extradenticle

DEVELOPMENTAL BIOLOGY

Embryonic

With the exception of pole cells, where EXD is not found, EXD distribution during early embryogenesis is uniform. Protein levels decline prior to germ band extention. Levels increase again at stage 9 [Image]. Nonuniform expression patterns emerge and by stage 12, transcripts are detected in the head and thoracic regions, while levels decline posteriorly. exd is required for proper neurogenesis and is expressed in the CNS. Expression in the CNS and brain continues after expression is lost in the epidermis. exd is expressed in somatic and visceral mesoderm, where levels are high in anterior regions and low in posterior regions (Rauskolb 1993).

It is noteworthy that although Extradenticle protein's distribution is initially uniform but it is excluded from nuclei until gastrulation. During the extended germ band stage the protein remains predominantly cytoplasmic and does not accumulate in nuclei until germ band retraction. Nuclear accumulation occurs in a pattern that is highly regulated. For example, EXD is present at high levels within the nucleus of visceral mesoderm cells at the positions where the gastric caeca and all the midgut constrictions will form. EXD is also present throughout the underlying endoderm, but it is cytoplasmic at both ends of the midgut and only accumulates in nuclei in the central zone. The zone of nuclear accumulation of EXD in the endoderm, although broader than the region of labial expression, is similarly centered around parasegment 7. In the imaginal discs the nuclear accumulation of Extradenticle is also spatially regulated and, in the wing and leg discs, distal regions exhibit cytoplasmic Extradenticle, whereas proximally the protein is nuclear. In the wing imaginal disc, there are high levels of nuclear EXD in a ring around the area where the wing blade will form. This corresponds to the hinge region. In the notum EXD is nuclear in patches. EXD is cytoplasmic in the wing blade. It is suggested that this regulation of the sub-cellular localization of EXD is important for the interactions between EXD and the homeotic selector proteins and that EXD is not simply a ubiquitously available cofactor (Aspland, 1997).

Arthropod appendages are thought to have evolved as outgrowths from the body wall of a limbless ancestor. Snodgrass, in his Principles of Insect Morphology (1935), proposed that, during evolution, expansion of the body wall would originate the base of the appendages, or coxopodite, upon which the most distal elements that represent the true outer limb, or telopodite, would develop. The homeobox gene Distal-less (Dll), which is required in the Drosophila appendages for development of distal regions is now thought to promote formation of telopodite structures above the evolutionary ground-state of non-limb or body wall. Another homeobox gene, extradenticle, which is required for appropriate development of the trunk and the proximal parts of the appendages, represents a coxopodite gene. exd is transcribed in a pattern that surrounds and abuts Dll-expressing imaginal disc primordia in the ventrolateral epidermis of stage 14 Drosophila embryos. Early in embryogenesis, exd is broadly distributed throughout the embryo and colocalizes with Dll in the limb primordia. exd function is eliminated from the distal precursors in the developing limb and subsequently remains restricted to proximal precursors. This elimination is important because when ectopically expressed, exd prevents distal development and gives rise to truncated appendages lacking distal elements. This restriction of EXD protein to the peripheral parts of the disc is in contrast to its reported uniform mRNA distribution. EXD mRNA accumulates preferentially in the periphery of the leg disc, although lower levels are also detected in the central regions. This EXD mRNA in central regions may be responsible for the low levels of EXD protein detected in the cytoplasm, suggesting that the restriction of exd function to proximal leg parts may be controlled not only transcriptionally but also at the level of nuclear transport. The maintenance of exd expression during larval stages, contrary to Dll, does not require the hedgehog signaling pathway, suggesting that proximal regions of appendages develop independent of hh function. Targeting exd transcription to the distal limb, using a Dll promoter attached to the exd coding region, prevents distal leg development. Ectopic exd seems to interfere with cell proliferation in the central disc and results in cell death induction in the distal domain of the leg. Finally, in the crustacean Artemia, exd and Dll are expressed in comparable patterns as in Drosophila, suggesting a conserved genetic mechanism subdividing the arthropod limb (Gonz‡lez-Crespo, 1996).

Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues

The generation of distinct neuronal subtypes at different axial levels relies upon both anteroposterior and temporal cues. However, the integration between these cues is poorly understood. In the Drosophila central nervous system, the segmentally repeated neuroblast 5-6 generates a unique group of neurons, the Apterous (Ap) cluster, only in thoracic segments. Recent studies have identified elaborate genetic pathways acting to control the generation of these neurons. These insights, combined with novel markers, provide a unique opportunity for addressing how anteroposterior and temporal cues are integrated to generate segment-specific neuronal subtypes. Pbx/Meis, Hox, and temporal genes were found to act in three different ways. Posteriorly, Pbx/Meis and posterior Hox genes block lineage progression within an early temporal window, by triggering cell cycle exit. Because Ap neurons are generated late in the thoracic 5-6 lineage, this prevents generation of Ap cluster cells in the abdomen. Thoracically, Pbx/Meis and anterior Hox genes integrate with late temporal genes to specify Ap clusters, via activation of a specific feed-forward loop. In brain segments, 'Ap cluster cells' are present but lack both proper Hox and temporal coding. Only by simultaneously altering Hox and temporal gene activity in all segments can Ap clusters be generated throughout the neuroaxis. This study provides the first detailed analysis of an identified neuroblast lineage along the entire neuroaxis, and confirms the concept that lineal homologs of truncal neuroblasts exist throughout the developing brain. Also this study provides the first insight into how Hox/Pbx/Meis anteroposterior and temporal cues are integrated within a defined lineage, to specify unique neuronal identities only in thoracic segments. This study reveals a surprisingly restricted, yet multifaceted, function of both anteroposterior and temporal cues with respect to lineage control and cell fate specification (Karlsson, 2010).

