Deformed

Promoter Structure

Deformed has one transcript with no TATA sequence in the promoter.

A small 120 bp autoregulatory element called module E is part of a larger 2.7 kb autoregulatory enhancer that maintains Deformed transcription in the epidermis of the gnathal segments of Drosophila embryos. Module E contains only one DFD protein binding site. However, the DFD binding region alone is not sufficient to supply DFD-dependent expression. An additional region containing an imperfect inverted repeat sequence is also required for the function of this homeotic protein response element (Zeng, 1994).

A 3.2 kb DNA fragment containing an enhancer that mimics the expression of Dfd in the subesophageal ganglion of the embryonic central nervous system has been identified. This neural autoregulatory enhancer (NAE) maps in the large Dfd intron just upstream of the homeobox exon and requires DFD protein function for its full activity. A 608 bp NAE subfragment retains regulatory function that is principally localized in the subesophageal ganglion. There are numerous blocks of sequence conservation within a comparable region of D.hydei. A pair of conserved blocks of NAE sequence match a DFD protein binding site in the epidermal autoregulatory element (Lou, 1995).

The Extradenticle protein is ubiquitously expressed in early embryonic cells during the period when segmental identities are being determined. exd interacts genetically with partial loss-of-function Dfd mutant alleles, enhancing their lethality. Thus, exd function is required for the autoactivation phase of Deformed expression in the posterior head: Exd partners Dfd in Dfd-directed Dfd autoactivation. An epidermal autoregulatory enhancer (EAE) maps in a 2.7 kb fragment about 4 kb upstream of the Dfd start site. Mutations that change the affinity of the small autoactivation element (120 base pairs) for Exd protein result in corresponding changes in the element's embryonic activity. The Exd and Dfd proteins directly activate this element in maxillary cells without cooperative binding. Based on the types of homeotic transformations and changes in gene expression observed in exd mutant embryos, a new model for Exd/PBX action is proposed in which these proteins are required for HOX protein transcriptional activation functions, but dispensable for HOX transcriptional repression functions. Although the selection of a specific target gene activation site by one HOX protein versus another may be explained in some cases by Exd's selective modulation of HOX binding specificity, the favored idea in this instance is that Exd can interact with HOX proteins to switch them into a state where they are capable of transcriptional activation. The experiments provide no support for the idea that a specialized heterodimer-binding site that would bind a Dfd-Exd complex stably and tightly is required for the specific activation of the 120 bp element in embryos. In this case Exd functions as a coactivator and does not function to promote cooperative binding with Dfd. Binding of Exd without simultaneous binding of Dfd may mediate repression. At adjacent regions of the 120 bp element, a novel protein, DEAF-1 has been shown to bind. Mutant substitutions in these adjacent regions reduce their affinity for DEAF-1. It is possible the Exd and DEAF-1 collaborate in non-maxillary cells to prevent ectopic activation of this head-specific HOX element (Pinsonneault, 1997).

The homeodomain proteins encoded by the Hox complex genes do not bind DNA with high specificity. In vitro, Hox specificity can be increased by binding to DNA cooperatively with the homeodomain protein Extradenticle or its vertebrate homologs, the PBX proteins (when considered together, known as the PBC family). One of the best characterized Hox-PBC binding sites is present in a 20 bp oligonucleotide repeat 3, which was identified in the 5' promoter region of the mouse Hoxb-1 gene. Hoxb-1 protein or its Drosophila ortholog Labial are both able to bind cooperatively with Exd to the binding site whereas other Hox proteins, such as Ultrabithorax or Hoxb-4 cannot. A two basepair change in a Hox-PBC binding site, from GG to TA, switches the Hox-dependent expression pattern generated in vivo from labial to Deformed. The change in vivo correlates with an altered Hox binding specificity in vitro. Similar Deformed-PBC binding sites were identified in the Deformed and Hoxb-4 genes. The Deformed sites include well characterized epidermal (EAE) and neural (NAE) autoregulatory enhancers. Two repeats containing TA sequence binding sites were found in the 2.7 kb EAE and two were found in the 600 bp NAE. These sites generate Deformed or Hoxb-4 expression patterns in Drosophila and mouse embryos, respectively. These results suggest a model in which Hox-PBC binding sites play an instructive role in Hox specificity by promoting the formation of different Hox-PBC heterodimers in vivo. Thus, the choice of Hox partner, and therefore Hox target genes, depends on subtle differences between Hox-PBC binding sites (Chan, 1997).

A comparison of the activity of genetic elements from the regulatory region of the Drosophila Deformed gene during embryogenesis and adult life reveals important similarities and differences. The 2.7 kb epidermal autoregulatory enhancer (EAE) of the Deformed gene drives expression of a beta-galactosidase reporter in unique spatial and temporal patterns in the adult antennae; this pattern is insensitive to temperature effects. The Deformed regulatory region possesses distinct enhancer elements that can direct the expression of a beta-galactosidase reporter spatially and temporally. A 120 bp region can reproduce the general features of the larger EAE fragment. The Deformed binding site is essential for temporal and spatial expression of beta-galactosidase during embryogenesis but is not required in the adult (Hoopengardner, 2002).

Substitutional analysis of the 120 bp E fragment suggests that the regulatory elements involved in directing reporter gene expression during adult life are different from those important in embryogenesis. Some elements that are dispensable during embryogenesis are essential for adult reporter gene expression, while others that are important for embryonic expression are unnecessary for adult reporter gene expression. Random substitutional changes in regions 1, 2, and 4 of the E fragment (either singly or with all three combined) has no effect on the E fragment's embryonic reporter gene expression. In contrast to this, reporter gene expression in the adult is completely ablated by substitutions in regions 1, 2, or 4 alone. Remarkably, when all three substitutional mutations are combined in the same construct normal temporal and spatial reporter gene expression is restored in the adult. Furthermore, the temperature insensitivity is also preserved. This suggests an adult-specific mechanism of regulation requiring an interaction between regions 1, 2, and 4 in order for normal reporter gene expression to take place (Hoopengardner, 2002).

One of the two regions within the E fragment that is most critical for reporter gene expression during embryogenesis is region 3. Region 3 contains the Deformed protein binding site that is thought to be important in maintaining expression through an autoregulatory loop. Despite the importance of the Deformed protein binding site (DBS) for normal embryonic regulation, it does not appear to be important for adult reporter gene expression. Substitutional mutations in the Deformed protein binding site that enhance or eliminate embryonic expression have little effect on adult reporter gene expression. Furthermore, the Deformed F fragment, a 470 bp fragment containing six Deformed binding sites, lacks any adult transcriptional activity. Unlike during embryogenesis, reporter gene expression in adults of DNA fragments from the Deformed E fragment may not be dependent upon feedback from the Deformed protein through the Deformed protein binding site (Hoopengardner, 2002).

A second region that is critical for embryonic reporter gene expression is region 5-6, which is largely made up of an imperfect palindrome sequence. Substitutional mutations in the palindrome from the region 5 or region 6 halves both disrupt embryonic activity of the E region, either decreasing reporter gene expression or abolishing it, while changes that improve the palindrome increase expression during embryogenesis. However, all alterations in this region, either improving or disrupting the palindromic sequence, led to a complete loss of adult gene expression, suggesting that the palindrome itself is not necessarily the critical element in adult expression (Hoopengardner, 2002).

The function of Deformed gene expression in the adult antenna is not understood. Northern analysis and RT-PCR have demonstrated that the Deformed message is detectable in the adult. Adult antennal expression of a number of other genes besides Deformed and Distal-less has also been suggested using the enhancer-trap system. These include genes such as wingless, engrailed, Antennapedia, armadillo, hedgehog, and decapentaplegic. Why these well-known 'developmental' genes are expressed during adult life is not clear. Knowledge of the roles these genes serve during embryonic and larval-pupal development does not readily explain their spatially restricted and temporally complex pattern of expression during adult life. The adult antenna is made up of cells that are post-mitotic and fully differentiated. Had expression been limited to the first few days of adult life, then the expression of these developmental genes might have been rationalized as a relic of the developmental processes involved in the formation of the adult or an aspect of the maturational process. The continued well-regulated expression of these genes in adult life provides evidence that they may be playing other critical roles in the adult (Hoopengardner, 2002).

Transcriptional Regulation

The activation of Deformed is dependent on combinatorial input from at least three levels of the early hierarchy. The simplest activation code sufficient to establish Deformed expression, given the absence of negative regulators such as Fushi tarazu, consists of a moderate level of expression from the coordinate gene bicoid, in combination with expression from both the gap gene hunchback, and the pair-rule gene even-skipped. In addition, the activation code for Deformed is redundant; other pair-rule genes in addition to even-skipped can apparently act in combination with bicoid and hunchback to activate Deformed (Jack, 1990).

Ectopically activated Dfd results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear. This out-of-context activation occurs in cells belonging to the anterior compartments of the three thoracic and the A1 to A8 abdominal segments. It requires the normal function of the polarity genes wingless and engrailed. The WG product, in addition to that of DFD, appears to be sufficient to activate, albeit indirectly, the endogenous Dfd gene in many embryonic cells. SCR, ANTP, UBX and ABD-B repress Dfd both transcriptionally and at the phenotypic level, if their products are in sufficient amounts. In addition, the differentiation of cirri can be induced by Dfd-expressing cells in non-expressing neighboring cells. This interaction occurs across the parasegmental border (Gonzalez-Reyes, 1992).

In the anterior domain tailless exerts a repressive effect on the expression of fushi tarazu, hunchback, and Deformed (Reinitz, 1990).

Deformed, cubitus interruptus, and engrailed itself are all targets of engrailed. engrailed is involved in an auto-regulatory loop in posterior compartments, and both Dfd and ci are limited to anterior compartments by engrailed function in adjacent posterior compartments. Engrailed binding sites have been found in the promoters of both engrailed and ci (Saenz-Robles, 1995).

Ectopic orthodenticle expression also causes the loss of head structures derived from the maxillary segment, which lies posterior to the otd domain. Ubiquitous otd affects the cirri and maxillary sense organ. This effect is associated with otd repression of the homeotic selector gene Deformed (Dfd). While Dfd mutants lack mouth hooks, the maxillary sense organ, cirri, and the ventral organ, it has been shown that otd induction reduces the number of cirri and maxillary sense organ papillae, but does not eliminate them altogether (Gallitano-Mendel, 1998).

