Targets of Activity

There are reciprocal amounts of UBX and ABD-A antigens in segments of the abd-A domain. In the absence of abd-A the range of Ubx expression expands. Thus ABD-A is a repressor of Ubx expression (Marcias, 1990).

There are 30 common binding sites for the proteins encoded by the Ubx and abd-A genes within a negatively regulated target, the P2 promoter of the Antennapedia (Antp) gene. By systematically mutagenizing binding sites and observing the resulting P2 expression pattern in embryos, evidence has been found for cell-type-specific interactions that are mediated by these sequences. In certain neuronal cells, UBX and ABD-A proteins appear to repress by competing for common binding sites with another homeodomain protein, which may be ANTP acting to induce P2 transcription in an autoregulatory manner. In sets of cells that contribute to the tracheal system, UBX and ABD-A repress by counteracting the function of a factor acting at independent sites. The latter mechanism of repression requires only that multiple homeodomain binding sequences be present and is not dependent on any particular binding site (Appel, 1993).

Genes that limit where the homeotic gene Sex combs reduced is transcribed can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is repressed by the three bithorax complex homeotic genes, thus preventing it from inducing prothoracic structures. However, two of the BX-C homeotic genes, Ultrabithorax and abdominal-A, have no effect on the ability of SCR to direct the formation of salivary glands (Andrew, 1994).

In the Drosophila midgut the UBX protein activates and ABD-A represses transcription of the dpp gene. A 45bp fragment of DNA within the dpp midgut enhancer correctly responds to both UBX and ABD-A in a largely tissue-specific manner, thus representing the smallest in vivo homeotic response element identified to date (Manak, 1994).

teashirt is necessary for proper formation of anterior and central midgut structures. ANTP activates tsh in anterior midgut mesoderm. In the central midgut mesoderm Ubx, abd-A, dpp, and wg are required for proper tsh expression. The control of tsh by Ubx and abd-A, and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals as well as localized transcription regulators, the TSH transcription factor is produced in a spatial pattern distinct from any of the homeotic genes (Mathies, 1994).

An investigation was carried out into the mechanisms by which Hox genes compete for the control of positional identity. Functional dominance is often observed where different Hox genes are co-expressed, and frequently the more posteriorly expressed Hox gene is the one that prevails, a phenomenon known as posterior prevalence. To investigate functional dominance among Hox genes on a molecular basis, dpp674, a visceral mesoderm-specific enhancer of decapentaplegic was used. In the visceral mesoderm, dpp is expressed in parasegment 7 (PS7), where it is required for the formation of the second midgut constriction. Expression of dpp is positively regulated by Ubx in PS7, and negatively regulated by abd-A in PS8-12. Regulation of dpp by Ubx and abd-A takes place through a 680-bp visceral mesoderm-specific enhancer (dpp674). This enhancer contains Ubx/Abd-A protein binding sites defined by DNase I protection assays. Ubx regulation of dpp674 is direct, as shown in experiments in which expression from a mutated enhancer is reconstituted by compensatory changes in the UBX protein that alter its DNA-binding specificity (Capovilla, 1998 and references).

Posterior prevalence is not adequate to describe the regulation of dpp by Hox genes. Instead, abdominal-A dominates over the more posterior Abdominal-B and the more anterior Ultrabithorax. In the context of the dpp674 enhancer, abd-A functions as a repressor whereas Ubx and Abd-B function as activators. Thus, these results suggest that other cases of Hox competition and functional dominance may also be understood in terms of competition for target gene regulation, in which repression dominates over activation (Capovilla, 1998).

The idea that posterior prevalence is the dominance of repression over activation is supported by the observation that abd-A functions as an activator through the 5' portion of the dpp enhancer and as a repressor through its 3' portion. When these portions are fused together in the full enhancer, repression by abd-A prevails over activation. These findings suggest the possibility that other cases of functional dominance may be explained in terms of Hox proteins functioning as repressors and prevailing over Hox proteins functioning as activators. For example, in accordance with posterior prevalence, repression of Distal-less (Dll) by Ubx prevails over Dll activation by genes of the Antennapedia (Antp) complex. Similarly, apterous (ap) repression by Ubx prevails over Antp activation in the central nervous system. In contrast, and in violation of posterior prevalence, repression of centrosomin (cnn) by Antp dominates activation by Ubx in the visceral mesoderm. In another case, a phosphorylation-defective Antp protein (Antp [1,2,3,4]A ) has novel functions in addition to the wild-type Antp functions. At least some of its novel functions are a consequence of the ability of Antp [1,2,3,4]A to misregulate regulatory targets of other Hox genes. Antp [1,2,3,4]A violates posterior prevalence by repressing empty spiracles (ems) expression, which is normally activated by Abd-B. However it is interesting that the novel function of Antp [1,2,3,4]A as a dpp activator cannot overcome repression by abd-A, hence respecting the phenomenon of posterior prevalence. Thus the attractiveness of this model is that it explains the cases of posterior prevalence in which the posterior gene is the repressor, but it also explains other cases of functional dominance in which posterior prevalence is violated (Capovilla, 1998 and references).

Different Hox binding sites mediate different transcriptional activities. Repression by Abd-A is mediated only by certain binding sites. The ability of individual Abd-A binding sites to mediate repression does not correlate with their affinity for Abd-A measured in vitro. Specifically, a low-affinity site (binding site 4) is better able to mediate repression by Abd-A than a high-affinity site (binding site 2). These results suggest the existence of cofactors involved in the regulation of dpp674 by Abd-A. One candidate for such a factor is the product of extradenticle (exd); however, exd is not required for abd-A repression of dpp or dpp674lacZ (M. Capovilla and J. Botas, unpublished). Thus Hox specificity cannot be explained solely by Hox/Exd cooperative binding, and unidentified cofactors interacting differentially with Abd-A and other Hox products probably exist. These factors may alter Abd-A binding specificity and/or may function as corepressors or coactivators, altering Abd-A activity as a transcription factor. The above hypothesis on Hox functional dominance implies that in many cases posterior Hox genes function as repressors whereas anterior Hox genes function as activators of specific target genes. Posterior Hox genes would generally determine posterior body patterns by repressing target genes activated by more anterior Hox genes. However, it is of course unlikely that posterior Hox genes function exclusively as repressors. They probably also function as activators of some targets; the gene ems is a good candidate for direct activation by Abd-B. In these cases the cross-regulation between Hox genes (posterior Hox genes repress the expression of more anterior Hox genes) would ensure the dominance of posterior Hox genes. From the viewpoint of evolution, the easier way to create additional posterior patterns might be to generate ‘new’ Hox genes that repress existing targets rather than activate new targets or combinations (Capovilla, 1998).

