources: they will also experience this specific level of Wg and Dpp for a shorter period of time than more centrally located, presumptive distal cells. Consequently, the length of time a cell receives this specific level of Wg and Dpp may provide positional information along the P/D axis: the longer it receives it the more distal it becomes. It is proposed that the present results with Dll may provide some evidence for such a dynamic version of this model. These results suggest presumptive intermediate level cells express Dll early in development but it is lost later, whilst presumptive distal cells show continuous expression. If it is assumed that a cell expresses Dll above a certain threshold of Wg/Dpp, then its expression may be lost during development at the edge of its expression domain when these cells become situated further from the sources of Wg and Dpp, as the disc grows in size. One problem with this model is that maintenance of Dll expression does not appear to require continuous Wg and Dpp signaling. Dll expression is not lost in clones of a Dpp receptor, thick veins, or a Wg signal transducer, dishevelled, even when these are made during the second instar, i.e. at a time when the present results suggest Dll is still transiently expressed in some cells. There are at least two possible explanations for this: (1) the above model is correct but it is impossible to determine timing of gene function by making clones because this ignores the possibility of perdurance of gene products, or (2) the model is incorrect, but this may be because it assumes that Wg and Dpp are the only limiting factors controlling Dll expression (and patterning along the P/D axis): there may be an additional signal, possibly derived from the presumptive tip, which is also required for Dll expression. A temporal mechanism for axis formation is more evident in vertebrate appendages where positional identity along the P/D axis appears to be determined by such a mechanism: the longer a cell spends in the progress zone, the region behind the tip of the developing limb, the more distal it becomes. Consequently, the P/D axis is determined in a proximal to distal sequence. In Drosophila, there is contradictory evidence as to the order in which segments are specified along the P/D axis and further studies are required to resolve this question (Campbell, 1998 and references).

One function of Distal-less is marked by its absence. Homeotic genes Ultrabithorax and abdominal-A act on known elements of a distal enhancer of Distal-less to repress Dll expression in the abdomen, thereby effectively blocking induction of appendages in the posterior segments (Vachon, 1992).

One aspect of Distal-less research, perhaps the most exciting, has been almost completely neglected. distal-less is expressed in the optic lobe, targeted by Wingless (Kaphingst, 1994). The vertebrate homolog of Distal-less is expressed in premigratory and migratory neural crest cells and in the forebrain (Holland, 1996 and references). The role of Distal-less in Drosophila neurogenesis should be an exciting area of investigation. Whereas amphioxus (cephalochordata within the phylum Chordata) has only one Distal-less gene, craniates have five or six, which probably evolved from a single ancestral gene. The presence of a single Distal-less-related gene in amphioxus (phylum Urochordata) and invertebrates, and multiple members of this gene family in craniates is yet another indication that the size of developmental gene families increased abruptly early in craniate evolution. This increase may well have permitted the evolution of the complex features distinguishing the craniates from their invertebrate ancestors (Holland, 1966 and references).

Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system

Vertebrate Dlx genes have been implicated in the differentiation of multiple neuronal subtypes, including cortical GABAergic interneurons, and mutations in Dlx genes have been linked to clinical conditions such as epilepsy and autism. This study showed that the single Drosophila Dlx homolog, distal-less, is required both to specify chemosensory neurons and to regulate the morphologies of their axons and dendrites. distal-less was shown to be necessary for development of the mushroom body, a brain region that processes olfactory information. These are important examples of distal-less function in an invertebrate nervous system and demonstrate that the Drosophila larval olfactory system is a powerful model in which to understand distal-less functions during neurogenesis (Plavicki, 2012).