Deletion mutants of cap'n'collar coding sequences indicate that cnc functions are required for the normal development of both labral and mandibular structures (Mohler, 1995). In place of the missing mandibular structures, some maxillary structures - mouth hooks and cirri - are ectopically produced (Harding, 1995; Mohler, 1995). The genetic function of the homeotic gene Deformed (Dfd) is required in the cnc mutant background to produce ectopic mouth hooks, and Mohler (1995) have proposed that Dfd and cnc function in combination to specify mandibular identity. A protein isoform (CncB) from the Drosophila cap ‘n’ collar locus has been characterized that selectively represses cis-regulatory elements that are activated by the Hox protein Deformed. Analysis of the cnc gene reveals the presence of three isoforms: cncA, cncB, and cncC. The expression patterns of the three transcript isoforms were analyzed using exon-specific probes both on wild-type and EMS-induced cnc mutants. In wild-type embryos, a cncB probe detects cytoplasmic transcripts limited to the mandibular and labral segments from cellular blastoderm to the end of embryogenesis. The cncB transcripts are expressed throughout both anterior and posterior regions of the mandibular lobes. In contrast, a cncA-specific probe detects a ubiquitous distribution of presumably maternal RNA at syncytial and early cellular blastoderm stages. After cellular blastoderm, cncA transcripts are not detectable until stage 14, when the level of ubiquitous cytoplasmic transcript increases and remains high for the remainder of embryogenesis. cncC-specific probes also detect a ubiquitous distribution of mRNA in syncytial stage embryos and a low level ubiquitous expression pattern in embryos after stage 14 (McGinnis, 1998).

Based on the above results, the labral and mandibular stripes of transcription that were detected by Mohler, (1991) using a probe including the cnc common exons (A2 and A3), correspond primarily to cncB transcripts. Since cncB is the transcript isoform that is expressed throughout the entire mandibular segment during mid-embyronic stages, cncB is likely to encode the principal function that modulates Dfd function in the mandibular segment (Harding, 1995). To further test this hypothesis, an assay was carried out to see whether cncB transcript or protein abundance is altered in embryos homozygous for the cnc^2E16 and cnc^C7, mutations known to reveal interaction with Dfd. The pattern of zygotic RNA expression detected with a cncB probe is unaltered in the EMS-induced cnc mutant embryos. The signal due to cncA and cncC transcripts is also unchanged in these mutants. However, the use of polyclonal antiserum raised against the common domain of the cnc isoforms (anti-Cnc) indicates that CncB protein expression is strikingly reduced in both cnc^2E16 and cnc^C7 mutant embryos. In wild-type embryos, the anti-Cnc antiserum exhibits a low-level ubiquitous staining in syncytial embryos, presumably due to maternally deposited CncA and CncC isoforms. From cellular blastoderm (stage 5) until stage 14, the staining detected by the anti-Cnc antiserum is localized in the nuclei of mandibular and labral cells. Although the anti-Cnc antiserum used in these experiments cross-reacts with all three Cnc proteins, only cncB RNA expression is localized in mandibular and hypopharyngeal regions from stages 6 through 14. cnc^2E16 mutants (and cnc^C7 mutants) accumulate much lower levels of Cnc antigen in both mandibular and labral cells of stage 11 embryos. These results provide further evidence that the cnc^2E16 and cnc^C7 mutations result in a loss of cncB function, and is consistent with the idea that CncB protein is required to prevent the maxillary-promoting function of Dfd from being active in mandibular cells (McGinnis, 1998).

In another test of the functions of the Cnc protein isoforms, each of the cncA, cncB and cncC open reading frames were placed under the control of the heat-shock promoter in P-element vectors and transgenic fly strains were generated carrying these constructs. Using the Cnc common-region antiserum to stain heat-shocked embryos, it appears that all three isoforms are produced at similar levels, localized in nuclei and possess similar stabilities after ectopic expression. However, their morphogenetic and regulatory effects are quite dissimilar. Heat-shock-induced ectopic expression of CncA during embryogenesis has no effect on embryonic morphology. Nearly all of the hs-cncA embryos hatch and proceed through larval development, and many eclose as viable adults. In contrast, ectopic expression of CncB at mid-stages (4-10 hours) of embryonic development is lethal. When ectopic expression is induced at 6 to 8 hours after egg lay, a defective embryonic head phenotype, which resembles the mutant phenotype of strong Dfd hypomorphs is produced. These hs-cncB embryos develop with rudimentary mouth hooks, H-piece and cirri. In addition, the anterior portion of the lateralgräten are truncated. All of these structures are components of the head skeleton that are absent or abnormal in Dfd mutant embryos. The head defects seen in the hs-cncB embryos also include an absent or abnormal dorsal bridge, a structure that is usually unaffected in Dfd mutant embryos. Many other head structures that develop in a Dfd-independent manner, such as the antennal sense organ, vertical plates and T-ribs develop normally in the hs-cncB embryos. The hs-cncB head defects are produced at high penetrance (>95%) by heat shocks in mid-embryogenesis (4-10 hours). In 10%-70% of these embryos, depending on the stage of heat shock, abdominal denticles near the ventral midline are replaced with naked cuticle. Ectopic induction of hs-cncC at 6-8 hours of development also results in highly penetrant defects in head development that include the loss of maxillary mouth hooks and cirri as well as head involution defects that are more profound than those induced by hs-cncB. In addition to the morphological defects described for CncB, ectopic CncC induces the formation of an abnormal head sclerite that develops as an extension of the normal lateralgräten. The position and appearance of this extra fragment of head skeleton suggests that it might correspond to ectopic production of lateralgräten or longitudinal arms of the H-piece (McGinnis, 1998).

Since CncB encodes a function that is required and sufficient to antagonize the maxillary-promoting effects of the Hox gene Dfd, it is reasonable to ask if CncB protein acts upstream to repress Dfd transcription, or in parallel to inhibit Dfd protein function? It is possible for CncB to do both, since Dfd protein function is required to establish an autoactivation circuit that provides persistent Dfd transcription in maxillary and mandibular cells. In wild-type embryos at stage 9, both Dfd and CncB proteins are expressed throughout the entire mandibular segment. By stage 11, Dfd protein is present at lower levels in the anterior, when compared to posterior mandibular nuclei, while CncB protein persists at relatively high levels throughout the segment. Finally, at stage 13, Dfd protein expression is no longer detected in anterior mandibular nuclei, although it is still abundant in posterior nuclei. cnc is required for this progressive repression of Dfd expression in the anterior mandibular segment, since cnc null mutants as well as the EMS-induced mutants show inappropriate persistence of Dfd transcripts and protein after stage 11 in anterior mandibular cells. All of these data suggest that CncB is not capable of repressing Dfd expression before stage 11. But after this stage, CncB represses the maintenance phase of Dfd transcription in mandibular cells, perhaps by repressing the autoactivation circuit that is normally established during stages 9 and 10 (Zeng et al., 1994). CncB is found to be sufficient to repress Dfd transcription outside the mandibular segment. When CncB is ectopically expressed in embryos, Dfd transcript levels in the maxillary segment are reduced, especially in the anterior region of the segment. Only the CncB isoform is capable of repressing Dfd transcription. Neither the ectopic expression of CncA nor CncC have an effect on the abundance or pattern of Dfd transcripts in the maxillary epidermis. Since the phenotypic effect of hs-cncC in epidermal cells strongly resembles that of hs-cncB, this indicates that the effect of Cnc gene products on maxillary epidermal development may not require repression of Dfd transcription per se. However, various experiments show that the maxillary-promoting function of Dfd protein is reduced in the presence of CncB; this could either be due to CncB-mediated repression of the Dfd autoactivation circuit in ectopic positions or to CncB repression of downstream target elements of Dfd protein, or to both of these effects. It is concluded that CncB provides a mechanism to modulate the specificity of Hox morphogenetic outcomes, which results in an increase in the segmental diversity in the Drosophila head (McGinnis, 1998).

The cnc locus encodes three transcript and protein isoforms. The cncB transcript is expressed in an embryonic pattern that includes the labral, intercalary and mandibular segments, while cncA and cncC are expressed ubiquitously. CncB suppresses the segmental identity function of the Hox gene Deformed (Dfd) in the mandibular segment of Drosophila embryos. Evidence has been provided that the CncB-mediated suppression of Dfd requires the Drosophila homolog of the mammalian small Maf proteins, Maf-S, and that the suppression occurs even in the presence of high amounts of Dfd protein. Interestingly, the CncB/Maf-S suppressive effect can be partially reversed by overexpression of Homothorax (Hth), suggesting that Hth and Extradenticle proteins antagonize the effects of CncB/Maf-S on Dfd function in the mandibular segment. In embryos, multimers of simple CncB/Maf-S heterodimer sites are transcriptionally activated in response to CncB, and in tissue culture cells the amino-terminal domain of CncB acts as a strong transcriptional activation domain. There are no good matches to CncB/Maf binding consensus sites in the known elements that are activated in response to Dfd and repressed in a CncB-dependent fashion. This suggests that some of the suppressive effect of CncB/Maf-S proteins on Dfd protein function might be exerted indirectly, while some may be exerted by direct binding to as yet uncharacterized Dfd response elements. Ectopic CncB is sufficient to transform ventral epidermis in the trunk into repetitive arrays of ventral pharynx. The functions of CncB are compared to those of its vertebrate and invertebrate homologs, p45 NF-E2, Nrf and Skn-1 proteins, and it is suggested that the pharynx selector function of CncB is highly conserved on some branches of the evolutionary tree (Veraksa, 2000).