odd paired is positively regulated by Antennapedia and Abdominal-A at the location of the first and third midgut constrictions respectively. Between these domains opa is negatively regulated by Ultrabithorax and Decapentaplegic (Cimbara, 1995).

unplugged expression occurs in portions of the tracheal system that penetrate the CNS, including the cerebral branch specific to T1. To test the possibility that genes in the BX-C play a role in regulating unpg expression, the distribution of unpg transcript was examined in Ultrabithorax and abdominal-A mutants, and in Ubx, abd-A, Abd-B triple mutants. In Ubx mutant embryos additional unpg expression is observed in cells surrounding the tracheal pits of T2 and T3, indicative of a role for Ubx in repression of unpg in the posterior segments and consistent with homeotic transformation in Ubx mutants of posterior T2 and T3 toward a T1 identity. In abd-A mutants embryos extra patches of unpg-expressing cells around the tracheal pits extend posteriorly to A7, indicating a role for Abd-A in the repression of unpg expression in the abdominal segments. The homeotic gene abd-B probably contributes to the repression of unpg expression in A7, since slightly elevated expression in A7 is observed in the triple mutants (C. Chiang, 1995b).

There are independent enhancers for Distal-less, arrayed over 40 kb of DNA. Head and leg synthesis is independently regulated. One distal 5' enhancer sequence, an early enhancer regulating Dll expression in germ band extended embryos, is subject to repression by Ultrabithorax and abdominal-A (Vachon, 1992).

Segmental modulation of 18 wheeler expression later in the tracheal system is dependent upon the function of the homeotic genes of the bithorax complex. In Ultrabithorax mutant embryos, a larger, more intense patch of 18w extends to cells surrounding the tracheal pits in T2 and T3, indicative of a role for Ultrabithorax protein in repression of 18w in T2 and T3 tracheal pits and consistent with a homeotic transformation in Ubx mutants of posterior T2 and T3 towards a T1 identity. Expanded 18w similarly extends posteriorly to A6 in flies lacking both Ubx and abd-A functions and to A7 in a triple mutant also deficient in Abd-B, indicating a role for abd-A and Abd-B in the repression of 18w in the posterior abdominal segment. In the triple mutant, loss of intense posterior spiracle staining suggests that Abd-B may also be required in A8 for positive regulation of 18w. It is not known whether BX-C regulation of 18w is direct or indirect (C. Chiang 1995a). Scabrous is a target for UBX. Parts of the last intron and exon of the scabrous gene contain five ATTA sequences, the core sequence shared by most homeodomain binding sites. Mutation of Ubx results in the ectopic transcription of sca in the first abdominal segment. Transcript localization in several combinations of deficiencies in the bithorax complex indicates that sca is downregulated by Abdominal-A and Abdominal-B, and suggests that it is a common target of the three genes of BX-C (Graba, 1992).

Muscle diversification in the Drosophila embryo is manifest in a stereotyped array of myofibers that exhibit distinct segment-specific patterns. The homeotic genes of the bithorax complex control the identities of abdominal somatic muscles and their precursors by functioning directly in cells of the mesoderm. Whereas Ultrabithorax and abdominal-A have equivalent functions in promoting the formation of particular muscle precursors in the anterior abdominal segments, Abdominal-B suppresses the development of these same myogenic cells in the posterior region of the abdomen. When expressed in the same mesodermal cells, however, either Ubx or abd-A can override the inhibitory influence of Abd-B, suggesting that these factors may compete in the regulation of common downstream genes. Furthermore, targeted ectopic expression of Ubx or abd-A indicates that these homeotic genes influence muscle cell fates by autonomous action in mesodermal cells. Muscle identity also appears to be sensitive to the level of Ubx expression in myogenic precursors. Homeotic cues specific to both the mesoderm and the ectoderm cooperate to specify the pattern of muscle attachment sites (Michelson, 1994).

Homeotic genes are expressed in the mesoderm, and are regulated in a segment-specific pattern analogous to, but different from, that seen in the ectoderm. Moreover, the effects of homeotic mutations on the muscles do not always mirror transformations seen in the epidermis. abdominal-A may be expressed ectopically in the mesoderm without altering its expression in the overlying ectoderm. The pattern of adult muscle precursor cells characteristic of the thorax can be converted to that seen in the abdomen by expressing abdominal-A specifically in the mesoderm (Greig, 1993).

Connectin is a cell-surface molecule containing leucine-rich repeats. Connectin can mediate cell-cell adhesion, suggesting a direct link between homeotic gene function and processes of cell-cell recognition. A 4 kb restriction enzyme fragment of Connectin, encompassing the 100 base pair clone of the Connectin promoter immunopurified with anti Ultrabithorax antibody, gives a consistent pattern of expression corresponding to a subset of the total Connectin expression pattern. High levels of expression are detected in a small group of cells in the gnathal and thoracic segments and in a posterior segment. This fragment does not produce CNS expression. The reduced levels of expression of the 4 kb fragment suggest a down regulation by Ubx and the abdominal homeotic genes. In Ubx mutants, expression is derepressed in the abdominal segments A1 and A2. Derepression is more dramatic in a Ubx/abd-A double mutant indicating that both genes repress the 4 kb construct. Antp is required for the high levels of expression found in T2 and T3 of wild type embryos (Gould, 1992).

clift/eyes absent is expressed within somatic gonad precursor cells (SGP) as these cells first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in gonadogenesis. In abdominal-A mutants, clift fails to be expressed, and in abd-A overexpressors, clift is likewise expressed ectopically. Despite the early expression of clift, SGP's are specified in the absence of clift function. However, they fail to maintain their fate; as a consequence, germ cells do not coalesce into a gonad. In addition, using clift as a marker, it is shown that the anteroposterior and dorsoventral position of the somatic gonadal precursor cells within a parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with a gene regulatory hierarchy involving abd-A within the mesoderm (Boyle, 1997).

During gastrulation, the Drosophila mesoderm invaginates and forms a single cell layer in close juxtaposition to the overlying ectoderm. Subsequently, particular cell types within the mesoderm are specified along the anteroposterior and dorsoventral axes. The exact developmental pathways that guide the specification of different cell types within the mesoderm are not well understood. The developmental relationship between two mesodermal tissues in the Drosophila embryo, the gonadal mesoderm and the fat body, has been analyzed. Both tissues arise from lateral mesoderm within the even-skipped domain. Whereas in the eve domain of parasegments 10-12 gonadal mesoderm develops from dorsolateral mesoderm and fat body from ventrolateral mesoderm, in parasegments 4-9 only fat body is specified. The cell fate decision between gonadal mesoderm and fat body identity within dorsolateral mesoderm along the anteroposterior axis is determined by the combined actions of genes including abdA, AbdB and srp; while srp promotes fat body development, abdA allows gonadal mesoderm to develop by repressing srp function. Genetic analysis suggests that before stage 10 of embryogenesis, gonadal mesoderm and the fat body have not yet been specified as different cell types, but exist as a common pool of precursor cells requiring the functions of the tin, zfh-1 and cli genes for their development (Moore, 1998).