To understand segment-specific neuronal subtype specification, this study focused on the Drosophila neuroblast 5-6 lineage and the thoracic-specific Ap cluster neurons born at the end of the NB 5-6T lineage. The thoracic appearance of Ap clusters was shown to result from a complex interplay of Hox, Pbx/Meis, and temporal genes that act to modify the NB 5-6 lineage in three distinct ways (see Summary of Hox/Pbx/Meis and temporal control of NB 5-6 development). In line with other studies of anterior-most brain development, it was found that the first brain segment (B1) appears to develop by a different logic. These findings will be discussed in relation to other studies on spatial and temporal control of neuroblast lineages (Karlsson, 2010).

In the developing Drosophila CNS, each abdominal and thoracic hemisegment contains an identifiable set of 30 neuroblasts, which divide asymmetrically in a stem-cell fashion to generate distinct lineages. However, they generate differently sized lineages -- from two to 40 cells, indicating the existence of elaborate and precise mechanisms for controlling lineage progression. Moreover, about one third of these lineages show reproducible anteroposterior differences in size, typically being smaller in abdominal segments when compared to thoracic segments. Thus, neuroblast-specific lineage size control mechanisms are often modified along the anteroposterior axis (Karlsson, 2010).

Previous studies have shown that Hox input plays a key role in modulating segment-specific behaviors of neuroblast lineages. Recent studies have resulted in mechanistic insight into these events. For instance, in the embryonic CNS, Bx-C acts to modify the NB 6-4 lineage, preventing formation of thoracic-specific neurons in the abdominal segments. This is controlled, at least in part, by Bx-C genes suppressing the expression of the Cyclin E cell cycle gene in NB 6-4a. Detailed studies of another neuroblast, NB 7-3, revealed that cell death played an important role in controlling lineage size in this lineage: when cell death is genetically blocked, lineage size increased from four up to 10 cells. Similarly, in postembryonic neuroblasts, both of these mechanisms have been identified. In one class of neuroblasts, denoted type I, an important final step involves nuclear accumulation of the Prospero regulator, a key regulator both of cell cycle and differentiation genes. In 'type II' neuroblasts, grh acts with the Bx-C gene Abd-A to activate cell death genes of the Reaper, Head involution defective, and Grim (RHG) family, and thereby terminates lineage progression by apoptosis of the neuroblast. This set of studies demonstrates that lineage progression, in both embryonic and postembryonic neuroblasts, can be terminated either by neuroblast cell cycle exit or by neuroblast apoptosis. In the abdominal segments, it was found that the absence of Ap clusters results from a truncation of the NB 5-6 lineage, terminating it within the Pdm early temporal window, and therefore Ap cluster cells are never generated. These studies reveal that this truncation results from neuroblast cell cycle exit, controlled by Bx-C, hth, and exd, thereafter followed by apoptosis. In Bx-C/hth/exd mutants, the neuroblast cell cycle exit point is bypassed, and a thoracic sized lineage is generated, indicating that these genes may control both cell cycle exit and apoptosis. However, it is also possible that cell cycle exit is necessary for apoptosis to commence, and that Bx-C/hth/exd in fact only control cell cycle exit. Insight into the precise mechanisms of the cell cycle exit and apoptosis in NB 5-6A may help shed light on this issue (Karlsson, 2010).

Whichever mechanism is used to terminate any given neuroblast lineage -- cell cycle exit or cell death -- the existence in the Drosophila CNS of stereotyped lineages progressing through defined temporal competence windows allows for the generation of segment-specific cell types simply by regulation of cell cycle and/or cell death genes by developmental patterning genes. Specifically, neuronal subtypes born at the end of a specific neuroblast lineage can be generated in a segment-specific fashion 'simply' by segmentally controlling lineage size. This mechanism is different in its logic when compared to a more traditional view, where developmental patterning genes act upon cell fate determinants. But as increasing evidence points to stereotypic temporal changes also in vertebrate neural progenitor cells (Okano, 2009), this mechanism may well turn out to be frequently used to generate segment-specific cell types also in the vertebrate CNS (Karlsson, 2010).

These findings of Hox, Pbx/Meis, and temporal gene input during Ap cluster formation are not surprising -- generation and specification of most neurons and glia will, of course, depend upon some aspect or another of these fundamental cues. Importantly however, the detailed analysis of the NB 5-6T lineage, and of the complex genetic pathways acting to specify Ap cluster neurons, has allowed this study to pin-point critical integration points between anteroposterior and temporal input. Specifically, cas, Antp, hth, and exd mutants show striking effects upon Ap cluster specification, with effects upon expression of many determinants, including the critical determinant col. Whereas Antp plays additional feed-forward roles, and exd was not tested due to its maternal load, it was found that both cas and hth mutants can be rescued by simply re-expressing col. This demonstrates that among a number of possible regulatory roles for cas, hth, Antp, and exd, one critical integration point for these anteroposterior and temporal cues is the activation of the COE/Ebf gene col, and the col-mediated feed-forward loop. Both col and ap play important roles during Drosophila muscle development, acting to control development of different muscle subsets. Their restricted expression in developing muscles has been shown to be under control of both Antp and Bx-C genes. Molecular analysis has revealed that this regulation is direct, as Hox proteins bind to key regulatory elements within the col and ap muscle enhancers. The regulatory elements controlling the CNS expression of col and ap are distinct from the muscle enhancers, and it will be interesting to learn whether Hox, as well as Pbx/Meis and temporal regulatory input, acts directly also upon the col and ap CNS enhancers (Karlsson, 2010).