Mammalian homologs of Cnc (p45 NF-E2 and Nrf proteins) have been shown to bind DNA as obligate heterodimers with small Maf proteins (MafK/p18, MafF and MafG). An apparent Drosophila ortholog of the mammalian small Mafs was identified in a yeast two-hybrid screen for genes encoding peptides capable of interacting with the common b-Zip domain of Cnc proteins. BLAST searches with a corresponding cDNA sequence detect only one small maf gene, corresponding to CG9954, in the near complete Drosophila genome. The gene has been called Drosophila maf-S (S for Small), and the protein Maf-S. In the basic region that contacts DNA, Maf-S shares a domain of perfect identity with mammalian small Maf proteins, and partial identity in the leucine zipper and other protein regions. In situ hybridizations show that maf-S RNA is maternally deposited in the Drosophila egg, and the transcripts are present in all or nearly all embryonic cells throughout development, making the Maf-S protein available for potential interactions with all Cnc isoforms. In addition, there is an enrichment in maf-S transcripts in certain areas of the embryo, against the ubiquitous expression background. At stage 8, slightly higher levels are detected around cephalic furrow and in the mesoderm. At stage 11 and beyond, there is an enrichment in maf-S transcripts in the anterior and posterior midgut, in the nervous system, as well as in the involuting intercalary segment that will give rise to ventral pharynx (Veraksa, 2000).

Double stranded RNA interference was used in an attempt to test the phenotypic effects of loss of maf-S function. After injection of maf-S dsRNA into the head region of precellular blastoderm embryos, only 1.5% of embryos hatched as first instar larvae. The cuticles of injected embryos show head defects that are remarkably similar to those found in cncB mutants: duplications of mouth hooks, shortened lateralgraten, missing or deformed median tooth and truncated ventral pharynx. Immunostaining of maf-S dsRNA-injected embryos with Cnc-specific antibody reveals that the levels of CncB protein are not significantly different from wild type. Results of the dsRNA interference experiment suggest that Maf-S is an obligate functional partner of CncB. Without the small Maf subunit, CncB is unable to promote the development of intercalary, mandibular and labral head structures and to repress the function of Dfd. The maf-S gene, which maps to chromosomal segment 57A, is uncovered by Df(2R)exu². Homozygotes for this deletion, which lack the zygotic dose of maf-S and other nearby genes, exhibit mild head defects that include disruptions of the pattern of ventral pharyngeal transverse ribs (T-ribs). The weakness of this phenotype relative to the RNAi result may be due to the maternal contribution from maf-S, which is unaffected in the zygotic deletion mutants (Veraksa, 2000).

Previous genetic and molecular data have established that CncB has several functions in the developing Drosophila embryo: it is required for the formation of labral and intercalary structures such as median tooth and pharynx, and it is required to inhibit Dfd function in the mandibular segment. A summary model has been proposed in which these functions are mediated in part by the transcriptional activator properties of CncB, and are achieved through specific DNA binding with Maf-S to an 11-bp CncB/Maf-S heterodimer binding site. In the intercalary and anterior mandibular segments of the embryo, heterodimerization of CncB with Maf can activate some downstream target genes, and the heterodimers are required to develop ventral pharyngeal structures (T-ribs and ventral arms). Furthermore, CncB, in the context of the ventral epidermis of other head and trunk segments, is sufficient to promote pharynx identity, and thus cncB functions as a pharynx selector gene (Veraksa, 2000).

The function of CncB in suppressing the maxillary-promoting function of Dfd is carried out in the mandibular cells. Early in development CncB and Dfd are coexpressed throughout the mandibular segment, but by stage 13 Dfd expression retracts and becomes localized to a row of cells in the posterior of the segment. In these cells, CncB and Dfd are both required to specify lateralgraten and the base of the mouth hook. Although Dfd function is required for the normal structures that derive from the posterior mandibular segment, the maxillary-promoting function of Dfd is inhibited in these cells. In the anterior mandibular segment, CncB represses both the function and expression of Dfd. Since Dfd protein activates its own transcription, inhibition of Dfd function inevitably results in silencing the endogenous transcription of the Dfd gene. Persistent expression of Dfd in the posterior mandibular cells is likely to be mediated by regulatory regions that are different from the autoactivation enhancers and are insensitive to CncB-mediated repression. Based on the present data, it is suggested that CncB-mediated repression of Dfd function is at least in part indirect, for several reasons. (1) There are no close matches to the consensus CncB/Maf-S binding site in known Dfd response elements, despite the fact that these elements can be repressed by ectopic expression of CncB, e.g. modules C, E, and F from the 2.7 kb Dfd epidermal autoregulatory enhancer. (2) The CncB-specific amino terminal region, crucial for the suppression of Dfd function, contains a strong transcriptional activation domain. (3) A CncA fusion protein with a simple heterologous transcriptional activation domain from VP16 can also partially repress Dfd-dependent maxillary structures upon overexpression. (4) Chimeric Dfd response elements containing Cnc/Maf binding sites adjacent to Dfd binding sites are not repressed by overexpression of CncB, as might be expected if the action of CncB on Dfd were direct. Instead, the endogenous Dfd-dependent maxillary expression of 4CE is enhanced in HS-CncB embryos when compared to non-heat-shocked controls. Suppression of Dfd function by CncB may therefore be achieved by transcriptional activation of an intermediate gene encoding a repressor or corepressor that would interfere with the function of Dfd. Another possibility is that CncB might compete for a common cofactor or coactivator (such as Exd, Hth or CBP), or it might activate a specific Dfd modifying enzyme such as a kinase or phosphatase. It is still possible that the suppressive effect that CncB protein exerts on Dfd function may involve CncB mediated transcriptional repression on as yet uncharacterized response elements. A binding interaction between Dfd and CncB proteins has been observed in GST pull-down assays, but the biological significance of this result is as yet unclear (Veraksa, 2000).

Repression of Dfd function by CncB is analogous to the phenomenon of phenotypic suppression (also known as posterior prevalence), which denotes the ability of more posterior Hox proteins to suppress the function of more anterior Hox proteins in the same cells. 'Anterior prevalence' of CncB over Dfd provides additional morphological diversity in the mandibular and maxillary segments. The indirect way by which CncB acts on Dfd maxillary function has a precedent in Hox phenotypic suppression. The inhibitory effects of posterior Hox proteins on Antennapedia function can apparently be exerted via an indirect pathway involving phosphorylation of Antp protein by casein kinase II. Dfd protein is also phosphorylated in vitro by CKII, but whether this phosphorylation is relevant to the suppression by CncB is unknown (Veraksa, 2000).

trithorax encodes a positive regulatory factor required throughout development for normal expression of multiple homeotic genes of the bithorax and Antennapedia complexes (BX-C and ANTP-C). To determine how trx influences homeotic gene expression, the expression of the BX-C genes Ultrabithorax, abdominal-A, Abdominal-B and the ANTP-C genes Antennapedia, Sex combs reduced and Deformed were examined in trx embryos. Each of these genes have different requirements for trx in different spatial contexts in order to achieve normal expression levels (Breen, 1993).

Dissecting the functional specificities of two Hox proteins

Hox proteins frequently select and regulate their specific target genes with the help of cofactors like Extradenticle (Exd) and Homothorax (Hth). For the Drosophila Hox protein Sex combs reduced (Scr), Exd has been shown to position a normally unstructured portion of Scr so that two basic amino acid side chains can insert into the minor groove of an Scr-specific DNA-binding site. This study provides evidence that another Drosophila Hox protein, Deformed (Dfd), uses a very similar mechanism to achieve specificity in vivo, thus generalizing this mechanism. Furthermore, it was shown that subtle differences in the way Dfd and Scr recognize their specific binding sites, in conjunction with non-DNA-binding domains, influence whether the target gene is transcriptionally activated or repressed. These results suggest that the interaction between these DNA-binding proteins and the DNA-binding site determines the architecture of the Hox-cofactor-DNA ternary complex, which in turn determines whether the complex recruits coactivators or corepressors (Joshi, 2010).

Previous work on Scr's ability to specifically regulate its target gene, fkh, revealed that the N-terminal arm of its homeodomain and preceding linker region are positioned in such a manner as to allow the insertion of two basic side chains into the minor groove of the target DNA, fkh250 (Joshi, 2007). Importantly, the correct positioning of these residues depends on an interaction between Scr's YPWM motif and the cofactor Exd. This study shows that an analogous mechanism is required for Dfd to bind productively to a Hox-Exd-binding site in the EAE element and to activate EAE-lacZ in vivo. Specifically, it was found that Dfd's YPWM motif is required for cooperative binding to EAE's site I in vitro, and for executing Dfd-specific functions in vivo. Like Scr, Dfd has the same two basic residues -- a histidine (likely to be protonated when bound to DNA) and an arginine -- at the equivalent positions relative to its YPWM motif and homeodomain. Moreover, these residues are also required for Dfd to execute its specific functions in vivo. Thus, the activation of fkh by Scr and the activation of Dfd by Dfd appear to use analogous mechanisms, whereby linker and N-terminal arm residues are used to bind paralog-specific binding sites in an Exd-dependent manner (Joshi, 2010).

The YPWM-to-YPAA mutation severely impaired Dfd's ability to carry out its specific in vivo functions, such as activation of EAE-lacZ and production of cirri. Thus, the YPWM motif of Dfd is critical for Dfd function in vivo. This situation contrasts with other apparently more complex scenarios. For example, mutation of the YPWM motif of the Hox protein Ultrabithorax (Ubx) did not significantly impair some of its in vivo functions. In this case, it appears that other sequence motifs, in particular a domain C-terminal to the Ubx homeodomain, are important for Ubx to carry out its specific functions in vivo. These Ubx sequences also appear to help recruit Exd to DNA, and therefore may be used for binding site selection in conjunction with YPWM at a subset of Ubx target-binding sites. Interestingly, a sequence motif immediately C-terminal to Dfd's homeodomain also plays a role in in vivo specificity, although its impact on DNA binding has not been examined. As these sequences are still present in Dfd^ScrSMδ23, it may explain why this chimera retains some Dfd-specific functions, such as the formation of cirri and ability to activate EAE-lacZ. The picture that emerges from all of these data is that Hox proteins may use different motifs to interact with cofactors such as Exd, depending on the specific in vivo function and target gene being regulated (Joshi, 2010).

In general, the sequences surrounding Hox YPWM motifs and the N-terminal arms of their homeodomains are highly conserved, from invertebrates to vertebrates, in a paralog-specific manner (Joshi, 2007). Thus, based on the results presented in this study, it is hypothesized that these sequences, which are referred to as Hox specificity modules, may in general be used for the recognition of specific DNA-binding sites in a cofactor-dependent manner. In the case of Scr binding to fkh250, an X-ray crystal structure revealed that the histidine and arginine side chains recognize an unusually narrow minor groove that is an intrinsic feature of the fkh250-binding site. Without the benefit of a Dfd-Exd-site I crystal structure, it cannot be know with certainty if Dfd's His-15 and Arg3 also read the shape of a narrow minor groove. However, the fact that the same two basic residues are required for both Scr and Dfd suggests the possibility that this is the case for Dfd binding to EAE site I as well (Joshi, 2010).