Abd-A regulates the segmental identity of neural elements in the peripheral nervous system. Anti-POXN stains cells in the PNS that give rise to poly-innervated sensory organs. Some of these stain-accepting cells produce structures that are homologous to one another but are still different from one another, depending on their location ( thorax or abdomen). A dorsal row of POXN-positive cells become kölbchen in the thorax (dorsal pits), but become small sensory hairs in the abdomen. These sense organs differ in both their position and in their differentiation. In thoracic segments T2 and T3, the dorsal POXN-positive cells migrate to a more ventral position than do those in the abdomen. Both differential migration and the terminal differentiation of these precursors are determined by abdominal-A (Castelli-Gair, 1994).

homothorax expression is regulated by genes of the bithorax complex. Starting at germband extension, and throughout the rest of embryonic development, hth expression is modulated in a segment-specific fashion. Most notable is the repression of hth expression in the ectoderm and VNC of abdominal segments during late stages of embryonic development. The genes of the homeotic complexes are the major regulators of segmental identity. hth expression has therefore been analyzed in embryos deficient for the abd-A gene, or the abd-A and Ubx genes together. In the absence of abd-A activity, hth expression is derepressed in the abdominal ectoderm in cells along the segment boundaries. In the absence of both abd-A and Ubx, the derepression is more prominent; hth is expressed in ectodermal cells throughout the segment. In addition, a uniform level of the Hth protein is observed in all the thoracic and abdominal neuromers of Df(3R)Ubx 109 homozygous embryos (Kurant, 1998).

An immunopurification method has been used to clone target genes of the Antennapedia protein (ANTP). centrosomin (cnn) is expressed in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS). In the VM, Antp and abdominal-A negatively regulate cnn, while Ultrabithorax shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut construction (Heuer, 1995).

Signaling by the Epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. High EGFR signalling activity depend on bithorax complex gene function. In mutants lacking abdominal-A and Ultrabithorax, rhomboid expression is very weak. In these mutants, there is very little expression of argos. These homeotic genes account for the main difference in shape between abdominal and thoracic denticle belts (Szuts, 1997).

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).

Ser transcripts in the trunk are first detected at the extended germband stage in ventral patches in the middle of abdominal segments A2-A8 and in offset lateral patches. The ventral regions of thoracic segments do not exhibit Ser expression at this stage. As the germband retracts, the abdominal stripes intensify and develop sharp anterior borders. The first abdominal segment (A1) is unique: Ser expression begins later than in the other abdominal segments and forms a narrower stripe after germband retraction. After germband retraction, Ser transcripts can also be detected in the ventral regions of thoracic segments in broad, faint patches. Embryos mutant in all genes of the Bithorax Complex (BX-C), Ubx, abd-A and Abdominal-B (Abd-B), develop thoracic-type denticles throughout the trunk region. Consistent with this transformation, stage 11 and 12 BX-C mutant embryos have no Ser expression in ventral regions. Ventral Ser expression does begin in BX-C mutants after germband retraction, but the location and level of expression matches that of the thoracic segments. As expected, Ubx mutant embryos show a transformation of abdominal- to thoracic-type Ser expression only in A1; abd-A mutants show Ser transcript stripes in A2-A8 that are similar to the wild-type A1 pattern, and Abd-B mutants display no change in A1-A8 ventral Ser transcription. Thus Ubx function is sufficient to activate some Ser expression in the center of each segment, but abd-A function is required for the earlier, broader pattern of Ser transcription in A2-A8, a transcript pattern that correlates with complete diversification of denticle belts. Embryos lacking all trunk Hox functions express Ser at the margins of the anterior part of each trunk segment and at lower levels in the center of this region, a pattern almost the inverse of that seen in wild type. Transcription of Ser in the posterior-most region of each segment, probably corresponding to the posterior compartment, is completely suppressed. The delimitation of Ser expression to reiterated subsegmental stripes in the embryonic metameres suggests that segment polarity genes also regulate the Ser transcript pattern. ptc mutants lack ventral abdominal Ser transcripts, correlating with the loss of denticle diversity and number in ptc denticle belts. Ser transcription in wingless (wg) mutants appears in broad stripes, while hedgehog (hh) and engrailed (en) mutant embryos exhibit Ser transcription throughout almost the entire ventral epidermis of the abdominal segments. Broadened patterns of Ser transcription in these segment polarity mutants correspond to expanded fields of denticles that lack significant diversity of denticle type (Wiellette, 1999).

A model is presented for the roles of Ser, rho and Hox genes in the generation of denticle belt patterns in the thorax and abdomen. Three Hox genes (Antp, Ubx, and abdA) serve to establish the segmentally specific levels of Ser expression in the third abdominal segment and in the first two thoracic segments respectively. Ser is activated at stage 11 in abdominal parasegments by Ubx and abd-A functions but not in thoracic parasegments where Antp is the principal Hox function. Ubx function is required for the A1-type abdominal expression pattern of Ser, which is narrower and fainter than the pattern in other abdominal segments. This pattern correlates with a narrower, less complex denticle pattern in A1 than in more posterior segments. abd-A function is required for the wider, more abundant Ser stripes in A2-A8. Expression of Ser in the embryonic epidermis results in context-dependent responses, including rho expression, denticle belt patterning and normal development of the mouth hooks. These embryonic roles of Ser are apparently different from its roles in wing margin determination and wing outgrowth. One similarity is the short range over which Ser function is exerted, either at the anterior border of its ventral A2-A8 expression pattern, or at the dorsal/ventral margin of its expression boundary in the wing pouch. The spitz-group gene rho can potentiate Egfr activation via the Spitz (Spi) ligand. Egfr activation is required from late stage 11 to early stage 13 for patterning of the denticle belts, and rho, unlike spi and Egfr, has a spatially and temporally regulated expression pattern. Abdomen-specific rho expression is required for patterning of abdominal denticle rows 1 through 4, probably by allowing secretion of Spitz protein from denticle row 2 and 3 cells, which activates Egfr in neighboring cells. Ser function is required for activation of the abdomen-specific posterior row of rho transcription, expression of which is also dependent on Ubx/abd-A. The evidence presented in this paper suggests that Ser provides a critical intermediate that translates broad Hox and segment polarity domains into narrow stripes of rho expression, which then specify diversification at the single cell level (Wiellette, 1999 and references).