One particularly surprising finding pertains to the instructive role of Hth levels in NB 5-6T. At low levels, Hth acts in NB 5-6A to block lineage progression, whereas at higher levels, it acts in NB 5-6T to trigger expression of col within the large cas window. It is interesting to note that the hth mRNA and Hth protein expression levels increase rapidly in the entire anterior CNS (T3 and onward). In addition, studies reveal that thoracic and anterior neuroblast lineages in general tend to generate larger lineages and thus remain mitotically active for a longer period than abdominal lineages. On this note, it is tempting to speculate that high levels of Hth may play instructive roles in many anterior neuroblast lineages. In zebrafish, Meis3 acts to modulate Hox gene function, and intriguingly, different Hox genes require different levels of Meis3 expression. In the Drosophila peripheral nervous system, expression levels of the Cut homeodomain protein play instructive roles, acting at different levels to dictate different dendritic branching patterns in different sensory neuron subclasses. Although the underlying mechanisms behind the levels-specific roles of Cut, Meis3 or Hth are unknown, it is tempting to speculate that they may involve alterations in transcription factor binding sites, leading to levels-sensitive binding and gene activation of different target genes (Karlsson, 2010).

The vertebrate members of the Meis family (Meis1/2/3, Prep1/2) are expressed within the CNS, and play key roles in modulating Hox gene function. Intriguingly, studies in both zebrafish and Xenopus reveal that subsequent to their early broad expression, several members are expressed more strongly or exclusively in anterior parts of the CNS, in particular, in the anterior spinal cord and hindbrain. Here, functional studies reveal complex roles of the Meis family with respect to Hox gene function and CNS development. However, in several cases, studies reveal that they are indeed important for specification, or perhaps generation, of cell types found in the anterior spinal cord and/or hindbrain, i.e., anteroposterior intermediate neural cell fates. As more is learned about vertebrate neural lineages, it will be interesting to learn which Meis functions may pertain to postmitotic neuronal subtype specification, and which may pertain to progenitor cell cycle control (Karlsson, 2010).

In anterior segments -- subesophageal (S1-S3) and brain (B1-B3) -- a more complex picture emerges where both the overall lineage size and temporal coding is altered, when compared to the thoracic segments. Specially, whereas all anterior NB 5-6 lineages do contain Cas expressing cells, expression of Grh is weak or absent from many Cas cells. The importance of this weaker Grh expression is apparent from the effects of co-misexpressing grh with Antp -- misexpression of Antp alone is unable to trigger FMRFa expression, whereas co-misexpression with grh potently does so. It is unclear why anterior 5-6 lineages would express lower levels of Grh, since Grh expression is robust in some other anterior lineages (Karlsson, 2010).

In the B1 segment two NB 5-6 equivalents have been identified. However, the finding of two NB 5-6 equivalents is perhaps not surprising, since the B1 segment contains more than twice as many neuroblasts as posterior segments. Due to weaker lbe(K)-lacZ and -Gal4 reporter gene expression, and cell migration, these lineages could not be mapped. However, irrespective of the features of the B1 NB 5-6 lineages, bona fide Ap cluster formation could not be triggered by Antp/grh co-misexpression in B1. Together, these findings suggest that the B1 segment develops using a different modus operandi, a notion that is similar to development of the anterior-most part of the vertebrate neuroaxis, where patterning and segmentation is still debated. On that note, it is noteworthy that although Hox genes play key roles in specifying unique neuronal cell fates in more posterior parts of the vertebrate CNS, and can indeed alter cell fates when misexpressed, the sufficiency of Hox genes to alter neuronal cell fates in the anterior-most CNS has not been reported -- for instance, Hox misexpression has not been reported to trigger motoneuron specification in the vertebrate forebrain. Thus, in line with the current findings that Antp is not sufficient to trigger Ap cluster neuronal fate in the B1 anterior parts, the anterior-most part of both the insect and vertebrate neuroaxis appears to be 'off limits' for Hox genes (Karlsson, 2010).

The Hox, Pbx/Meis, and temporal genes are necessary, and in part sufficient, to dictate Ap cluster neuronal cell fate. However, they only do so within the limited context of NB 5-6 identity. Within each abdominal and thoracic hemisegment, each of the 30 neuroblasts acquires a unique identity, determined by the interplay of segment-polarity and columnar genes. In the periphery, recent studies demonstrate that anteroposterior cues, mediated by Hox and Pbx/Meis genes, are integrated with segment-polarity cues by means of physical interaction and binding to regulatory regions of specific target genes. It is tempting to speculate that similar mechanisms may act inside the CNS as well, and may not only involve anteroposterior and segment-polarity integration, but also extend into columnar and temporal integration (Karlsson, 2010).

Effects of Mutation or Deletion

The varying results presented by mutant exd-clones has been studied to determine the role of EXD in adult patterns for different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax, labial, or Antennapedia. In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia (Gonzalez-Crespo, 1995).

Looking at genetic mosaics and a hypomorphic exd allele caused by a point mutation in the homeodomain furthered study of the role of exd in adult development. Loss of exd results in homeotic transformations: abdominal segments take on A5 or A6 segmental identity, antenna and arista transform to leg, and head capsule to dorsal thorax or notum. Proximal leg structures are particularly sensitive to the loss of exd, although exd does not influence the allocation of proximal positional values of the leg imaginal disc (Rauskolb, 1995).

Sex combs reduced (Scr) activity is proposed to be required cell nonautonomously for determination of tarsus identity, and Extradenticle (Exd) activity is required cell autonomously for determination of arista identity. Using the ability of Proboscipedia to inhibit the Scr activity required for determination of tarsus identity, it was found that loss-of-Exd activity is epistatic to loss-of-Scr activity in tarsus vs. arista determination. That is, loss-of-Exd activity produces tarsus when there is no Scr activity, suggesting that Exd functions downstream of Scr. This suggests that in the sequence leading to arista determination, Scr activity is OFF while Exd activity is ON, and in the sequence leading to tarsus determination Scr activity is ON, which turns Exd activity OFF. Immunolocalization of Exd in early third-instar larval imaginal discs reveals that Exd is localized in the nuclei of antennal imaginal disc cells and localized in the cytoplasm of distal imaginal leg disc cells. It is propose that Exd localized to the nucleus suppresses tarsus determination and activates arista determination. It is further proposed that in the mesodermal adepithelial cells of the leg imaginal discs, Scr is required for the synthesis of a tarsus-inducer, which, when secreted, acts on the ectoderm cells inhibiting nuclear accumulation of Exd, such that tarsus determination is no longer suppressed and arista determination is no longer activated (Percival-Smith, 1998).