Dfd^ScrSMδ23, which has the specificity module of Scr in place of Dfd's, exhibited clear Scr-like functions in vivo, as assayed by fkh250-lacZ activation and larval cuticle transformation. Other attempts to swap Hox specificities by generating chimeric Hox proteins have had variable success. For example, when the linker and N-terminal arm of Scr is used to replace the equivalent region of Antennapedia (Antp), the chimera behaved like Scr. This finding supports the importance of specificity modules in conferring Hox specificity. When the homeodomain and C-terminal region of Ubx were replaced by the equivalent domains from Antp, the chimera behaved like Antp, suggesting that the identity of the linker region may not be critical in all cases. Other Hox chimeras have generated less clear changes of specificity. For example, chimeras between Ubx and Dfd generated a cuticle phenotype that was dissimilar to that produced by either parent protein. Similarly, a chimera between Ubx and Abd-B had novel properties that were unlike those produced by either parent protein. It is noteworthy that the cleanest changes in specificity occurred when the chimera was generated between Hox genes that are adjacent to each other in the Hox complex. This correlation may be due to the fact that adjacent Hox genes are more similar to each other, both in sequence and in function, than nonadjacent Hox genes. This higher degree of similarity is likely a consequence of how these genes are thought to have duplicated during evolution (Joshi, 2010).

Previous work on the regulation of fkh by Scr, the reporter gene used to study the activity of the Exd-Scr-binding site had a multimerized version of the minimal 37-bp fkh250 element. In contrast, in the work described in this study, an intact regulatory element from the Dfd gene was characterized, revealing significantly more complexity. In particular, the 570-bp modC element contains a single 'classical' Exd-Hox composite site, but also four additional Dfd sites and several additional Exd-Hth-binding sites. Mutagenesis studies suggest that all of these inputs are important for the full activity of this enhancer. Also noteworthy is that there are additional Dfd-Exd-binding sites in the larger 2.7-kb EAE element that, in principle, could also be used in vivo. Thus, the picture that emerges from this analysis is that native enhancer elements may use a combination of classical Exd-Hox-binding sites together with additional arrangements that may not always conform to the classical spacing of the Exd and Hox half-sites. This picture raises the question of how the linker and N-terminal arm residues are positioned correctly in these nonclassical arrangements. The answer may lie in the fact that, in vivo, the assembly of the complete multiprotein complex -- which is likely to include factors in addition to Dfd, Exd, and Hth -- promotes the recognition of Dfd-binding sites in ways that are not fully revealed by experiments that examine binding to individual or small groups of binding sites in isolation (Joshi, 2010).

Depending on the context, most transcription factors have the capacity to activate and repress transcription. In most cases, it is not understood how this choice is made. One established scenario is that other proteins that get recruited to an enhancer element determine the sign of the regulation. However, this type of model is not sufficient to explain the results presented in this study. The results suggest that the DNA-binding properties of the Exd-Hox complex influence the regulatory output of the bound protein-DNA complex. Deletion of two motifs (γ23) from the N-terminal region of Dfd^ScrSM converted this protein from a repressor of fkh250-lacZ to an activator of fkh250-lacZ, while deletion of the same motifs from Dfd^WT did not change the regulatory output: The protein retained its ability to repress fkh250-lacZ. The only difference between Dfdγ23 (represses) and Dfd^ScrSMγ23 (activates) is the specificity module, and the only difference between Dfd^ScrSMγ23 (activates) and Dfd^ScrSM (represses) is the presence or absence of motifs 2 and 3. These results imply that the relevance of motifs 2 and 3, which are far from the DNA-binding domain, depends on the identity of the specificity module. These findings lead to a suggestion that the DNA-binding site, together with how it is read by the specificity module, plays an important role in determining the overall conformation of the Hox-Exd complex, which eventually determines whether there will be recruitment of a coactivator or corepressor. This idea fits well with a DNA allostery model that was supported recently by cell culture experiments with the glucocorticoid receptor. In these experiments, it was discovered that small differences in the DNA-binding site lead to differences in conformation and the degree of transcriptional activation. This study extends this idea by showing that Hox proteins with different specificity modules, and therefore with slightly different DNA recognition properties, result in unique regulatory outputs in an in vivo context. Furthermore, in these experiments, a complete change was observed in the sign of the regulation from repression to activation, instead of a more subtle change of activation amplitude. Thus, the transcriptional output of a Hox-cofactor complex depends both on the ability of these complexes to bind to their binding sites with high specificity, in part by reading structural features of the DNA, and on the three-dimensional architecture of the bound complex, which is a consequence of both protein-DNA and protein-protein interactions. An important goal for the future will be to use structural biology methods to see how different Hox specificity modules result in distinct conformations of Exd-Hox complexes (Joshi, 2010).

Targets of Activity

Distal-less is a downstream gene of the homeotic selector (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 (O’Hara, 1993).

The genetic function of the homeotic gene Deformed (< I>Dfd) is required in the cnc mutant background to produce ectopic mouth hooks, and it has been proposed that Dfd and cnc function in combination to specify mandibular identity. One of the downstream genes that is activated by Dfd in maxillary cells is Distal-less (Dll). Dll is required for the formation of the larval appendage primordia and the distal regions of adult appendages. In the maxillary segment, Dll is expressed in two patches of cells: a dorsal patch that gives rise to the maxillary sense organ and a ventral patch that consists of the primordia for the maxillary cirri. The dorsal maxillary domain of Dll expression is largely independent of Dfd function, while the ventral maxillary patch of Dll is activated by Dfd through a 3' enhancer (O’Hara, 1993). In cnc^2E16 mutant embryos, Dll is ectopically expressed in ventral mandibular cells, suggesting that cncB, one of the transcripts coded for by cnc, represses Dll transcription in mandibular cells. In hs-cncB embryos 30 minutes after heat shock, when Dfd protein abundance is normal, Dll expression is repressed in the ventral maxillary segment but other domains of Dll expression in the head and thorax are relatively unaffected, indicating that CncB selectively represses the Dfd-dependent portion of the Dll expression pattern. In hs-cncC and hs-cncA embryos, the ventral maxillary expression of Dll is not selectively repressed. Reporter gene expression from the 3' enhancer follows the expression of Dll as the enhancer is ectopically activated in the ventral mandibular region in cnc mutants and repressed in hs-cncB embryos (McGinnis, 1998).

Diversification of Drosophila segmental morphologies requires the function of Hox transcription factors. However, little information is available that describes pathways through which Hox activities effect the discrete cellular changes that diversify segmental architecture. Serrate is a Hox gene target. Serrate acts in many segments as a component of such pathways. In the embryonic epidermis, Serrate is required for morphogenesis of normal abdominal denticle belts and maxillary mouth hooks, both Hox-dependent structures. The Hox genes Ultrabithorax and abdominal-A are required to activate an early stripe of Serrate transcription in abdominal segments. In the abdominal epidermis, Serrate promotes denticle diversity by precisely localizing a single cell stripe of rhomboid expression, which generates a source of EGF signal that is not produced in thoracic epidermis. In the head, Deformed is required to activate Serrate transcription in the maxillary segment, a region where Serrate is required for normal mouth hook morphogenesis. However, Serrate does not require rhomboid function in the maxillary segment, suggesting that the Hox-Serrate pathway to segment-specific morphogenesis can be linked to more than one downstream function (Wiellette, 1999).

A single allele of the Serrate gene (Ser^5A29 ) has been isolated in a screen for mutations that enhanced hypomorphic phenotypes of mutations in the Hox gene Dfd. The Ser^5A29 allele reduces the survival of Dfd hypomorphs to 40% of normal levels. To test whether this interaction with Dfd is allele-specific, null mutants of Ser were tested. The interaction strength of two such alleles with Dfd is 20-30% of the control chromosomes. Therefore, it is concluded that the function of Dfd is sensitive to the dose of wild-type Ser activity (Wiellette, 1999).

To explore the connection between Ser and Dfd functions, the embryonic/larval phenotypes of Ser mutants were examined. The Ser larval phenotype consists of blunt mouth hooks, anterior spiracle malformations and small imaginal discs in larvae of indeterminate age. Some animals lacking Ser die as embryos without emerging from the chorion, while others survive for variable periods as larvae. In both classes, the mouth hooks are blunt, lacking the sclerotic material that forms a curved hook in the normal structure. Since the mouth hook is a Dfd-dependent structure that is formed principally from maxillary cells, the requirement for Ser in normal mouth hook development is consistent with the genetic interaction between Ser and Dfd. Additionally, Ser mutant larvae have abnormally patterned abdominal denticle belts. Within each wild-type denticle belt from A2 to A8, the row 3 and 4 denticles have similar sizes and shapes but the hooks of row 3 point to the posterior, and those of row 4 point to the anterior. In Ser mutants, these two rows of denticles are combined into one row, the 3/4 row. The denticles in the 3/4 row have no regular polarity and about half of the embryos develop small denticles in this row. To determine whether the 3/4 row of denticles in Ser mutant larvae is a fusion of the two rows or a loss of one, denticles were counted, using the fourth abdominal segment (A4) as an example. The results are inconsistent with complete loss of either row 3 or row 4. Previous experiments have failed to detect any gross morphological defects in the cuticular features of embryos when Ser is ubiquitously expressed throughout Drosophila embryos. Using a Ser cDNA under Gal4-UAS control, the phenotypic effects of ectopic Ser were also assayed, focusing on denticle morphology. Ubiquitous expression of Ser induces no detectable changes in the shape, size or pattern of denticles. However, when Ser is ubiquitously expressed in a Ser heterozygous or null background, significant morphological defects are observed. The mouth hooks develop additional sclerotic material in the middle and base of the structure, and about a quarter of the embryos show duplication of mouth hook tips. Excessive sclerotic material also develops in the dorsal pouch. The expressivity of this phenotype varies from an extended and fragmented dorsal bridge to extreme sclerotization of the dorsal pouch and shortening of the lateralgräten. About half of the denticle belts in embryos of this genotype display the wild-type pattern but only rarely do embryos develop extra denticles around row 3. An additional result of ectopic Ser expression in a reduced-dosage background is excessive sclerotization of the proventriculus and gut. Both the dorsal pouch and the proventriculus are responsible for secretion of specialized cuticle to make head sclerites and gut lining, respectively (Wiellette, 1999).