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

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

Two types of GAL4/UAS assays were used to test the involvement of genes in the formation of oenocytes. The ectopic assay tested whether gene products can trigger oenocyte formation in the T1-T3 thoracic segments while the rescue assay tested the potential of genes to overcome the oenocyte deficit in abdA mutants. en-GAL4 was used to express AbdA or Ubx in ectodermal stripes that include the oenocyte precursors. In this ectopic assay, only AbdA was able to produce oenocytes in T1-T3. Together with the preceding results, this indicates that abdA provides a highly selective patterning input and is both necessary and sufficient for the oenocyte fate (Brodu, 2002).

At the time of oenocyte induction during stage 11, a transient burst of AbdA expression is found in both C1 and oenocyte precursors. To ascertain where abdA function is required, two drivers were used that, unlike en-GAL4, have complementary expression in the oenocyte precursors (sal-GAL4, or in the C1 lineage (ato-GAL4). Driving AbdA with ato-GAL4 is sufficient to induce a late oenocyte marker in thoracic segments and also to rescue oenocyte formation in abdA mutants. In each assay, using sal-GAL4 to drive AbdA in the dorsal ectoderm fails to produce oenocytes and using both drivers together does not augment the numbers of oenocytes formed with ato-GAL4 alone. These experiments demonstrate clearly that abdA is required in the C1 lineage but not in the presumptive oenocyte itself, despite being transiently expressed there. It therefore follows that although abdA switches on an extensive hierarchy of early-to-late differentiation genes within the oenocyte, all this regulation must be indirect (Brodu, 2002).

Potential abdA targets were sought from among the genes known to play a role in the specification or function of C1. This particular sensory organ precursor produces a type of stretch receptor, the chordotonal organ, that is defined by the proneural gene atonal (ato). ato is also required for oenocyte formation but it is similarly expressed in thoracic and abdominal C1, is not regulated by abdA and is downregulated prior to oenocyte induction. rho, a gene downstream of ato and rate-limiting for the production of sSpi by cleavage from an inactive membrane-bound precursor (mSpi) in the Golgi apparatus, was examined. Like ato, rho is also required for oenocyte formation. Rho protein is first expressed in C1 at stage 10, after it has delaminated from the dorsal ectoderm. As with Ato at this stage, early Rho is present at similar levels in thoracic and abdominal C1 precursors and is not under abdA control. During stage 11, however, thoracic Rho becomes extinguished while abdominal Rho persists at a similar level in the C1 lineage. Unlike the early expression, this late phase correlates with the time of oenocyte induction and is missing in abdA mutants. Furthermore, driving AbdA in the C1 lineage during stage 11, either in the thorax of a wild-type embryo, or in the abdomen of an abdA mutant, is sufficient to prolong Rho expression. Together, these results indicate that the maintenance but not the establishment of Rho expression is under abdA control. Analysis of a rho-lacZ line, expressed at stage 11 but not stage 10, suggests that this late regulation is at the transcriptional level and is mediated by a different enhancer than that controlling the early phase of expression (Brodu, 2002).

Next, it was asked whether the rather simple Rho timing difference between the thorax and the abdomen is responsible for deciding whether a segment is going to form oenocytes. en-GAL4 or ato-GAL4 were used to extend the time-window of Rho expression in the thorax. Remarkably, using either driver results in the formation of bona fide oenocytes, albeit that they are frequently unclustered and dorsally misplaced. The sufficiency of rho in the absence of AbdA can be clearly demonstrated since ato-GAL4 driven expression of Rho rescues oenocyte formation in abdA mutants. Hence, prolonging the expression of Rho in the C1 lineage is all that is needed to reconstitute the oenocyte identity function of abdA. Next, ato-GAL4 was used to drive AbdA or Rho expression in Scr;Antp double homozygotes. Since ectopic oenocytes are formed in both cases in the mutant T1 segment, representing the ground state, any redundant requirement in the responding ectoderm arising from functional equivalence of Hox proteins can be ruled out (Brodu, 2002).

The above experiments do not reveal whether C1 also produces some other oenocyte signal that is normally present in both thorax and abdomen. Addressing this issue, en-GAL4 was used to express Rho in a genetic background lacking ato, and therefore missing a functional C1 cell. In this mutant context, oenocytes can still be induced, indicating that the only role that C1 plays during oenocyte specification is to express Rho and thus provide a source of sSpi signal (Brodu, 2002).

Maintenance of Rho expression by abdA is predicted to extend the period of Spi secretion, so that the abdominal C1 lineage signals for longer than its thoracic homolog. To test directly whether prolonging active ligand production could induce oenocytes, ato-GAL4 was used to drive a constitutively active form of sSpi, in the C1 lineage after stage 10. This resulted in ectopic oenocytes in the thorax and, more importantly, rescued oenocyte formation in abdA mutants. Providing sSpi prematurely, from stage 9 onwards using en-GAL4, also produces thoracic and rescued abdominal oenocytes but the onset of induction remains restricted to the normal time window during stage 11. Together, these results demonstrate that the oenocyte specification function of abdA can be rescued by adding back either Rho or sSpi in C1 during the period of ectodermal competence. Given that the oenocyte role of abdA is synonymous with prolonging Rho and thus sSpi synthesis in C1, then activating the Egfr in the dorsal ectoderm at the appropriate time would be expected to have the same effect. Consistent with this prediction, expressing constitutively active Egfr (EgfrACT) under the control of sal-GAL4 is sufficient to trigger oenocyte formation in abdA mutants, completely rescuing their number, position and clustering (Brodu, 2002).

Oenocyte formation is under the positive control of AbdA and its co-factor Exd. The temporally restricted pulse of AbdA expression in C1 reflects a transient function in prolonging the oenocyte-inducing signal during stage 11. This type of hit-and-run Hox function appears to be widespread and has previously been observed for other ectodermal derivatives. The misexpression experiments clearly indicate that the oenocyte-promoting role of AbdA is highly selective and can not be substituted for by Ubx. This is explained in molecular terms, since only AbdA is capable of maintaining the transcription of rho in the C1 lineage. Such selectivity contrasts with the equivalent biological activities of Ubx and AbdA proteins in promoting haltere formation. In this regard, it is noted that exd is required to make an oenocyte but not a haltere and therefore may allow these two Hox proteins to discriminate between different targets (Brodu, 2002).