Salivary gland formation in the Drosophila embryo is dependent on Scr. When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, SCR mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that Scr also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Two homeodomain-containing proteins encoded by the extradenticle and homothorax genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. Scr regulates the nuclear localization of Exd in the salivary gland primordia through repression of homothorax expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands (Henderson, 2000).

To determine if Exd cooperates with Scr to control salivary gland gene expression, the accumulation of two early salivary gland proteins, CrebA and Trh, was examined in embryos lacking exd function. Zygotic loss of exd function results in a reduction in the number of salivary gland cells expressing CrebA and Trh, as well as a decrease in the level of protein made in these cells. This reduced level of salivary gland protein expression is not as severe as the one seen in Scr mutant embryos. Unlike SCR, EXD mRNA is supplied maternally and, thus, the maternal contribution may partially compensate for the loss of zygotic function. To test this possibility, the maternal contribution of exd was removed using the FLP-FRT system. In embryos lacking maternal exd function, salivary gland expression of CrebA and Trh is at wild-type levels. However, salivary gland expression of CrebA and Trh is completely absent in embryos lacking both the maternal and the zygotic contributions of exd. Thus, exd is required for embryonic salivary gland gene expression. Moreover, zygotically provided exd can rescue the loss of maternally provided exd and maternally provided exd can partially compensate for zygotic loss of exd (Henderson, 2000).

Since Scr, exd, and hth are required for salivary gland formation, the mRNA and/or protein expression patterns of these genes during normal salivary gland formation were examined. During stages 9 and 10, when salivary gland gene expression is established, Scr and hth are expressed in the salivary gland primordia, as well as other tissues, and Hth and Exd are nuclear. During stage 11, after the establishment of early salivary gland gene expression, the salivary glands begin to invaginate. At this stage, there are several changes in the expression and/or localization of these genes and/or proteins in the salivary gland cells: Scr and hth transcripts disappear, Hth protein disappears, and Exd protein becomes cytoplasmic (Henderson, 2000).

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using Fasciclin II, an axonal marker, the activities of the extradenticle (exd) and homothorax (hth) genes have been shown to be essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level, including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of eyeless is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent of one another (Nagao, 2000).

In the course of embryonic brain development both EXD and HTH proteins became clearly detectable by early stage 12 in many of the delaminating cephalic neuroblasts. Strong nuclear expression is particularly evident in the deuto- and trito-cerebrum neuroblasts, but less prominent expression is also detectable in most of the protocerebrum neuroblasts. These patterns are maintained in almost identical manners since the brain neuromeres were formed by division of the cephalic neuroblasts. As development proceeds further, EXD and HTH localize in several domains in the brain: high level expression is maintained for both proteins in most of the neural cells in the deuto- and trito-cerebrum anlagen (neuromeres b₂ and b₃); in the mediolateral regions of the b₁ neuromere and most of the cells of the subesophageal ganglia. Both EXD and HTH localize in the nucleus in the developing brain neurons, as confirmed by colocalization with nuclear transcription factors. The apparent identical expression of EXD and HTH in the developing brain has been confirmed by double staining with anti-EXD and anti-HTH antibodies or a HTH-lacZ reporter. Moreover, EXD immunoreactivity in the brain is lost in the hth mutant whereas cytoplasmic EXD is still detectable in the epidermis. Likewise, HTH expression is dependent on the activity of exd, since virtually all the HTH immunoreactivity is lost in both the epidermis and the brain in exd mutant (Nagao, 2000).

The expression patterns of EXD and HTH in developing brain neuromeres are partly reminiscent of the patterns of fiber tract founder clusters. Examinations of embryos double stained with anti-HTH antibody and anti-FAS II antibody demonstrate that many of the cells in the fiber tract founder clusters indeed express the HTH protein. This coexpression is already seen by the middle of stage 12 when the first set of the Fas II clusters in the brain becomes evident. Significant coexpression is seen in the fiber tract founder cluster D/T, which is located in neuromere b₃, the tritocerebrum anlage: this stage is marked by the lab gene. Despite the fact that the HTH pattern becomes more restricted in later stages, the HTH expression in the fiber tract founder clusters is largely maintained. In particular, HTH is expressed at significant level in the D/T and P1 clusters. Similar overlapping expression in the fiber tract founder clusters is detected for the EXD protein. Coexpression of FAS II, HTH, and EXD is also seen in the developing optic lobe primordia (Nagao, 2000).

In order to gain insights into their functions, the expression patterns of EXD and HTH in the developing brain were further examined in conjunction with known neuraxial patterning genes. In the proto- and deuto-cerebrum anlagen, the immunoreactivity of the EXD protein only partially overlaps with otd transcripts except for the dorsally located cells in neuromere b₁, which express both genes at high levels. In contrast to otd, the EXD immunoreactivity largely overlaps with the EMS immunoreactivity in neuromeres b₂ and b₃). EMS is predominantly expressed in the anterior parts of neuromeres b₂ and b₃. EMS and EXD colocalize in many of the b₂ and b₃ cells with the exception of some of the most anterior cells of each neuromere, which clearly express EMS but EXD only faintly. Coexpression of the two genes is also detected in neuroblasts. In the tritocerebrum anlagen, EXD immunoreactivity overlaps with the lab-lacZ expression, which localizes in the posterior part of the b₃ neuromere. EXD immunoreactivity also overlaps with the DFD immunoreactivity in the mandibular and the anterior half of the maxillary neuromeres. Similarly, the hth-lacZ expression, which is identical to the endogenous hth and exd expression patterns in double staining, overlaps with the SCR immunoreactivity in the posterior half of the maxillary neuromere and the anterior half of the labial neuromere (Nagao, 2000).