Ser expression begins during embryonic stage 11, and eventually is transcribed in regions of the epidermis, tracheal trunks, foregut and hindgut, central nervous system and salivary ducts. Because of the phenotypic effect on mouth hooks, Ser expression was examined in the embryonic head. The transcript pattern in the head region at stage 12 includes the mandibular segment, the anterior and posterior lateral borders of the maxillary segment, and the anterior lateral and posterior ventral borders of the labial segment. A subset of cells in the clypeolabrum and dorsal head also accumulate Ser transcripts. The dependence of Ser expression on Dfd function and vice versa was tested and an epistatic relationship was found consistent with the genetic interaction. While there is no detectable change of Dfd expression in Ser mutants, Dfd and other head homeotic genes are required for the normal pattern of Ser transcription in the head. In Dfd mutant embryos, Ser transcripts are not expressed in cells in the anterior maxillary segment, which will eventually secrete part of the mouth hook. The posterior maxillary pattern is unchanged. When ectopic expression of Dfd protein is generated by heat shock (hs-Dfd), ectopic mouth hooks form in the labial and thoracic segments. These segments also express ectopic Ser on the lateral anterior and posterior borders. Thus, regulation of Ser by Dfd correlates with the segments where both normal and ectopic mouth hooks develop; it is concluded that Ser is one of the Dfd target genes that mediate mouth hook development. In Sex combs reduced mutant embryos, Ser transcripts are not expressed in the posterior maxillary segment or the anterior labial segment (Wiellette, 1999).

Homeotic genes of Drosophila encode transcription factors that specify segment identity by activating the appropriate set of target genes required to produce segment-specific characteristics. Advances in understanding target gene selection have been hampered by the lack of genes known to be directly regulated by the HOM-C proteins. Evidence is presented that the gene 1.28, coding for a glycine-rich domain/Proline-rich domain protein, is likely to be a direct target of Deformed in the maxillary segment. A 664-bp Deformed Response Element (1.28 DRE) has been identified that directs maxillary-specific expression of a reporter gene in transgenic embryos. The 1.28 DRE contains in vitro binding sites for Deformed and DEAF-1. The Deformed binding sites do not have the consensus sequence for cooperative binding with the cofactor Extradenticle, and no cooperative binding to these sites is detected, though an independent role for Extradenticle cannot be ruled out. Removing the four Deformed binding sites renders the 1.28 DRE inactive in vivo, demonstrating that these sites are necessary for activation of this enhancer element, and supporting the proposition that 1.28 is activated by Deformed. Comparisons of the 1.28 DRE with other known Deformed-responsive enhancers indicate that there are multiple ways to construct Deformed Response Elements (Pederson, 2000).

To function as a maxillary enhancer the 120 bp module E requires at least one sequence in addition to the Deformed and Exd binding sites. This sequence is found in an imperfect inverted repeat. Site-directed mutagenesis of this imperfect inverted repeat abolishes module E enhancer function. Though it seemed noteworthy that a similar imperfect inverted repeat sequence is located within the 1.28 DRE, that sequence is not required for 1.28 DRE enhancer function, and deletion of this repeat has no consequence on expression of the endogenous 1.28 gene. Attaching the module E inverted repeat sequence to the Deformed binding portion of the 1.28 DRE does increase activity of the 1.28 DRE, indicating that this sequence can function in a heterologous enhancer. The idea is favored that the module E inverted repeat region contains a binding site or sites for other unknown factors, and that these factors act to enhance maxillary-specific expression. Such a site is likely to be within the inverted repeat sequence of module E. It has been proposed that factors bind to sequences GGC and AAAGC of the module E repeat. This sequence is not present in the 1.28 DRE, suggesting again that regulation through these two enhancers uses different mechanisms (Pederson, 2000 and references therein).

The DEAF-1 protein was initially hypothesized to be an activator involved in Deformed autoregulation because it binds tightly to the inverted repeat region of module E. However, accumulating evidence indicates that this may not be the case. The DEAF-1 binding site is located in region 6 of module E. Though this region is necessary for maxillary enhancer function, eliminating the DEAF-1 binding does not alter the ability of this fragment to be a maxillary enhancer. The DEAF-1 binding region of the 1.28 DRE does not enhance maxillary expression of either the 1.28 DRE or the module E Deformed binding sites, and furthermore, in both cases this region suppresses the weak, endogenous activity often observed for the pHZ-white reporter alone. The DEAF-1 binding region appears to, at least under some circumstances, act as a negative element. DEAF-1 perhaps does play a role in expression, as a repressor (Pederson, 2000).

Hox proteins control morphological diversity along the anterior-posterior body axis of animals, but the cellular processes they directly regulate are poorly understood. During early Drosophila development, the Hox protein Deformed (Dfd) maintains the boundary between the maxillary and mandibular head lobes by activating localized apoptosis. Dfd accomplishes this by directly activating the cell death promoting gene reaper (rpr). One other Hox gene, Abdominal-B (Abd-B), also regulates segment boundaries through the regional activation of apoptosis. Thus, one mechanism used by Drosophila Hox genes to modulate segmental morphology is to regulate programmed cell death, which literally sculpts segments into distinct shapes. This and other emerging evidence suggests that Hox proteins may often regulate the maintenance of segment boundaries (Lohmann, 2002).

Several lines of evidence -- the effects of manipulating rpr expression in embryos, in vitro DNA binding studies with Dfd protein and mutagenesis of Dfd binding sites in the rpr enhancer, the phenocopy of the Dfd mutant boundary defect with an apoptosis inhibitory gene, its rescue with an apoptosis promoting gene, and the phenotype of rpr mutants -- show that the Hox protein Dfd is a direct transcriptional activator of rpr in the anterior maxillary segment, and that rpr expression and apoptosis are necessary to maintain the maxillary/mandibular boundary. At least in part, this Dfd-dependent, anterior maxillary expression of rpr is conferred by a 674 bp enhancer (rpr 4-S3) that maps 3.1 kb upstream of the rpr transcription start. This demonstrates that a Hox protein directly regulates a cell biological effector gene that mediates a morphological subroutine for that Hox function. Therefore, rpr qualifies as a directly regulated realizator. Interestingly, although Dfd is expressed in nearly all maxillary cells, the loss of Dfd function does not influence rpr expression in the posterior maxillary segment, indicating that other activators and/or repressors of rpr are distributed in maxillary cells that influence the transcriptional activity of Dfd protein on this locus. In the tail region, Abd-B is also required for the formation of normal boundaries between the abdominal segments A6/A7 and A7/A8, and their maintenance correlates, as in the case of the maxillary/mandibular boundary, with the localized activation of rpr. Thus, at both termini of the Drosophila body, Hox control of apoptosis is used for segment boundary maintenance (Lohmann, 2002).

Hox proteins may have a wider role in the programming of segmental boundaries than is currently believed. There is strong evidence that two Drosophila homeobox genes that are used to control segment number, even-skipped and fushi-tarazu, are independently derived from genes that still possess Hox segment identity functions in most insects and other arthropods. In addition, mutants in the mouse Hoxa-2 gene have segmentation defects in the hindbrain. Although a segmental boundary is normally established between rhombomeres 1 and 2 in the Hoxa-2 mutants, it is not maintained, which is reminiscent of the defect in boundary maintenance observed in Dfd mutant embryos (Lohmann, 2002).

Surprisingly, although rpr is required for maxillary/mandibular boundary maintenance during embryogenesis, flies lacking rpr function survive to adulthood with only minor defects. One possible explanation is that other cell death activators can compensate for the absence of rpr at later stages of development, since other apoptosis genes, like hid, grim, and sickle, are expressed in overlapping patterns with rpr and share IAP binding motifs in their N-terminal protein sequence. This may also explain why the maxillary/mandibular segmentation defect is less severe in Dfd null mutants and in XR38/H99 mutants when compared to homozygous Df(3L)H99 mutants. Since rpr and hid, but not grim, are expressed in anterior maxillary cells at many developmental stages, and since the combined activities of hid and rpr dictate the probability of a cell to undergo apoptosis, it is suggested that in wild-type embryos the combination of rpr and hid are required to kill cells at the maxillary/mandibular boundary (Lohmann, 2002).

Many Drosophila genes are known to be regulated in a Hox-dependent manner, but most encode either transcriptional regulators or cell signaling molecules. These Hox effectors presumably act both independently and/or in parallel to Hox genes to indirectly influence cell type identity and morphology. For example, the Hox target gene Distal-less (Dll) is required for the development of embryonic appendages and is directly repressed in abdominal segments by the Hox proteins Ubx and Abd-A. However, at some point the Hox proteins, their downstream effectors, and other cofactors must affect cellular changes by means of the class of realizer genes (Lohmann, 2002).

There are three good candidates for realizer genes in Drosophila: connectin, centrosomin, and ß-tubulin. connectin encodes an extracellular cell surface protein with leucine-rich repeats. It mediates cell-cell adhesion in cell culture assays and acts as a homophilic cell adhesion molecule in the lateral transverse muscles. In the nervous system, connectin expression is under the control of Ubx; a small regulatory fragment that mediates portions of connectin expression has been isolated by its affinity for Ubx in coimmunoprecitation assays. In some tissues, connectin is under the direct control of Ubx protein, but it is still unclear which of the morphogenetic subfunctions of Ubx require connectin function. centrosomin is a subunit of the centrosome and is necessary for the proper development of the CNS, PNS, and midgut. During the formation of the second midgut constriction, the functions of both Ubx and centrosomin are required, and centrosomin is lost in the visceral mesoderm cells of Ubx mutants. The ß-tubulin gene encodes a major component of microtubules and contains a cis-regulatory element that is regulated by Ubx in the visceral mesoderm (Lohmann, 2002).