The phenotype exhibited by dll-null embryos is more severe than that of sc, amos, or ato single mutants or amos;ato double mutants, most closely resembling that of the sc;amos;ato triple mutants, indicating that dll might lie upstream of the proneural genes and regulate their expression in precursors of the Dorsal organ (DO), the larval olfactory organ (see Schematic of the larval chemosensory system). However, ato is expressed in the antennal segments of dll-null embryos, and dll is expressed in ato¹ and amos¹ mutants. Altogether, these data indicate that dll is likely to act in parallel with the proneural genes during DO development (Plavicki, 2012).

prospero (pros) is also required for DO development. pros encodes a homeodomain transcription factor that is asymmetrically distributed during SOP division. In other contexts, pros represses neuronal stem-cell proliferation while promoting neuronal differentiation. Mutations in pros result in gustatory behavioral deficits and disrupt axon and dendrite outgrowth from both DO and terminal organ (TO) neurons. The axon pathfinding defects exhibited by pros mutants resemble those seen in dll-null embryos, although it is unclear whether the same subsets of neurons are affected (Plavicki, 2012).

The axon scaffolding associated with the MBs in the embryonic brain is disrupted in dll-null embryos. Specifically, projections from the MB Kenyon cells across the supraesophageal commissure appear to be missing in late-stage embryos. MB defects also were observed in the brains of larvae in which postmitotic drivers such as elav-GAL4 and OR83b-GAL4 were used in conjunction with dll-RNAi to knock down activity. This finding indicates that dll also may be necessary for the later specification and/or differentiation of larval-born Kenyon cells. Because elav-GAL4 is not active until neurons are specified, the disruptions in the larval MBs observed in elav-GAL4;UAS-dll-RNAi animals are consistent with a role for dll in either axon guidance or viability of the postmitotic neurons (Plavicki, 2012).

The brain phenotypes detected in dll mutants resemble those of cephalic gap gene mutants. Specifically, loss of otd, ems, or btd results in embryonic brain segmentation defects and disrupts the formation of brain commissures and axon tracts. In otd mutants, protocerebral neuroblasts, including the MB precursors, are missing. In ems and/or btd mutants, subsets of neuroblasts are lacking in the deutocerebral neuromere, which harbors the larval antennal lobe (LAL), and the tritocerebral neuromere, which receives gustatory inputs. It therefore is possible that dll is a key effector of cephalic gap gene function during brain development (Plavicki, 2012).

pros mutants exhibit DO axon defects similar to those of dll. It therefore is possible that Dll and Pros collaborate to regulate other genes needed for axon pathfinding by DOG neurons. In the brain, but not in the DO Ganglion (DOG) or TO ganglion (TOG), mislocalization of Futsch protein was observed in dll mutants. Futsch is a microtubule-associated protein with functions in both axonogenesis and dendritogenesis. It therefore is possible that some of the defects observed in dll mutant MB lobes (which consist of axon tracts) and calyces (which contain Kenyon cell dendrites) are caused by misregulation of futsch. Other likely effectors of dll function during axon pathfinding in both DO and MB are Pak3 and Down syndrome cell adhesion molecule (Dscam). Both play important roles in axon guidance, including the targeting of adult Drosophila ORNs. Both also have been identified as putative downstream targets of vertebrate Dlx1/2. The loss-of-function Drosophila dll phenotypes in both DOG and MB are reminiscent of DSCAM phenotypes and consistent with dll regulation of DSCAM in multiple neuronal subtypes (Plavicki, 2012).

Given the dramatic reduction in Kenyon cell number in the dll hypomorphs, it might be expected that the peduncles and lobes would be even thinner than observed. However, a similar reduction in Kenyon cell number without concomitant thinning of the lobes has been observed in ey^R mutants. In this case, ablation studies were used to demonstrate that, at late third instar, recently born Kenyon cells have not yet contributed to MB lobes. It is therefore anticipated that MB defects may be more pronounced in dll mutant adults (Plavicki, 2012).

Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors

In eukaryotes, members of transcription factor families often exhibit similar DNA binding properties in vitro, yet orchestrate paralog-specific gene regulatory networks in vivo. The serially homologous first (T1) and third (T3) thoracic legs of Drosophila, which are specified by the Hox proteins Scr and Ubx, respectively, offer a unique opportunity to address this paradox in vivo. Genome-wide analyses using epitope-tagged alleles of both Hox loci in the T1 and T3 leg imaginal discs, the precursors to the adult legs and ventral body regions, show that ~8% of Hox binding is paralog-specific. Binding specificity is mediated by interactions with distinct cofactors in different domains: the Hox cofactor Exd acts in the proximal domain and is necessary for Scr to bind many of its paralog-specific targets, while in the distal leg domain, the homeodomain protein Distal-less (Dll) enhances Scr binding to a different subset of loci. These findings reveal how Hox paralogs, and perhaps paralogs of other transcription factor families, orchestrate alternative downstream gene regulatory networks with the help of multiple, context-specific cofactors (Feng, 2022).

This study used a combination of whole-genome and mechanistic approaches to understand how serially homologous appendages, such as the fly T1 and T3 legs, obtain their unique morphologies due to the activities of parallel Hox gene networks. The very similar transcriptomes in the three pairs of leg discs suggest that the different morphologies are largely a consequence of changing the expression patterns of the same sets of genes. By comparing the genome-wide DNA-binding profiles of the two relevant Hox paralogs, Scr and Ubx, in their native physiological contexts, this study found hundreds of paralog-specific Hox targets, accounting for ~8% of all binding events for these two Hox proteins. Next, differences in chromatin accessibility and Hox monomer binding preferences were shown to be unlikely to account for paralog-specific binding. Instead, it was demonstrated that interaction with the Hox cofactor Exd explains a large fraction of Scr's paralog-specific binding events. Finally, this study identified Dll as a Hox cofactor in the complementary distal domain of the leg disc. Results from RNA-seq, CBP ChIP, and reporter assays suggest that about 1/3 of the paralog-specific Scr-binding events are functional and lead to tissue-specific gene regulation. Thus, paralog-specific Hox-DNA binding, which is mediated by multiple cofactors including Exd and Dll, contribute significantly to paralog-specific Hox gene networks (Feng, 2022).

Previous in vitro studies provided compelling evidence that the DNA-binding specificities of different Hox-Exd dimers are more divergent from each other than those of Hox monomers, a phenomenon termed latent specificity. There have also been several in vivo examples in which paralog-specific Hox-DNA binding and target regulation was shown to depend on an interaction with Exd. This study shows that, on a genome-wide scale, the interaction with Exd explains a significant fraction of paralog-specific Hox binding, which often leads to paralog-specific gene regulation (Feng, 2022).

Earlier work also suggested that there is a tradeoff between specificity and affinity for Hox-Exd-binding motifs, where high affinity binding motifs are more likely to have low specificity for different Hox-Exd heterodimers. The paralog-specific, Exd-dependent CRMs characterized in this study (ac^-1, h^-1, and fj<^-1), have higher affinity Hox-Exd-binding motifs than those previously described in the shavenbaby (svb) gene: the major Scr-Exd motif in the fj^-1 CRM has an affinity of about 0.06 relative to the optimal motif in the genome, while the motifs in ac^-1-1 and h^-1 have even higher relative affinities of nearly 0.15 and 0.2, respectively. In contrast, the Ubx-Exd-binding motifs in CRMs from svb have a relative affinity of <0.0118. One possible explanation for this difference is that the svb CRMs are active in embryos, which have many different cell types, while the CRMs characterized in this study are active in leg discs, which have significantly less cell-type complexity. Embryonic CRMs may require especially low-affinity binding motifs to distinguish their activities in a context with many cell types. Consistent with this idea, the fkh250 CRM, which is also active in embryos, uses an Scr-Exd-binding motif with a low relative affinity of 0.01718,36. Notably, the relative affinities for the Scr-Exd-binding motifs in ac^-1, h^-1, and fj^-1 are at least eightfold higher than for Ubx-Exd. Manual inspection of other intergenic and intronic loci with Scr_T1 > Ubx_T3 binding suggests that there are many other CRMs that follow this same rule. Thus, for specificity to occur, the most relevant feature may be that the affinity for the 'correct' TFs, in this case Scr-Exd, must be significantly greater compared to the affinity for other 'incorrect' TFs that are co-expressed in the same or homologous cells (Feng, 2022).