Thus exd and hth genes are coexpressed in many of the neurons of the fiber tract founder clusters, suggesting that the activities of these genes are intrinsically required for axonal programming of the tract founder cluster neurons. This is particularly evident for the D/T cluster, in which Fas II expression is largely dependent on exd and hth. Most of the Eyeless patterns, including those that partially overlap with the fiber tract founder clusters, are suppressed in the mutants. Given these results, it is likely that the intrinsic axonal programs of the fiber tract founder clusters are altered in the exd and hth mutants. Intriguingly, in addition to the apparent defects in the primary axonal scaffolds, mutations in the exd and hth genes result in gross anatomical defects in the developing brain. Notably, both mutations cause abnormal positioning of the brain commissure at more posterior positions (in neuroaxis), suggesting widespread regional patterning defects in the mutant brains. In support of this notion, molecular neuroanatomical analyses have revealed alterations to the expression patterns of many of the regional patterning genes, including stimulation of otd and suppression of ems, in the developing brain neuromeres. Similarly, in accordance with the anatomical defects at the cervical junction, expression of the anterior HOM-C genes lab, Dfd and Scr are significantly suppressed in the mutant brains. Furthermore, consistent with the anatomical abnormalities, both engrailed expressing cells en-b₁ and Brain-specific homeobox (Bsh) are up-regulated in the mutant brains with ectopic cell clusters in more posterior positions. Thus the strong defects in the embryonic axonal scaffolds in the exd and hth mutant brains are likely to be caused by combined defects in intrinsic neural programming of the fiber tract founder neurons and in extrinsic patterning of the brain neuromeres that provide the substrate for the axonal extension of the fiber tract founder clusters (Nagao, 2000).

The Hox/homeotic genes encode transcription factors that generate segmental diversity during Drosophila development. At the level of the whole animal, they are believed to carry out this role by regulating a large number of downstream genes. This study addresses the unresolved issue of how many Hox target genes are sufficient to define the identity of a single cell. Focus was placed on the larval oenocyte, which is restricted to the abdomen and induced in response to a non-cell autonomous, transient and highly selective input from abdominal A (abdA). Hox mutant rescue assays were used to demonstrate that this function of abdA can be reconstituted by providing Rhomboid (Rho), a processing factor for the EGF receptor ligand, secreted Spitz. Thus, in order to make an oenocyte, abdA regulates just one principal target, rho, that acts at the top of a complex hierarchy of cell-differentiation genes. These studies strongly suggest that, in at least some contexts, Hox genes directly control only a few functional targets within each nucleus. This raises the possibility that much of the overall Hox downstream complexity results from cascades of indirect regulation and cell-to-cell heterogeneity (Brodu, 2002).

Oenocytes are present in clusters of approximately six cells in each of the abdominal segments A1-A7. In the thorax, there is no Egfr induction around the chordotonal organ precursor called C1 and no specific serial homolog of the oenocyte. In order to score unambiguously the presence of oenocytes in a range of different genetic backgrounds, a panel of seven immediate-early, early and late markers were identified. To determine why oenocyte formation is restricted to the abdomen, embryos lacking various Hox genes or extradenticle (exd), which encodes a Hox co-factor, were examined. These experiments indicate that oenocyte formation requires exd and abdA but not two other Hox genes that are also expressed in the abdomen: Antp and Ubx. To assess whether oenocytes form in the absence of all Hox functions, the T1 segment was examined in embryos lacking Sex combs reduced (Scr) and Antp activities. No oenocytes are produced in this context, and therefore these cells are not part of the ground state. However, the ground state does contain both the signaling and responding cell types involved in oenocyte induction: C1 and the Sal-positive dorsal ectoderm (Brodu, 2002).

Genetic analysis shows that Engrailed has both negative and positive targets. Negative regulation is expected from a factor that has a well-defined repressor domain but activation is harder to comprehend. VP16En, a form of En that has its repressor domain replaced by the activation domain of VP16, has been used to show that En activates targets using two parallel routes, by repressing a repressor and by being a bona fide activator. The intermediate repressor activity has been identified as being encoded by sloppy paired 1 and 2 and bona fide activation is dramatically enhanced by Wingless signaling. Thus, En is a bifunctional transcription factor and the recruitment of additional cofactors presumably specifies which function prevails on an individual promoter. Extradenticle (Exd) is a cofactor thought to be required for activation by Hox proteins. However, in thoracic segments, Exd is required for repression (as well as activation) by En. This is consistent with in vitro results showing that Exd is involved in recognition of positive and negative targets. Moreover, genetic evidence is provided that, in abdominal segments, Ubx and Abd-A, two homeotic proteins not previously thought to participate in the segmentation cascade, are also involved in the repression of target genes by En. It is suggested that, like Exd, Ubx and Abd-A could help En recognize target genes or activate the expression of factors that do so (Alexandre, 2003).

Wg signaling contributes to the activation of En's positive targets. The temporal aspect of this requirement has not been investigated, but earlier results suggest that it is probably transient. Note that Wg signaling is irrelevant to repression by En and that, even in cells that are within the range of Wg, repression and activation (of distinct targets) coexist. For example, in the normal domain of en expression, ci is repressed and hh is activated. Therefore, Wg signaling does not convert En from an activator to a repressor. Perhaps Wg signaling helps the recruitment, on specific targets, of a cofactor needed to mask the repressor domain of En, while at the same time providing an activation domain. One candidate cofactor that could be regulated by Wg is the homeodomain protein encoded by exd, a known cofactor of Hox gene activity in vivo. However, Exd is not an activation-specific cofactor and more work is therefore needed to understand how Wg signaling contributes to the activating function of En (Alexandre, 2003).