In Drosophila, as in vertebrates, programmed cell death is used for the sculpting of morphological structures. For example, limb formation in amniotes is accompanied by massive cell death in almost all the interdigital mesenchymal tissue located between the chondrifying digits, eliminating the cells located between the differentiating cartilages and thus sculpting the shape of the limb. Interestingly, in Hoxa13 heterozygous mutant mice, the apoptosis that normally occurs in the interdigital regions is reduced, leading to a partial fusion of digits II and III in adult mice. In Hoxa13 homozygous mutant mice, there is no interdigital apoptosis and no digit separation in 14-day-old embryos. Although it remains to be seen whether Hoxa13 and other Hox genes are direct regulators of apoptotic genes in amniotes and other animals, one intriguing possibility is that Hox-dependent regulation of apoptosis is a more general mechanism used to generate and maintain metameric pattern during animal development (Lohmann, 2002).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Rusch, 2000).

Cross-regulation of Homeotic Complex (Hox) genes by ectopic Hox proteins during the embryonic development of Drosophila was examined using Gal4 directed transcriptional regulation. The expression patterns of the endogenous Hox genes were analyzed to identify cross-regulation while ectopic expression patterns and timing were altered using different Gal4 drivers. Evidence is provided for tissue specific interactions between various Hox genes and the induction of endodermal labial (lab) by ectopically expressed Ultrabithorax outside the visceral mesoderm (VMS). Similarly, activation and repression of Hox genes in the VMS from outside tissues seems to be mediated by decapentaplegic gene activation. Additionally, it has been found that proboscipedia (pb) is activated in the epidermis by ectopically driven Sex combs reduced (Scr) and Deformed (Dfd); however, mesodermal pb expression is repressed by ectopic Scr in this tissue. Mutant analyses demonstrate that Scr and Dfd regulate pb in their normal domains of expression during embryogenesis. Ectopic Ultrabithorax and Abdominal-A repress only lab and Scr in the central nervous system (CNS) in a timing dependent manner; otherwise, overlapping expression in the CNS in tolerated. A summary of Hox gene cross-regulation by ectopically driven Hox proteins is tabulated for embryogenesis (Miller, 2001).

The pb gene is normally expressed during embryogenesis but mutants have no apparent embryonic phenotype. However, ectopic Pb protein in the embryo does produce homeotic transformations in embryos. These observations suggest that the regulation of pb expression during embryogenesis may be important for proper development. Both Scr and Dfd are necessary for establishing the proper expression patterns of pb during embryogenesis. Although ectopic Scr and Dfd function equivalently to activate pb in the antennal segment epidermis, they have an opposite effect on native pb expression in the mandibular mesoderm (Mn). Ectopic Dfd accumulation has no significant effect on pb expression in the Mn, which is part of the Dfd expression domain. However, ectopic expression of Scr by the prd and 69B drivers represses pb expression in the Mn, demonstrating the opposite tissue specific regulation of pb by Scr (Miller, 2001).

Genetic analyses demonstrate native regulatory interactions between pb, Dfd and Scr during embryogenesis. The reduction of pb expression in Dfd and Scr mutant backgrounds shows that normal pb expression is dependent on these genes. Dfd is required in the mandibular mesoderm (Mn) and anterior maxillary (Mx) segments (Miller, 2001).

Similarly, Scr is necessary in the posterior Mx and Labial (Lb) segments. The functional significance of these regulatory interactions is debatable due to the lack of mutant embryonic phenotypes in pb nulls; however, the evolutionary implications are perhaps more interesting. Positive cross-regulation between Hox genes has not been previously demonstrated in Drosophila except through signal transduction. Nevertheless, vertebrate enhancers that are responsible for direct positive cross-regulation exhibit similar activity when tested in Drosophila suggesting that these differences are probably due to evolutionary changes at cis-regulatory elements. Since there is no clear mutant pb embryonic phenotype, these cis-regulatory elements apparently direct positive Hox regulatory interactions between Scr, Dfd and pb may be atavistic and non-functional in derived insects such as Drosophila. However, these cis-regulatory elements may also be necessary for proper Hox gene expression later in development when pb is essential for the morphogenesis of adult structures (Miller, 2001).

A summary of Hox cross-regulation demonstrates the tendency of the posteriorly expressed genes to repress those with more anterior domains. However, there are clear exceptions to this generalization. For example, prd=>Antp and 69B=>Antp show no apparent repression of Dfd and Scr. Similarly, ectopic Scr and Dfd proteins have no effect on lab gene expression while positive regulation of pb by Dfd and Scr is found (Miller, 2001).

Epidermal Hox interactions seem to fit the posterior dominance model best. That is, the observed effects on resident Hox gene expression caused by ectopic Hox protein accumulation usually exhibit repression of the more anteriorly expressed gene. Antp represents the predominant exception to this hierarchy. Ectopic Antp protein represses lab expression in epidermal cells but has no significant effect on pb, Dfd or Scr expression in this tissue. However, Antp normally restricts the posterior domain of Scr in the VMS in a manner that appears to be mediated through short-range signaling. Since there is such a clear effect on the most anteriorly expressed Hox gene lab, while the three Hox genes (expressed more posterior to lab yet anterior to Antp's domain) appear to be refractory to Antp's negative control, it would appear these indifferent Hox genes or some other factor is negating Antp's influence. However, a resolution of the underlying cause of this observation awaits further experimentation (Miller, 2001).

In summary, cross-regulation of the Hox genes cannot be described effectively by generalized models such as posterior prevalence. Among the Hox genes, different tissues exhibit unique characteristics and timing dependent interactions. Moreover, signal transduction pathways complicate interpretations of cross-regulation between Hox genes in these ectopic expression experiments since these pathways are not always subject to current regulatory paradigms. Observations that Scr and Dfd positively regulate pb in a tissue specific manner suggests that some interactions may be atavistic or perhaps, only significant in other developmental stages. Posterior prevalence occurs frequently but specific interactions demonstrate extensive variability; however, much of this variability is likely due to indirect signaling cascades set up by the Hox genes themselves (Miller, 2001).

The Drosophila genes disconnected and disco-related are redundant with respect to larval head development and accumulation of mRNAs from Deformed target genes

HOM-C/hox genes specify body pattern by encoding regionally expressed transcription factors that activate the appropriate target genes necessary for differentiation of each body region. The current model of target gene activation suggests that interactions with cofactors influence DNA-binding ability and target gene activation by the HOM-C/hox proteins. Currently, little is known about the specifics of this process because few target genes and fewer cofactors have been identified. A deficiency screen in Drosophila melanogaster was undertaken in an attempt to identify loci potentially encoding cofactors for the protein encoded by the HOM-C gene Deformed (Dfd). A region of the X chromosome was identified that, when absent, leads to loss of specific larval mouthpart structures producing a phenotype similar to that observed in Dfd mutants. The phenotype is correlated with reduced accumulation of mRNAs from Dfd target genes, though there appears to be no effect on Dfd protein accumulation. These defects are due to the loss of two functionally redundant, neighboring genes encoding zinc finger transcription factors, disconnected and disco-related. The role of these genes during differentiation of the gnathal segments is discussed and, in light of other recent findings, it is proposed that these regionally expressed zinc finger proteins may play a central role with the HOM-C proteins in establishing body pattern (Mahaffey, 2001).

Presence of either gene product is sufficient for normal development of the mandibular, maxillary, and labial lobes, but absence of both gene products disrupts development in these regions. The phenotype of terminal larvae lacking these two genes is strikingly similar to that of larvae lacking the HOM-C genes Dfd and Scr. disco was identified earlier as encoding a protein required for the formation of certain neural connections during embryonic and adult development of Drosophila. This does not appear to be a redundant function, because the phenotype was no more severe in Df(1)19 hemizygous embryos that lack both disco and disco-r. At present, it is not known whether disco-r also has an independent role (Mahaffey, 2001).

disco and disco-r encode proteins containing paired zinc finger domains, Disco with one pair while Disco-r has two pairs. The near identity of the Disco zinc finger pair and the first pair in Disco-r indicates that these proteins may bind to the same DNA sequence. This, along with overlapping distribution of mRNAs, likely explains the redundancy. However, the putative Disco-r protein contains a second pair of zinc fingers, and it is possible that these also influence DNA binding. If so, there may be some differences in the recognition site of these two proteins and, possibly, differences in their roles during development. It is worth noting that a mammalian gene, basonuclin, has been identified that encodes a protein with zinc finger domains similar to those in Disco; Basonuclin contains three pairs of zinc fingers, so in this respect it is more similar to the Disco-r protein. An ORF is also present in the Caenorhabditis elegans genome that encodes a peptide containing a single pair of zinc fingers quite similar to those in Disco; however, at this time little is known of the gene. Finding similar proteins in animals widely divergent from Drosophila indicates that at least some functions of Disco and/or Disco-r may be conserved during evolution (Mahaffey, 2001).

One may wonder whether disco and disco-r are head gap genes. The early distribution of disco mRNA may be suggestive, it is unlikely for the following reasons. Loss of disco and disco-r does not appear to cause a gap phenotype. No loss of segments is observed; the gnathal lobes form as expected. In addition, no change was observed in the distribution of the Engrailed protein in the gnathal cells until head involution is underway, and then the changes appear to be due to improper migration of the gnathal lobes in the mutant embryos. Further, disco-r function is sufficient for normal gnathal development, yet accumulation of disco-r mRNA in gnathal cells occurs well after segmentation. Finally, the process of segmentation in the gnathal region follows that of the trunk, relying on the gap, pair rule, and segment polarity functions. Taking this into consideration, it seems unlikely that disco and disco-r are head gap genes (Mahaffey, 2001).

However, it is suggested that disco/disco-r and btd may have similar roles. The Btd protein has been shown to be required along with the homeodomain-containing protein Empty spiracles (Ems) to specify intercalary identity. Ectopic Ems is capable of transforming regions only where Btd is present, indicating that Btd is necessary for Ems activity. Btd and Ems proteins can interact, and this can occur at the Btd zinc finger domain as well as elsewhere in the protein. It is concluded that Btd and Ems together specify intercalary identity, and that Btd represses phenotypic suppression of Ems. This supports the contention that Ems is an escaped HOM-C gene (Mahaffey, 2001 and references therein).