In the absence of any Hox input, oenocytes are completely missing and therefore are not an overt part of the ground state. At first sight, it might seem that for cell types that have no morphological representation in the ground state, such as oenocytes, Hox genes must necessarily play a classic instructive role in defining the appropriate pathway of differentiation. However, as will now be argued, this is not the only way that Hox genes can direct the formation of segment-specific cell types. Two lines of evidence suggest that the sSpi signal from C1 is permissive in the sense that it does not itself contain any oenocyte specificity information: (1) providing ectopic sSpi signal outside of a restricted dorsal zone around C1 fails to induce oenocytes; (2) the degree of sSpi signaling influences the number of induced cells rather than their identity. In contrast, it has been demonstrated that all of the cell-type specificity information is encoded in the dorsal ectoderm as an oenocyte prepattern. One crucial component of this prepattern is encoded by sal. The Sal zinc-finger transcription factor acts to prime the Egfr response in favour of the oenocyte fate. In its absence, there is a fate switch and sSpi signaling now induces secondary chordotonal organs. Thus, it has been shown that oenocyte specificity is provided by the sal-dependent prepattern and not by the sSpi-inducing signal (Brodu, 2002).

The segmental restriction of oenocyte induction has been analysed and evidence is provided supporting a model where there is no Hox input into the prepattern but the timing of the sSpi-inducing signal is controlled by abdA. Together with the previous finding that sSpi signaling is permissive, it is now concluded that abdA does not directly specify the oenocyte identity, rather it determines which segments will form oenocytes. This involves modifying the signaling properties of C1, a serially reiterated cell type that is part of the ground state. In turn, this provides a permissive trigger that uncovers a cryptic oenocyte identity also present in the ground state. Hence ato and sal, two of the genes that contribute to the ground state, are essential for specifying the C1 cell type and the complete oenocyte prepattern, respectively. Another important feature of this model is that the dorsal ectoderm is not competent for oenocyte induction until stage 11. This implies that if competence were to be acquired earlier, when C1 expresses Rho in both the thorax and abdomen, then oenocytes would be produced in all trunk segments independent of Hox genes (Brodu, 2002).

The 26 bp bx1 element from the regulatory region of Distal-less is capable of imposing control by the homeotic genes Ultrabithorax and abdominal-A on a general epidermal activator in Drosophila. This provides an assay to analyze the sequence requirements for specific repression by these Hox genes. Both the core Hox binding site, 5'-TAAT, and the adjacent Exd 5'-TGAT core site are required for repression by Ultrabithorax and abdominal-A. The Distal-less bx1 site thus fits with the model of Hox protein binding specificity based on the consensus PBX/HOX-family site TGATNNAT[g/t][g/a], where the key elements of binding specificity are proposed to lie in the two base pairs following the TGAT. A single base pair deletion in the bx1 sequence generates a site, bx1:A-mut, which on the consensus PBX/HOX model would be expected to be regulated by the Deformed Hox gene. It has been observed, however, that the bx1:A-mut site is regulated predominantly by Sex combs reduced, Ultrabithorax and abdominal-A. The analysis of this site indicates that the specificity of action of Hox proteins may depend not only on selective DNA binding but also on specific post-binding interactions (White, 2000).

A homeotic response element that mediates repression by the Hox genes Ubx and abd-A has been built. The element is based on two short sequence modules; the 21 bp binding site Grainyhead binding site element (Gbe) for the transcription factor GRH and the 26 bp UBX/ ABD-A bx1 footprint site from the Dll regulatory sequences. On its own the Gbe mediates uniform epidermal activation. However, the combination of the Gbe and the bx1 element produces a homeotically modulated response. Specifically in the domain of expression of the Hox genes Ubx and abd-A the epidermal expression is repressed. This homeotic element thus successfully recapitulates the features of endogenous regulatory elements from homeotic target genes giving tissue-specific regulation by multiple homeotic genes (e.g. connectin). However, while endogenous target gene regulatory elements tend to be large (typically covering several kb) and are correspondingly difficult to analyze, this constructed element is appealingly simple and provides a manageable system for analyzing the specificity of the homeotic response (White, 2000).

The bx1 element contains the notable sequences 5' - TAAT (the core Hox binding site) and 5' -TGAT (the core Exd binding site). These two sequences are contained within the 5' -TGATTTAATT which is similar to the proposed PBX/HOX consensus site 5' -TGATNNAT[g/t][g/a]. Mutation of the 5' -TAAT site within bx1 abolishes the abdominal repression conferred by this element, presumably by reducing the affinity of the site for Ubx and Abd-A proteins. Thus, both Ubx and Abd-A appear to bind in vivo to the same site in the bx1 element. Mutation of the 5' -TGAT putative Exd binding site also abolishes the abdominal repression, suggesting that both Ubx and Abd-A require the binding of the cofactor Exd in order to function as repressors on this construct (White, 2000).

Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. This study describes a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. Fifteen deficiencies were found that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways; this suggests an extensive use of signaling in the control of Hox gene function (Marabet, 2002).

This study surveyed 60% of the genome and 11 genomic regions were found acting as recessive activators of ems HRE; 4 were found acting as recessive repressors of ems HRE, and 18 were found acting as recessive repressors of wg HRE. So far, the only known gene in addition to AbdB required for ems activation is lines. Df(2R)H3E1, which uncovers lines, has been recovered from the screen for AbdB modifiers. A search for discrete mutations that reproduce the deficiency phenotypes allowed identification of four ems HRE modifier genes: dally, ds, scw, and ttk. Although ttk and scw have already been linked to filzkörper development, none of the four genes had previously been involved in the control of ems expression in posterior spiracles. The screen for AbdA modifiers was restricted to genomic regions leading to ectopic activation of the wg HRE; these response elements relate to functions that repress the enhancer. Accordingly, genomic regions or genes already known to play a role in wg activation, such as abdA, exd, hth, or genes coding for components of the Dpp signaling pathway, were not recovered. Five mutations at specific loci reproduce the phenotypes caused by original deficiencies. Four of these mutations identify tsl, ttk, and genes encoding a putative MPK and a putative CBP as candidate modifiers of wg HRE. None of these genes has so far been involved in the regulation of wg in the visceral mesoderm (Marabet, 2002).