Two types of activities have been ascribed to Exd. According to the selective binding model, Exd could help En recognize positive targets and assemble a transcription complex. Alternatively, or in addition, Exd could mask the repressor domain of En and, at the same time, recruit an activator (the so-called activity regulation model). Adding a functional activation domain to En (as in VP16En) does not override the need for Exd. This gives in vivo support to the selective binding model and is consistent with in vitro studies, which have shown that Exd and En can dimerize and bind DNA cooperatively. Cooperativity requires the eh2 domain of En, a domain that is left intact in VP16En. Because VP16En requires Exd for in vivo activity, it is concluded that the N-terminal half of En, which is absent in VP16En, is not required for the interaction with Exd (Alexandre, 2003).

In thoracic segments, VP16En requires exd to act on all En targets, positive and negative. This is the first indication that Exd could be involved in negative (as well as positive) target recognition by En. Indeed in thoracic segments wild-type En requires Exd for repression of its natural targets. This had presumably not been noticed previously because endogenous expression of En is lost in the absence of Exd. That Exd could be involved in repression is consistent with in vitro studies with PBX proteins and earlier suggestions from in vivo work with Drosophila. Because Exd is required for both repression and activation, the issue of what distinguishes activated targets from repressed ones remains unresolved. Throughout the present study, it has been found that the two En-positive targets, en and hh, are expressed identically in a variety of experimental conditions. It may therefore be that the regulatory regions of these two genes might contain unique features that make them positive targets (Alexandre, 2003).

En must be capable of activating transcription in the appropriate context. Because En harbors a robust repressor domain, it is likely that one or several cofactor(s) mask this domain and recruit an activation function and, it is unlikely that Exd alone provides such an activity. Nevertheless, the possible role of Hth is worth discussing. In vitro, Hth binds DNA as a part of a ternary complex with Exd and a Hox protein. Intriguingly, overexpression of an activator form of Hth (VP16Hth) phenocopies the overexpression of wild-type Hth (VP16Hth mimics overactive Hth). This suggests that the normal role of Hth is to bring an activation domain to a complex -- a conclusion that contradicts the observation that Hth is required for both repression and activation by En. One way to resolve this paradox would be to suggest that Hth has two distinct roles: to help target recognition on negative and positive targets and, in addition, to bring an activation domain onto positive targets. Of course activation by En could also involve as yet unidentified activating cofactors. Further progress will require the identification, within natural targets, of enhancers that confer either activation or repression. Comparing these sites and subsequent mutational and biochemical analysis could lead to a molecular understanding of what distinguishes negative from positive targets (Alexandre, 2003).

The most unexpected aspect of these results is that, in abdominal segments, the Hox proteins Ubx and Abd-A are involved in repression by En. In formal genetic assays, Ubx and Abd-A can substitute for Exd in helping En act on negative targets. In the absence of Ubx, Abd-A and Exd, En can no longer repress target genes. By contrast, two other Hox proteins (Antp and Abd-B) appear not to be involved in En function. Antp does not help En repress targets in vivo even though its homeodomain differs from that of Abd-A at only five positions. Likewise, Abd-B, a more distantly related Hox protein, is also unlikely to participate in En function. It is concluded that the role of Ubx and Abd-A in repression by En is specific (Alexandre, 2003).

How could ectopic Ubx or Abd-A allow En to repress targets in the absence of Exd? It could be that this is mediated by wholesale transformation of segmental identity [although such transformation would have to be exd/hth-independent. Alternatively, Ubx and Abd-A could have a more immediate involvement in En function. One can envisage that they could regulate an as yet unidentified corepressor of En (although such regulation would not require Exd). Alternatively, and more speculatively, Ubx and Abd-A could serve as cofactors themselves in regions of the embryo where Exd levels are low. Again, molecular analysis of negative targets will be needed to discriminate these possibilities (Alexandre, 2003).

Homeotic genes have not been previously implicated in En function despite many years of genetic analysis of the Bithorax complex. It is suggested that the role of Ubx and Abd-A in En function has been overlooked previously because, in the absence of these two genes, Exd is upregulated in the presumptive abdomen and thus takes over as a repression cofactor. However, the present results establish that homeotic genes do participate in the segmentation cascade and link two regulatory networks previously thought to be independent (Alexandre, 2003).

TALE-class homeodomain transcription factors, homothorax and extradenticle, control dendritic and axonal targeting of olfactory projection neurons in the Drosophila brain

Precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. In the Drosophila brain, olfactory projection neurons convey odor information from the antennal lobe to higher order brain centers such as the mushroom body and the lateral horn. This study shows that Homothorax (Hth), a TALE-class homeodomain transcription factor, is expressed in many of the antennal lobe neurons including projection neurons and local interneurons. In addition, HTH is expressed in the progenitors of the olfactory projection neurons, and the activity of hth is required for the generation of the lateral but not for the anterodorsal and ventral lineages. MARCM analyses show that the hth is essential for correct dendritic targeting of projection neurons in the antennal lobe. Moreover, the activity of hth is required for axonal fasciculation, correct routing and terminal branching of the projection neurons. Another TALE-class homeodomain protein, Extradenticle (Exd), is required for the dendritic and axonal development of projection neurons. Mutation of exd causes projection neuron defects that are reminiscent of the phenotypes caused by the loss of the hth activity. Double immunostaining experiments show that Hth and Exd are coexpressed in olfactory projection neurons and their progenitors, and that the expressions of Hth and Exd require the activity of each other gene. These results thus demonstrate the functional importance of the TALE-class homeodomain proteins in cell-type specification and precise wiring of the Drosophila olfactory network (Ando, 2011).

In addition to the regulatory functions as homeotic cofactors, hth and exd have important functions in the determination of the developmental identity of the antennal segment; removing the function of exd or hth transforms the antenna into leg-like structures, and ectopic expression of hth can trigger antennal development elsewhere in the fly. In addition, the activities of hth and exd are required for the development of the embryonic axonal tracts that pioneer the projections between the deutocerebrum and the dorsal parts of the protocerebrum. Consequently, loss of hth or exd results in marked perturbations of the axonal scaffolds in the developing brain (Ando, 2011).