Though repression of phenotypic suppression may occur, it is proposed that there is a more fundamental role for the proteins encoded by btd and disco/disco-r. It is proposed that these zinc finger-containing proteins are required along with the HOM-C proteins to activate the appropriate target genes necessary to establish segment identity. In the case of disco and disco-r, this is with Dfd and Scr during differentiation of the gnathal lobes. disco and disco-r have a lot in common with the HOM-C genes. They encode spatially restricted transcription factors. Absence of these genes causes a similar phenotype to loss of Dfd and Scr, suggesting a loss of segment identity. It is suggested that, as with the HOM-C genes, disco and disco-r are needed to establish the appropriate transcriptional environment for gnathal segment identity. In an analogous manner, Btd and Ems are required for intercalary identity. Further, since Btd interacts directly with Ems, it seems possible that similar interactions may occur between other HOM-C proteins and zinc finger cofactors. It is tempting to speculate that this occurs with Disco/Disco-r and Dfd and Scr, but this may be a bit premature. Additional studies are necessary to determine if this model is correct, but the similarity of larvae lacking these genes to those lacking Dfd and Scr implies that the disco and disco-r function is crucial for normal pattern formation in the gnathal lobes (Mahaffey, 2001).

Hox proteins coordinate peripodial decapentaplegic expression to direct adult head morphogenesis in Drosophila

The Drosophila BMP, decapentaplegic (dpp), controls morphogenesis of the ventral adult head through expression limited to the lateral peripodial epithelium (P e) of the eye-antennal disc by a 3.5 kb enhancer in the 5' end of the gene. A 15 bp deletion mutation within this enhancer was recovered that identified a homeotic (Hox) response element that is a direct target of labial and the homeotic cofactors homothorax and extradenticle. Expression of labial and homothorax are required for dpp expression in the peripodial epithelium, while the Hox gene Deformed represses labial in this location, thus limiting its expression and indirectly that of dpp to the lateral side of the disc. The expression of these homeodomain genes is in turn regulated by the dpp pathway, as dpp signalling is required for labial expression but represses homothorax. This Hox-BMP regulatory network is limited to the peripodial epithelium of the eye-antennal disc, yet is crucial to the morphogenesis of the head, which fate maps suggest arises primarily from the disc proper, not the peripodial epithelium. Thus Hox/BMP interactions in the peripodial epithelium of the eye-antennal disc contribute inductively to the shape of the external form of the adult Drosophila head (Stultz, 2012).

dpp expression in the lateral PE of the eye-antennal disc is necessary for correct morphogenesis of the adult Drosophila head. This study shows that dpp expression related to ventral head formation is part of a Hox/BMP genetic network restricted to the PE of the eye-antennal disc. The homeotic gene lab, and its cofactors hth and exd positively regulate PE dpp expression. This is supported by the observation that Lab, Exd and Hth bind in vitro to the dpp^hc enhancer and the consensus sites for these factors are required in vivo for expression. In addition, individually, lab and hth are both genetically necessary and together demonstrate sufficiency for expression from dpp^hc enhancer, as shown from both LOF and GOF clonal analyses. lab exerts positive control over Dfd expression, as indicated by loss of Dfd expression in lab LOF clones. In contrast, Lab is ectopically expressed in Dfd LOF clones, demonstrating that Dfd represses lab in domains of its expression. Dpp signalling is genetically required for the transcription of lab, as expression from a lab reporter construct is reduced in tkv LOF clones. Expression of a hth enhancer trap increases in tkv LOF clones, and is reduced when activated Tkv is ectopically expressed, indicating that hth transcription is negatively regulated by Dpp signalling. Finally, dpp directly autoregulates its own expression (Stultz, 2006), and may be spatially limited to domains of signalling by repression by brk, as demonstrated by the ability of ectopically expressed Brk to repress expression from the dpp^hc enhancer. Lab and Hth (acting with Exd) activate the expression of dpp. Lab also contributes to the activation of Dfd, which when expressed, represses lab, acting as a switch to limit the extent of lab expression. It is envisioned that during disc development, lab initiates both dpp and Dfd, and when Dfd reaches a certain threshold level, it turns off lab, establishing the boundary between the two Hox proteins. However, while loss of Dfd is capable of derepressing lab throughout the disc, it does not do so to dpp, so further negative regulation must exist. brk may provide this repression to further ensure the lateral boundary of PE dpp through a potential AE element in the enhancer. These inputs collaborate to define the sharp boundary of PE dpp expression. The level of dpp transcription is positively modulated by feedback between lab and dpp and autoregulation of dpp, presumably through Mad/Med binding to the AE element. Negative feedback between dpp and hth provides a brake on expression; others may exist. For example, the inhibitory Smad protein, daughters against dpp is a target of peripodial Dpp expression (Stultz, 2006). It is presumed these interactions activate dpp expression rapidly but shut it down when a certain expression level is reached (Stultz, 2012).

The Hox response region represents one of what will likely be many inputs into the expression of this 3.5 kb enhancer. Another input, opa, is homologous to the Zinc Finger Protein of the Cerebellum or Zic family of transcription factors, and was identified due to its genetic interaction with dpp^s-hc mutations. Other transcription factors and signalling pathways display genetic interactions, and their contribution to PE dpp expression is being actively investigated, although it is noteworthy that lab, Dfd, hth, and exd are not among them. It is expected that many transcription factors and signalling pathways impinge on the dpp^hc enhancer. In this regard, the dpp^hc enhancer may resemble the dpp visceral mesoderm enhancer, another identified Hox target, where direct Ubx, Abdominal A, Exd, and Hth homeodomain inputs collaborate with the Fox-F-related factor binou, as well as Dpp and Wingless signalling to control gene expression. Enhancers that respond to signalling pathways often demonstrate characteristic behaviours: 'activator insufficiency', 'cooperative activation', and 'default repression', and the dpp^hc enhancer conforms to this model. No single activator is able to induce expression over the entire disc, as shown by GOF experiments. Ectopically expressed Lab produced activation only in close proximity to the domain of endogenous dpp, while Hth activated only in the PE of the posterior eye disc. Addition of two inputs together (Lab and Hth or Lab and Dpp signalling, activated over a much broader area. Only Opa has broad ability to activate on its own over the PE but only in concert with Lab was it able to activate outside the PE. Thus each activator is insufficient individually; the enhancer requires simultaneous cooperative inputs of multiple factors to produce correct spatial expression. Brk would provide the default repression, preventing Lab and Hth individually from successfully activating in the middle of the disc, away from domains of dpp activity (Stultz, 2012).

Based on the transcriptional inputs so far identified, it is proposed that activation is controlled on the lateral side at a minimum by Lab, Hth, Exd, Mad, Med, and Opa. In the middle of the disc, the presence of only Hth and Exd is insufficient to activate the enhancer, particularly over resident default repression provided by Brk. On the medial (future dorsal) side of the disc, Dpp and phosphorylated Mad expression are observed, controlled by an unknown area of the dpp gene. Lab, Hth, Exd, and Opa are expressed there as well, so an additional repressor was hypothesised to be needed that limits expression driven by the dpp^hc enhancer to the lateral side. In this model, Lab is the activity required for peripodial specificity, with its cofactors Hth and Exd, while Mad/Med and Opa act as necessary collaborative activators of the enhancer (Stultz, 2012).

At the nucleotide level, the Hox response element in and adjacent to the dpp^s-hc1 deficiency bears sequence homology to previously identified Lab response elements: the mouse Hoxb1 autoregulatory enhancer (b1-ARE), which also generates a lab-like pattern, dependent on lab and exd activity, in Drosophila, and the lab autoregulatory enhancer. Both these enhancers have binding sites for Hox (Hoxb1, Lab), PBC (Pbx, Exd) and MEIS (Prep, Hth) proteins. The orientation of the bipartite Exd/Lab site relative to the MEIS site is the same in these elements as seen in the dpp^hc Hox response element, and the relative spacing between the PBC/Hox and MEIS components is very similar. However, the dpp^hc Hox response element has a cluster of three overlapping Hth sites, two residing on the opposite strand, and an additional functional Exd site downstream of the Hth sites, as determined by its requirement for expression in vivo). The expression of mutated reporter constructs in vivo, as well as LOF analyses of lab and hth, indicate that Hth/Exd plays a more critical role in enhancer activity than does Lab, as neither mutations in the Lab binding site nor Lab loss-of-function within somatic clones completely extinguished expression. This suggests that there may be multiple ways that homeodomain transcription factors activate the enhancer, depending on the cellular context. It is noted that the expression driven by the dpp^hc enhancer actually manifests as two separate lines {see also Stultz, 2006b). The level of Lab associated with each of these lines is not equivalent, therefore the control of expression may be specific to each line. This would be reminiscent of a situation seen within dpp itself, where the Ubx responsive visceral mesoderm enhancer is activated by Ubx/Exd/Hth in parasegment seven, but only requires Hth/Exd for activation in parasegment three. The in vitro EMSA data further support this, as Hth and Exd bind synergistically to more locations within the enhancer than Lab. The TALE family homeodomain proteins function independently of Hox proteins in many contexts. An additional explanation for the apparent primacy of hth may be because it plays both direct and indirect roles on enhancer expression. Hth acts with the transcription factor Yorkie (Yki) as part of the Hippo signalling pathway, and the nuclear activity of Yki and Hth are required to specify the PE of the eye-antennal disc. In the absence of hth, the PE is incorrectly fated. This may effect early gene expression upstream of the Hox/BMP interactions described in this study, magnifying the genetic affect of hth (Stultz, 2012).

The Hox/BMP network described in this study plays a prominent role in the external appearance of the adult head, yet is restricted completely to the PE of the eye-antennal disc. The terminal mutant phenotypes of dpp^s-hc, Dfd, and lab have similarities, but are sufficiently distinct that additional targets for each must exist, and for the cell autonomous Dfd and lab, these targets must reside in the PE. Other signalling proteins such as Wingless and Hedgehog, and the Notch pathway ligands Serrate and Delta, are expressed in the PE of the eye-antennal disc. While some adult structures derive from the PE, and PE cells likely contribute to other adult structures, it is likely that much of the effect of the PE on head morphogenesis is via inductive interactions with the DP, either through secreted signalling molecules, or targeted cell protrusions. Based on the cuticular alterations seen in dpp^s-hc, Dfd, and lab mutations, such interactions are capable of exerting structural modifications on the final head shape. Dipterans demonstrate great variety in the external morphology of their heads often with sexually dimorphic alterations within a species. Much of this variety involves changes in the relative proportions of eye and head capsule tissue. BMP expression has been implicated in shaping the jaws of cichlid fish and the beak shape of finches, while dpp expression itself is correlated with the growth of beetle horns, a specialized cuticular structure of the head. It is speculated that the PE specific Hox/BMP network described in this study could be a motor for such types of shape change in the Drosophila species (Stultz, 2012).