Three of the four candidate genes identified from the ems screen encode molecules acting in or acting in connection with signal transduction pathways. The Scw protein is a secreted factor of the TGF-ß family. The loss of ems expression induced by Brk, a potent repressor of the Dpp/TGF-ß target gene, strongly supports this hypothesis. The involvement of additional signaling pathways in the regulation of ems is more indirectly suggested by the identification of ds and dally that act in connection with several signaling pathways. ds codes for a calcium-dependent cell adhesion molecule of the cadherin superfamily and genetically interacts with shotgun and rhomboid, two genes involved in epidermal growth factor (EGF) signaling, as well as with armadillo (arm), which produces a nuclear effector of the Wg transduction pathway. dally encodes a heparin sulfate proteoglycan involved in the reception of Wg. Although additional experiments are required to firmly establish the involvement of the Wg and EGF pathways, the integration of multiple signals seems to be required for accurate ems regulation by AbdB (Marabet, 2002).

Two modifier genes obtained from the wg screen are presumably involved in the signal transduction cascade. The first, tsl, encodes a ligand for the RTK Torso receptor and the second encodes a putative MKP. Signaling by Ras/MAPK could thus be part of the genetic network that controls wg expression in the midgut, which has been confirmed by showing that wg transcription is impaired by a constitutive active form of Ras. Interestingly, the Ras/MAPK pathway has been implicated in regulation of the Ubx and lab enhancer in the central midgut, and the ETS-domain-containing transcription factor Pointed, which acts as a nuclear effector of the Ras/MAPK pathway, is expressed in the third midgut chamber (Marabet, 2002).

Several modifiers of wg and ems HRE activities identified in this study encode molecules acting in signal transduction cascades. This indicates that signaling processes play important roles in the control of Hox gene function and extends previous observations from a screen for modifiers of a dominant Pb phenotype. Understanding how cell signaling and transcriptional control by Hox protein are mechanistically integrated requires further study (Marabet, 2002).

Tgfß signaling acts on a Hox response element to confer specificity and diversity to Abdominal A protein function

Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. The Dpp/Tgfß signal controls Abdominal A function, and Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity (Grienenberger, 2003).

AbdA is expressed and is active in the third and fourth compartments of the midgut (PS8-PS12), and yet it activates the wg target gene only in PS8. Dpp secreted from PS7 is shown to provide the spatial information required for PS8-localized wg activation and, acting through a newly identified 546 bp enhancer, AbdA and Mad, a transcriptional effector of the Dpp pathway, directly control wg transcription. The convergence of Hox function and Dpp signaling therefore occurs at the levels of DNA and transcription, and endows AbdA with PS8-specific regulatory properties (Grienenberger, 2003).

To identify the enhancer responsible for wg expression in the VM, subfragments of a 9kb genomic region known to drive wg embryonic expression were analyzed in transgenic lines transformed with lacZ reporter constructs. The smallest fragment that drives accurate expression in the VM is a 546 bp XhoI/ClaI (XC) restriction fragment. Its activity is first detected during germ-band retraction, when wg transcripts are visualized in the VM by in situ hybridization, and only in PS8 VM cells. During subsequent development, XC enhancer activity still mimics wg expression, and is associated with the site of central midgut constriction formation. Thus, from early on to the end of embryogenesis, the XC enhancer exclusively and accurately recapitulates wg spatiotemporal expression in the VM (Grienenberger, 2003).

To address whether AbdA and Dpp signaling could directly regulate wg, the sequence of the XC enhancer was examined for the presence of putative binding sites for AbdA and for Mad/Medea (referred to as DRS, for Dpp response sequence), the canonical transcriptional effectors of the Dpp signaling pathway known to recognize identical target sequences. Since genetic and molecular data led to the proposal that, in Drosophila, the CRE sequences to which Creb proteins bind are required to respond to Dpp in addition to DRSs, potential Creb binding sites were sought. Six TAAT core sequences and four sequences resembling the consensual Hox/Pbx binding sites (TGATNNATG/TG/A) were identified as potentially mediating AbdA function. The Hox/Pbx 3 and 2 sequences strongly match the consensus, with seven or six of the eight consensus nucleotides conserved, respectively. Hox/Pbx sequences 1 and 4 only have five of the eight consensus nucleotides conserved. The XC fragment contains three sequences matching DRSs and two potential CRE sites (Grienenberger, 2003).

To assess the evolutionary conservation of the XC enhancer, an homologous fragment from Drosophila virilis was isolated and analyzed for its in vivo activity by transgenesis in Drosophila melanogaster. The D. virilis fragment drives expression in a pattern very similar to that of the XC enhancer, suggesting that sequences conserved between these two enhancers may be important for wg regulation in the midgut. Sequence comparison, including sequences from D. pseudoobscura, revealed that a majority of the TAAT core motifs, the DRSs and the putative Creb-binding sequences are evolutionarily conserved, whereas sequences that match heterodimeric Hox/Pbx consensus binding sites are not. The existence of two large conserved sequences, Box 1 and 2, is noted. Since Box1 lies in a fragment that does not drive reporter gene expression in transgenic flies, particular attention was paid to Box2 (Grienenberger, 2003).

Hox signaling integration was examined to determine whether signaling pathways contribute towards specifying how AbdA, a widely expressed Hox selector protein, controls the development of distinct pattern elements at different locations. Dpp signal secreted from PS7 provides the positional cue responsible for localized activation of wg by AbdA. Biochemical and reverse genetics experiments have established that AbdA and Mad directly regulate wg transcription through the XC enhancer, which thus serves as an integrator of Hox and Dpp input. AbdA is impotent with respect to this enhancer in the absence of the Dpp signal, though it can function perfectly well on other genes without Dpp. Therefore, functional interactions between selector proteins and signaling pathways confer specificity to signaling pathways, and reciprocally confer functional diversity to selector proteins (Grienenberger, 2003).

This study provides a conceptual framework for understanding the molecular basis of regional Hox protein transcriptional activity. Dpp and Wg signaling subdivide the AbdA Hox domain, allowing activation of pointed (pnt) and opa target genes in the third and fourth midgut chambers, respectively. Based upon the data presented here, it is suspected that the localized activation of pnt and opa by AbdA also relies on direct enhancer integration of Hox and signaling inputs. Accordingly, a Hox/signaling combinatorial code functionally subdivides the domain where a single Hox protein is made, giving rise to discrete patterns of target gene activation. The structures of relevant cis-regulatory regions of AbdA target genes are instrumental for determining which signal is required to allow activation by AbdA. The pnt midgut enhancer would contain AbdA and Wg response elements and would be activated by AbdA specifically in the third midgut chamber through the combinatorial action of AbdA and the Drosophila Tcf/Arm transcriptional effector of Wg signaling. Similarly, the opa midgut enhancer would contain AbdA and Dpp response elements and would be activated only in the fourth gut chamber by AbdA, in this case because of an inhibitory effect of the Dpp-regulated transcription factor on AbdA activity (Grienenberger, 2003).