Because Exd is only nuclear in a subset of cells during Drosophila development, such as the proximal domain of the leg disc, it was unlikely that Exd was the only Hox cofactor. In fact, CRMs that are directly regulated by Ubx have been described in cells where Exd is not available to be a cofactor. However, it has remained an unresolved question whether non-Exd cofactors are used in these examples. More generally for the leg imaginal disc, the entire distal domain, extending from the trochanter to the tarsus, is without nuclear Exd, yet has Hox-dependent segment-specific morphological characteristics, such as the sex combs on the male T1 leg. Although several candidate TFs have been proposed to be Hox cofactors, none have been confirmed. This study provides evidence that Dll is a distally acting Hox cofactor in leg discs (Feng, 2022).

There are many differences between how Exd and Dll interact with Hox proteins when bound to DNA. The Scr-Exd-binding motif is comprised of two partially overlapping half-sites, while the Scr-Dll motif consists of two HD-binding motifs separated by a spacer of several base pairs. Another difference is that the amount of cooperativity observed for Hox-Exd is far greater than that observed for Hox-Dll. The overlapping nature of the Hox- and Exd-binding motifs may be important for latent specificity, which for Scr requires an Exd-induced conformational change of the homeodomain. In contrast, there is no evidence that latent specificity occurs as a consequence of Hox-Dll binding. Instead, the modest cooperativity observed for the Scr-Dll heterodimer is likely a consequence of increasing Scr-DNA-binding affinity via a protein-protein interaction and closely spaced Dll and Scr-binding motifs (Feng, 2022).

More generally, it is suggested that mode of DNA binding exhibited by Hox-Exd, which is highly cooperative and reveals latent specificity, may be the exception rather than the rule for TF-TF interactions within CRMs, and that the Scr-Dll example, with weak cooperativity between TFs stemming from a protein-protein interaction, may be the more common mode of interaction to distinguish the binding of paralogous TFs. In support of this notion, a systematic in vitro study identified 315 TF-TF interactions, only five of which exhibited latent specificity (Feng, 2022).

The TALE homeodomain proteins, which include Exd and Hth, are very ancient TFs that were present before the split of plants and animals, and TALE-mediated nuclear localization analogous to the Hth-Exd example in flies has been described in plants. In contrast, the Hox gene family is only present in metazoans, and Dll is specific to bilaterians. Moreover, it has been proposed that Dll initially functioned in the CNS, and was later co-opted to pattern the distal appendage. Based on these observations, it is plausible that the Hox-Dll interaction evolved more recently than the Hox-Exd interaction, accounting for why Exd interacts with all Hox paralogs, while Dll may be a more limited Hox cofactor. This is supported by the results from a small-scale bimolecular fluorescence complementation (BiFC) screen that revealed Dll interacts with some Hox proteins, but not others (Feng, 2022).

Notably, the combined activities of Exd and Dll still do not account for all ScrT1 > UbxT3-binding events genome-wide and reporter analysis suggests the presence of additional, yet to be identified Hox cofactors that have the capacity to promote Scr-specific binding. It is suggested that the Hox-Dll mode of binding uncovered in this study may be representative of additional TFs that also have the ability to promote paralog-specific Hox binding and activity at specific CRMs. Further, it is noted that the differentiation of the T1 and T3 leg fates is a continuous developmental process and that the observations described in this study are limited to the late 3rd instar stage. Nevertheless, it is expected that the principles governing Hox paralog specificity uncovered in this study will likely extend to other developmental stages and tissues. Finally, although this study focused on the role of paralog-specific TF-DNA binding, there may be additional mechanisms that do not depend on differences in DNA binding between paralogous TFs that also contribute to their specific functions (Feng, 2022).

GENE STRUCTURE

Bases in 5' UTR - greater than 741 bp

Introns - seven

Bases in 3' UTR - 7537 bp

PROTEIN STRUCTURE

Structural domains

DLL has a centrally located homeodomain (Vachon, 1992).

date revised: 20 December 97 

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