This study has shown that Hth is expressed in many of the AL neurons including local interneurons and PNs. Hth and Exd are coexpressed not only in postmitotic neurons but also in the progenitors of the anterodorsal and lateral AL lineages. Loss of either hth or exd causes loss of the lateral lineage, in which hth plays an essential role in suppression of apoptosis. Moreover, it has been shown that hth and exd are required for precise dendritic and axonal targeting of olfactory PNs. Mutations of hth result in profound targeting defects in many of the on-target glomeruli such as VA1lm, VA3 and D. They also cause ectopic innervation in many of the off-target glomeruli. The dendritic defect of exd¹ mutation is reminiscent of the phenotype of hth mutations, with reduced number of GH146-expressing cells yet exhibiting selective ectopic innervation in the off-target DA1 glomeruli. The functional importance of hth and exd was further confirmed in post-mitotic neurons by the analyses of single-cell clones of the anterodorsal DL1 and the lateral DA1 neurons. Apart from dendritic defects, hth and exd mutations cause severe axonal defasciculation, misrouting and aberrant ventrolateral extensions. As with hth mutant clones, exd mutant clones exhibit an increase in the numbers of the LH terminal branches. Moreover, it was shown that coexpression of Hth and Exd is essential for efficient expression of either protein in the olfactory PNs, recapitulating the interdependence of the expression of the two proteins in the embryonic brain. These results suggest that exd and hth could positively regulate each other expression in the olfactory PNs. Alternatively, stability of Hth and Exd in the olfactory PNs could depend on the interaction of the two proteins as suggested in the antennal and leg discs (Ando, 2011).

Although Hth is co-expressed with Acj6 and Lola in both developing and adult ad-PNs, loss of hth function fails to alter Acj6 and Lola expression. Conversely, mutation of neither acj6 nor lola abolishes Hth expression. In agreement with independent regulatory mechanisms, the dendritic phenotype of hth PNs diverges from the phenotypes of either acj6 or lola mutant PN clones. Thus, in contrast to the severe innervation defects in VA1lm, VA3, and D of the hth mutant clones, acj6⁶ mutant clones show only mild defects for VA1lm and D, and only VA3 is severely affected. Similar differences can be noted for single-cell clones, in which hth but not acj6 mutant clones exhibit complete switching of DL1 specificity (Ando, 2011).

With partial commonality with hth mutant PNs, lola mutant PNs exhibit severe defects in multiple glomeruli. Nonetheless, lola and hth mutant clones exhibit different degree and spectra of glomerular mistargeting. While both hth^P2 and hth^P1-K6-1 ad-NB clones fail to innervate the VA1lm glomerulus, most of the lola ad-NB clones completely innervate VA1lm. In addition, only 23% of the lola single-cell clones lack dendritic innervation in DL1 while the majority of the hth single-cell clones fails to innervate the correct target. It is also noteworthy that, unlike the mutations of hth or exd, loss of the lola activity dose not eliminate the lateral PN lineage. In addition to these LOF phenotypes, GOF mistargeting phenotypes are also different between hth, acj6, and lola mutations. Similar differences in dendritic phenotypes can be noted with mutations of other transcription factors that have been shown to be involved in PN specification. These results are thus consistent with the notion that different transcription factors control the targeting specificity of olfactory PNs via distinct intrinsic programs that regulate diverse repertoires of downstream genes, even though they are coexpressed during development (Ando, 2011).

In the development of vertebrate spinal motor neurons, homeodomain transcription factors play important roles in the generation and determination of diverse neuronal subtypes. In particular, Hox proteins act as central mediators of the intrinsic programs that shape motor neuron subtype identity and target muscle specificity. Hox proteins not only influence the identity of motor neuron columns but also control the initial specificity of motor axon projections via specific transcriptional cascades that determine the expression profiles of the cell surface guidance receptors such as Eph family proteins. Combinatorial expression of Hox proteins and a cofactor, Meis 1, the vertebrate homolog of Hth, determine the specificity of the motor neuron pool subtypes (Ando, 2011).

The result that Hth and Exd are expressed in many of the Drosophila AL neurons suggests that Hth and Exd are unlikely candidates as lineage specific or cell-type specific regulators by themselves. Rather, it is more likely that these TALE-class homeodomain transcription factors control the identity of the AL neuromere in collaboration with other transcription factors that regulate the wiring specificity of individual neurons. In the development of antenna, hth and exd genetically interact with Distalless, which encodes another homeodomain transcription factor. Although Distalless is expressed in the antennal olfactory neurons that innervate the AL glomeruli, it is expressed neither in the olfactory PNs nor in the local interneurons. Unlike the more posterior parts of the central nervous system, none of the homeotic proteins are expressed in the Drosophila deuto- and protocerebrum neuromeres except for Probosipedia that is expressed in a small number of cells at the posterior deutocerebrum. On the other hand, studies on the embryonic brain have shown that Ems plays an essential role in the development of the deutocerebrum primordia that give rise to the ALs. In addition, the activity of ems is required for the generation of the lateral PNs and for precise dendritic targeting of the ad-PNs. Intriguingly, the amino acid sequence Tyr-Pro-Trp, located in the immediate upstream of the Ems homeodomain, partially matches the YPWM motif of the homeotic proteins that are bound by Exd/Pbx proteins. Although the current data demonstrate that Ems expression is independent of the hth activity, the phenotypic commonality that both hth and ems are required for the generation of the lateral but not the ad-PNs suggests a cooperative interaction of the Hth, Exd and Ems proteins in the regulation of down stream programs that regulate the proliferation of the lateral progenitors. In addition, Hth and Exd could cooperatively function with non-homeodomain transcription factors in the AL neurons as demonstrated by the ternary interaction between the Pbx1, Meis1 and MyoD proteins on the downstream target genes that regulate myogenic differentiation (Ando, 2011).