Further studies are required to understand how Hox selector proteins functionally interact with nuclear effectors of signaling pathways to generate specific transcriptional patterns. In the control of wg by AbdA, several scenarios can be envisioned. In one, the effect of the Dpp transcriptional effector Mad on AbdA activity would be indirect, by antagonizing the function of a repressor that would otherwise act on the XC enhancer to prevent wg expression. The absence of a binding site for this hypothetical repressor in Box2 could explain how Box2 drives AbdA-dependent transcription even without Dpp transcriptional effector binding sites. In a second scenario, Dpp transcriptional effectors would more directly control the activity of AbdA by influencing its DNA binding or transregulatory properties. A direct interaction of HoxC8 and Smad1 has been reported to induce osteoblast differentiation in mammals, suggesting that the coordinate action of AbdA and Dpp signaling might rely on direct AbdA-Mad interaction. In wg regulation, the situation may be different, as additional regulatory inputs are involved. bin and hth are essential, and Wg signaling is required for accurate levels of wg expression. The contribution of Creb might indicate that the Ras/Mapk signaling pathway is involved as well. Ras signaling has been proposed to play a permissive role by acting on CRE sequences of the Ubx and lab enhancers. These observations suggest that AbdA and Hox proteins in general attain specificity and diversity by participating in a variety of protein interactions in enhancer-binding complexes (Grienenberger, 2003).

Direct integration of Hox and segmentation gene inputs during Drosophila development

During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).

The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior-posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior-posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior-posterior axis (Gebelein, 2004).

This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior-posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).

Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior-posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).

Exd and Hth, as well as their vertebrate counterparts, are used as Hox cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).

To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).

One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).

To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).

Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).

Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).

On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).

To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).

On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).

Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).

Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).

The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).

Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).

According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).

Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).

Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).

The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior-posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).

These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).

These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).

Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila; cycE is targeted by abd-A and Abd-B

This study examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. This study shows that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages (Berger, 2005).

In Drosophila, individual neuroblasts deriving from corresponding neuroectodermal positions among thoracic and abdominal segments generally acquire similar fates. However, some of these serially homologous neuroblasts produce lineages with segment-specific differences that contribute to structural and functional diversity within the CNS. The NB6-4 lineage was selected as a model to determine how this diversity evolves from a basic developmental ground state. As an experimental system, NB6-4 has an additional advantage, since Eagle (Eg) is expressed in all the cells of both thoracic and abdominal lineages and can thus be used as a lineage marker (Berger, 2005).

First the expression patterns of different homeotic genes were examined in thoracic and abdominal lineages of NB6-4. Antennapedia (Antp) is expressed in NB6-4t lineages of thoracic segments T1-T3. Abdominal A (Abd-A) is expressed in the NB6-4a lineage of abdominal segments A1-A6, whereas Abdominal B (Abd-B) is expressed in the NB6-4a lineage of segments A7-A8. Whereas loss of Antp function does not affect the NB6-4t lineage in any of the thoracic segments, loss-of-function mutations in abd-A and Abd-B cause NB6-4a-to-NB6-4t homeotic transformations in their corresponding segments. Interestingly, Ultrabithorax (Ubx), which is expressed in most of the cells of T3, is specifically absent in the NB6-4t lineage of that segment, and its loss-of-function alleles do not show any thoracic phenotypes. However, overexpression of Ubx as well as abd-A causes NB6-4t-to-NB6-4a transformations. Thus, it seems that the NB6-4t fate is the ground state and the NB6-4a state is imposed by the function of homeotic genes of the bithorax-complex (BX-C). This is consistent with previous reports that the T2 state is the ground state (for epidermis, including adult appendages) and other segmental identities are conferred by the function of homeotic genes (Berger, 2005).

The mechanism was examined by which abd-A or Abd-B specify the NB6-4a lineage compared with the NB6-4t lineage. As the mode and number of mitoses is the most obvious characteristic by which the NB6-4a lineage differs from NB6-4t, it was wondered whether factors regulating the cell cycle might be involved in controlling NB6-4 cell fate. One major factor that regulates the cell cycle is the G1 Cyclin CycE, which is needed for various aspects of the G1-to-S-phase transition (Berger, 2005).

To examine possible effects on cell fate decisions in the NB6-4t lineage, CycEAR95-mutant embryos were stained for gcm transcripts and Pros and Repo proteins. In wild-type embryos, gcm is initially distributed to both daughter cells during the first division of NB6-4t, but subsequently gets rapidly removed in the cell that functions as a neuronal precursor. Pros is transferred asymmetrically into only one cell, where it is needed to maintain and enhance the expression of gcm, thereby promoting glial cell fate. In CycEAR95 embryos, even at late stages (up to stage 14), gcm mRNA is strongly expressed in both daughter cells after the first division of NB6-4t. Even distribution of Pros was observed in both daughter cells, which could be the cause of continued expression of gcm. Furthermore, the glial marker Repo revealed that both cells differentiate as glial cells. The NB6-4a lineage is not affected in CycEAR95-mutant embryos, suggesting that the requirement for zygotic CycE is specific to NB6-4t (Berger, 2005).

Whether ectopic expression of CycE in abdominal lineages causes the opposite effect was tested. The sca-GAL4 line was used to drive UAS-CycE to achieve early expression in the neuroectoderm. An asymmetric distribution of Pros to one of the two progeny cells was observed just after the first division of NB6-4a. At later stages an increase was observed in the number of cells in the NB6-4a lineage (up to 5 cells). Some of these cells migrated medially, as NB6-4 glial cells normally do, maintaining Pros expression at a lower level. They also expressed Repo, which confirmed their glial identity. Other cells stayed in a dorso-lateral position and did not stain for Repo, suggesting neuronal identity (Berger, 2005).

To further investigate if ectopic CycE had indeed induced a neuronal sublineage in NB6-4a, and to test whether CycE can function cell-autonomously, a cell transplantation technique was employed. Single progenitor cells (stage 7) from the abdominal neuroectoderm of horseradish peroxidase (HRP)-labelled donor embryos overexpressing CycE were transplanted into the abdominal neuroectoderm of unlabelled wild-type hosts (at the same stage). The lineages produced by the transplanted cells were identified by morphological criteria. In all six cases, where cell clones were derived from NB6-4a, they were composed of both glial cells and neurons exhibiting their respective characteristic structures and positions. Because the clones are located in a wild-type abdominal environment, this experiment provides evidence that ectopic expression of CycE causes asymmetric division of NB6-4a and confers neuronal identity to one part of the lineage in a cell-autonomous manner. In these single-cell transplantation experiments, similar observations were made for NB1-1 and NB5-4, which also generate segment-specific lineages. Thus, CycE seems to have a general role in establishing segment-specific differences in neuroblast lineages (Berger, 2005).