Common origin of insect trachea and endocrine organs from a segmentally repeated precursor

Segmented organisms have serially repeated structures that become specialized in some segments. The Drosophila corpora allata, prothoracic glands, and trachea are shown to have a homologous origin and can convert into each other. The tracheal epithelial tubes develop from ten trunk placodes, and homologous ectodermal cells in the maxilla and labium form the corpora allata and the prothoracic glands. The early endocrine and trachea gene networks are similar, with STAT and Hox genes inducing their activation. The initial invagination of the trachea and the endocrine primordia is identical, but activation of Snail in the glands induces an epithelial-mesenchymal transition (EMT), after which the corpora allata and prothoracic gland primordia coalesce and migrate dorsally, joining the corpora cardiaca to form the ring gland. It is proposed that the arthropod ectodermal endocrine glands and respiratory organs arose through an extreme process of divergent evolution from a metameric repeated structure (Sanchez-Higueras, 2013).

The endocrine control of molting and metamorphosis in insects is regulated by the corpora allata (ca) and the prothoracic glands (pg), which secrete juvenile hormone and ecdysone, respectively. In Diptera, these glands and the corpora cardiaca (cc) fuse during development to form a tripartite endocrine organ called the ring gland. While the corpora cardiaca is known to originate from the migration of anterior mesodermal cells, the origin of the other two ring gland components is unclear (Sanchez-Higueras, 2013).

The tracheae have a completely different structure consisting of a tubular network of polarized cells. The tracheae are specified in the second thoracic to the eighth abdominal segments (T2-A8) by the activation of trachealess (trh) and ventral veinless (vvl) (Sanchez-Higueras, 2013).

The enhancers controlling trh and vvl in the tracheal primordia have been isolated and shown to be activated by JAK/ STAT signaling. While the trh enhancers are restricted to the tracheal primordia in the T2-A8 segments, the vvl1+2 enhancer is also expressed in cells at homologous positions in the maxilla (Mx), labium (Lb), T1, and A9 segments in a pattern reproducing the early transcription of vvl. The fate of these nontracheal vvl-expressing cells was unknown, but it was shown that ectopic trh expression transforms these cells into tracheae. To identify their fate, vvl1+2-EGFP and mCherry constructs were made (Sanchez-Higueras, 2013).

Although the vvl1+2 enhancer drives expression transiently, the stability of the EGFP and mCherry proteins labels these cells during development. It was observed that while the T1 and A9 patches remained in the surface and integrated with the embryonic epidermis, the patches in the Mx and Lb invaginated just as the tracheal primordia did. Next, the Mx and Lb patches fused, and a group of them underwent an epithelial-mesenchymal transition (EMT) initiating a dorsal migration toward the anterior of the aorta, where they integrate into the ring gland. To find out what controls the EMT, the expression of the snail (sna) gene, a key EMT regulator, was studied. Besides its expression in the mesoderm primordium, it was found that sna is also transcribed in two patches of cells that become the migrating primordium. Using sna bacterial artificial chromosomes (BACs) with different cis-regulatory regions, the enhancer activating sna in the ring gland primordium (sna-rg). A sna-rg-GFP construct labels the subset of Mx and Lb vvl1+2-expressing cells that experience EMT and migrate to form the ring gland. Staining with seven-up (svp) and spalt (sal) (also known as salm) markers, which label the ca and the pg, respectively, showed that the sna-rg-GFP cells form these two endocrine glands. The sna-rg-GFP-expressing cells in the Mx activated svp, and those in the Lb activated sal before they coalesced, indicating that the ca and pg are specified in different segments before they migrate (Sanchez-Higueras, 2013).

To test whether Hox genes, the major regulators of anteroposterior segment differentiation, participate in gland morphogenesis, vvl1+2-GFP embryos were stained, and it was found that the Mx vvl1+2 primordium expressed Deformed (Dfd) and the Lb primordium Sex combs reduced (Scr), while the T1 primordium expressed very low levels of Scr. Dfd mutant embryos lacked the ca, while Scr mutant embryos lacked the pg. Dfd and Scr expression in the gland primordia was transient, suggesting that they control their specification. Consistently, in Dfd, Scr double-mutant embryos, vvl1+2 was not activated in the Mx and Lb patches, and the same was true for vvl transcription. In these mutants, the sna-rg-GFP expression was almost absent, and the ca and pg did not form. In each case, Dfd controlled the expression of the Mx patch and Scr of the Lb patch (Sanchez-Higueras, 2013).

The capacity of different Hox genes to rescue the ring gland defects of Scr, Dfd double mutants was tested. Induction of Dfd with the sal-Gal4 line in these mutants restored the expression of vvl1+2 and sna-rg-GFP in the Mx and the Lb. However, in contrast to the wild-type, both segments formed a ca as all cells express Svp. Similarly, induction of Scr also restored the vvl1+2 and sna-rg-GFP expression, but both primordia formed a pg as they activate Sal and Phantom, an enzyme required for ecdysone synthesis. The capacity of both Dfd and Scr to restore vvl expression, regardless of the segment, led to a test of whether other Hox proteins could have the same function. Induction of Antennapaedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), or Abdominal-B (Abd-B) restored vvl1+2 expression in the Mx and Lb, but these cells formed tubes instead of migratory gland primordia. These cephalic tubes are trachea, as they do not activate sna-rg, they express Trh, and their nuclei accumulate Tango (Tgo), a maternal protein that is only translocated to the nucleus in salivary glands and tracheal cells, indicating that the trunk Hox proteins can restore vvl expression in the Mx and Lb but induce their transformation to trachea (Sanchez-Higueras, 2013).

To investigate whether vvl and trh expression is normally under Hox control in the trunk, focus was placed on Antp, which is expressed at high levels in the tracheal pits. In double-mutant Dfd, Antp embryos, vvl1+2 was maintained in the Lb where Scr was present, while the Mx, T1, and T2 patches were missing. In T3-A8, vvl1+2 expression, although reduced, was present, probably due to the expression of Ubx, Abd-A, and Abd-B in the posterior thorax and abdomen. Thus, Antp regulates vvl expression in the tracheal T2 primordium. Surprisingly, in Dfd, Antp double mutants, Trh and Tgo were maintained in the T2 tracheal pit, indicating that although Hox genes can activate ectopic trh expression, in the tracheal primordia they may be acting redundantly with some other unidentified factor, explaining why the capacity of Hox proteins to specify trachea had not been reported previously (Sanchez-Higueras, 2013).

sna null mutants were studied to determine sna's requirement for ring gland development, but their aberrant gastrulation precluded analyzing specific ring gland defects. To investigate sna function in the gland primordia, the sna mutants were rescued with the sna-squish BAC, which drives normal Sna expression except in the ring gland. These embryos have a normal gastrulation and activate the sna-rg- GFP; however, the gland primordia degenerate and disappear. To block apoptosis, these embryos were made homozygous for the H99 deficiency, which removes three apoptotic inducers. In this situation, the ca and pg primordia invaginated and survived, but they did not undergo EMT. As a result, the gland primordia maintain epithelial polarity, do not migrate, and form small pouches that remain attached to the epidermis. Vvl is required for tracheal migration. In vvl mutant embryos, sna-rg-GFP expression was activated, but the cells degenerated. In vvl mutant embryos also mutant for H99, the primordia underwent EMT and migrated up to the primordia coalescence; however, the later dorsal migration did not progress (Sanchez-Higueras, 2013).

This study has shown that the ca and pg develop from vvl-expressing cephalic cells at positions where other segments form trachea, suggesting that they could be part of a segmentally repeated structure that is modified in each segment by the activity of different Hox proteins. As the cephalic primordia are transformed into trachea by ectopic expression of trunk Hox, tests were performed to see whether the trachea primordia could form gland cells. Ectopic expression of Dfd with arm- Gal4 resulted in the activation of sna-rg-GFP on the ventral side of the tracheal pits. These sna-rg-GFP0-expressing cells also expressed vvl1+2 and Trh and had nuclear Tgo, showing that they conserve tracheal characteristics. These sna-rg-GFP-positive cells did not show EMT and remained associated to the ventral anterior tracheal branch. The strength of ectopic sna-rg-GFP expression increased when ectopic Dfd was induced in trh mutant embryos. However, migratory behaviors in the sna-rg-GFP cells were only observed if Dfd was coexpressed with Sal. Thus, sal is expressed several times in the gland primordia, first at st9-10 repressing trunk Hox expression in the cephalic segments and second from st11 in the prothoracic gland. It is uncertain whether the sal requirement for migration is linked to the first function or whether it represents an additional role (Sanchez-Higueras, 2013).

These results show that the endocrine ectodermal glands and the respiratory trachea develop as serially homologous organs in Drosophila. The identical regulation of vvl in the primordia of trachea and gland by the combined action of the JAK/STAT pathway and Hox proteins could represent the vestiges of an ancestral regulatory network retained to specify these serially repeated structures, while the activation of Sna for gland development and Trh and Tgo for trachea formation could represent network modifications recruited later by specific Hox proteins during the functional specialization of each primordium. This hypothesis or alternative possibilities should be confirmed by analyzing the expression of these gene networks in various arthropod species. The diversification of glands and respiratory organs must have occurred before the split of insects and crustaceans, as there is a correspondence between the endocrine glands in both classes, with the corpora cardiaca corresponding to the pericardial organ, the corpora allata to the mandibular organ, and the prothoracic gland to the Y gland. Despite their divergent morphology, a correspondence between the insect trachea and the crustacean gills can also be made, as both respiratory organs coexpress vvl and trh during their organogenesis. Divergence between endocrine glands and respiratory organs may have occurred when the evolution of the arthropod exoskeleton required solving two simultaneous problems: the need to molt to allow growth, and the need for specialized organs for gas exchange (Sanchez-Higueras, 2013).

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