Four Exd-related proteins (Pbx1, Pbx2, Pbx3, and Pbx4) and five Hth-related proteins (Meis1, Meis2, Meis3, Prep1 and Prep2) are found in vertebrates. Mutations of the Pbx/Meis genes cause homeotic transformations in the hindbrain, mimicking the LOF phenotypes of the Hox genes expressed in the anterior neuromeres. In addition, as with the Drosophila homologs, these vertebrate TALE-class homeodomain proteins are expressed in more anterior brain structures during development. In the mouse brain, Meis1/2 and Pbx1/2/3 are expressed in the developing telencephalon, and Meis2 and Pbx1/2 are expressed in the entire dorsal mesencephalon, regulating the expression of the cell-surface EphA8 receptor to a specific subset of cells. In zebrafish development, Pbx4 functionally interacts with Engrailed to pattern the midbrain-hindbrain and diencephalic-mesencephalic boundaries. Moreover, Pbx3 and Meis1 are coexpressed with Rnx, an orphan Hox protein, in the ventral medullary respiratory center to regulate the development and/or functions of inspiratory neurons. Given the cross-phylum commonality in the glomerular organization and neuronal connectivity between the Drosophila and vertebrate olfactory systems, it would be important to determine the functional significance of the TALE-class homeodomain transcription factors in the control of the cell type specificity in the vertebrate brain (Ando, 2011).

Extradenticle and homothorax control adult muscle fiber identity in Drosophila

This study had identified a key role for the homeodomain proteins Extradenticle (Exd) and Homothorax (Hth) in the specification of muscle fiber fate in Drosophila. exd and hth are expressed in the fibrillar indirect flight muscles but not in tubular jump muscles, and manipulating exd or hth expression converts one muscle type into the other. In the flight muscles, exd and hth are genetically upstream of another muscle identity gene, salm, and are direct transcriptional regulators of the signature flight muscle structural gene, Actin88F. Exd and Hth also impact muscle identity in other somatic muscles of the body by cooperating with Hox factors. Because mammalian orthologs of exd and hth also contribute to muscle gene regulation, these studies suggest that an evolutionarily conserved genetic pathway determines muscle fiber differentiation (Bryantsev, 2012).

These results demonstrate a dramatic effect upon muscle fiber identity of the two factors Exd and Hth. The fact that muscle fiber type can be profoundly influenced by the activity of the two genes defines a central mechanism for the control of fiber identity and begins to expose the entire fiber specification pathway (Bryantsev, 2012).

A recent study identified salm as a controller of transition from tubular leg muscle to the fibrillar fiber type. Tubular leg muscles could be transformed into the fibrillar type by ectopic expression of salm. This study has expand these observations to show that the mechanism of Salm action is less straightforward: salm is expressed in the tubular jump muscle, suggesting that its pro-fibrillar action may require cooperation with additional factors. The current data suggest that Salm cofactors could be Exd and Hth: their absence in the jump muscle prevents this muscle from acquiring a fibrillar fiber phenotype despite its expression of salm; also, ectopic expression of salm in leg muscles promotes fibrillar fate, perhaps because the leg muscles also express exd and hth (Bryantsev, 2012).

It is also noted that, in the flight muscles, Exd and Hth maintain their localization in the absence of Salm. Moreover, despite the sustained accumulation of Exd and Hth, loss of Salm nevertheless results in transformation of the flight muscles toward a tubular fate. This indicates that Exd and Hth have at least some requirement for Salm to promote fibrillar muscle fate, and it will be interesting in the future to identify the respective roles of these factors directly interacting with other fiber-specific enhancers (Bryantsev, 2012).

This study also provides a direct mechanistic link between the determinants of fibrillar fate, exd/hth, and the actin gene characteristic of the flight muscles, Act88F. Whether fibrillar muscle genes are direct targets of Exd/Hth or Salm, or both, is yet to be determined; nevertheless, the identification of fiber-specific enhancers will provide new mechanistic insight into this process (Bryantsev, 2012).

Since diverse fiber types are characteristic of many vertebrate muscles, these findings may relate directly to vertebrate myogenesis. In zebrafish, slow muscle fate is promoted by the activities of PBX and MEIS, which are the vertebrate orthologs of Exd and Hth, respectively (Maves, 2007). In mice, PBX and MEIS are cofactors for myogenic determination genes, where they facilitate transcription factor binding to nonconsensus target sites, and this effect might function to fine tune muscle fiber fate (Heidt, 2007). Thus, diverse lines of evidence suggest a robust and conserved mechanism for fiber type specification, acting through PBX1/Exd and MEIS/Hth (Bryantsev, 2012).

A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation

Trait development results from the collaboration of genes interconnected in hierarchical networks that control which genes are activated during the progression of development. While networks are understood to change over developmental time, the alterations that occur over evolutionary times are much less clear. A multitude of transcription factors and a far greater number of linkages between transcription factors and cis-regulatory elements (CREs) have been found to structure well-characterized networks, but the best understood networks control traits that are deeply conserved. Fruit fly abdominal pigmentation may represent an optimal setting to study network evolution, as this trait diversified over short evolutionary time spans. However, the current understanding of the underlying network includes a small set of transcription factor genes. This study greatly expands this network through an RNAi-screen of 558 transcription factors. Twenty-eight genes were identified, including previously implicated abd-A, Abd-B, bab1, bab2, dsx, exd, hth, and jing, as well as 20 novel factors with uncharacterized roles in pigmentation development. These include genes which promote pigmentation, suppress pigmentation, and some that have either male- or female-limited effects. Many of these transcription factors control the reciprocal expression of two key pigmentation enzymes, whereas a subset controls the expression of key factors in a female-specific circuit. Pupal Abd-A expression pattern was conserved between species with divergent pigmentation, indicating diversity resulted from changes to other loci. Collectively, these results reveal a greater complexity of the pigmentation network, presenting numerous opportunities to map transcription factor-CRE interactions that structure trait development and numerous candidate loci to investigate as potential targets of evolution (Rogers, 2014).

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date revised: 25 August 2023 

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