Next whether the requirement for CycE to specify the neuronal lineage in NB6-4t is due to altered cell-cycle phases was examined. In string mutants, NB6-4t (whose proliferation is blocked before its first division) expresses gcm mRNA, as well as Pros and hunchback protein, although it does not differentiate as a glial cell. The composition of NB6-4t and NB6-4a lineages were further analysed in embryos mutant for other factors that interact with CycE in cell-cycle regulation. dacapo (dap) is the Drosophila homologue of members of the p21/p27Cip/Kip inhibitor family, which specifically block CycE-cdk complexes. Interestingly, in dap-null-mutant embryos an additional glial cell was observed in the NB6-4a lineage, but the appearance of any neuron-like cells was not observed. Consistent with these results, overexpression of Dap resulted in a reduction in the number of neurons in the NB6-4t lineage (from the normal number of 6 to 2-4), but homeotic transformation of the lineage did not occur. The number of glial cells was never affected, neither in the thorax nor in the abdomen. Ectopic expression of p21, the human homologue of the Drosophila dacapo gene, generated similar phenotypes (Berger, 2005).

The influence of the transcription factor dE2F, which mediates the activation of several genes needed for the initiation of S phase, was tested. In dE2F-mutant embryos, unlike in CycE mutants, no homeotic transformation of NB6-4t to NB6-4a was observed, although the number of neurons was reduced from 5-6 to 2-4. Ectopic expression of dE2F resulted in an increase in cell number in some abdominal hemisegments. In only a small percentage of those embryos, cells at lateral positions in abdominal segments did not show expression of gcm or Repo, suggesting their neuronal identity. Thus, although dE2F activation in the CNS depends on CycE, ectopic expression of dE2F cannot fully bypass the requirement for CycE in a NB6-4a-to-NB6-4t transformation. Similarly, ectopic expression of Rbf, a potent inhibitor of E2F target genes, did not cause any changes in the segregation of gcm mRNA, Pros or Repo in the NB6-4t lineage (Berger, 2005).

Whether interfering with another checkpoint of the cell cycle, the transition from G2 to M phase, affects NB6-4 cell fate was tested. Previous studies show that loss of CycA function prevents further mitosis after the first division of NB6-4t. However, the first division of NB6-4t follows the normal pattern; it gives rise to one glial and one neuronal cell. Similar effects were observed in CycA mutants. The cyclin-dependent kinase cdc2 heterodimerizes with CycA and CycB, and high levels of cdc2 expression have been shown to be required for maintaining the asymmetry of neuroblast divisions. In a cdc2 loss-of-function background, NB6-4t generated a normal lineage consisting of two glial cells and five to six dorso-lateral neurons. These observations show that NB6-4 cell fates do not change after manipulation of the transition from G2 to M phase (Berger, 2005).

These results suggest a critical role for CycE per se in regulating the NB6-4t lineage. Therefore whether CycE itself is differentially expressed between thoracic and abdominal NB6-4 lineages was tested. In situ hybridization with CycE RNA on wild-type embryos revealed that CycE is expressed just before the first division in NB6-4t. After the first division, CycE mRNA was detected in the neuronal precursor only and not in the glial precursor. In abdominal segments, no CycE expression was detected in NB6-4a before or after the division. Consistent with the role of CycE in specifying the NB6-4t lineage, notable levels of CycE transcripts were detected in the homeotically transformed NB6-4a lineages in abd-A-mutant embryos. Conversely, overexpression of abd-A caused down-regulation of CycE levels in thoracic segments and homeotic transformation of NB6-4t to NB6-4a. The importance of CycE in generating neuronal cells in the NB6-4t lineage was confirmed in an epistasis experiment involving abd-A and CycE mutants. As described above, loss of abd-A leads to transformation of NB6-4a to NB6-4t. Such homeotic transformation was suppressed by mutations in CycE, suggesting an absolute requirement for CycE in specifying the NB6-4t lineage. Finally, nine potential AbdA-binding sites (five of which are evolutionarily conserved in Drosophila pseudoobscura) were identified in a 5.0-kb enhancer fragment of CycE that is known to harbour cis-acting sequences for driving CycE expression in the CNS (Berger, 2005).

It is concluded that, in addition to its role in cell proliferation, CycE is necessary and sufficient for the specification of cell fate in the NB6-4 lineage. These results suggest that the function of CycE in regulating cell fate in NB6-4 lineages is independent, albeit partially, of its role in cell proliferation. The absence of any cell fate changes in the loss-of-function mutants of string, dap, cdc2 or CycA and in the Dap or Rbf gain-of-function genetic background may be attributed to the presence of CycE, which is strongly expressed in the NB6-4t, but not the NB6-4a, lineage. However, CycE may still function by controlling cell-cycle progression. NB6-4a, which does not express CycE, divides once followed by a cell-cycle arrest, presumably in G1. After the first division in the thorax, one daughter cell expresses high levels of CycE and divides roughly three times to generate neuronal cells. Therefore, this daughter cell presumably progresses through S phase. Chromatin reorganization during S phase might allow cell fate regulators to access their target genes, driving neuronal differentiation. Contrary to this interpretation, the other daughter cell of NB6-4t, which does not express CycE, divides twice but still generates glial cells. Thus, it remains to be investigated whether the role of CycE in neuronal cell fate determination is entirely independent of its role in cell proliferation. The results on the role of CycE in specifying neuronal compared with glial cell fate in the CNS are consistent with data from Xenopus on the role of cyclin-cdk complexes in specifying neuronal cell fate, inhibition of which promotes glial cell fate. In addition, this study shows that homeotic genes contribute to regional diversification of cell types in the CNS through the regulation of CycE levels (Berger, 2005).

Hox and senseless antagonism functions as a molecular switch to regulate EGF secretion in the Drosophila PNS

Hox factors are key regulators of distinct cells, tissues, and organs along the body plan. However, little is known about how Hox factors regulate cell-specific gene expression to pattern diverse tissues. This study shows an unexpected Hox transcriptional mechanism: the permissive regulation of EGF secretion, and thereby cell specification, by antagonizing the Senseless transcription factor in the peripheral nervous system. rhomboid expression in a subset of sensory cells stimulates EGF secretion to induce hepatocyte-like cell development. A rhomboid enhancer was identified that is active in these cells; an abdominal Hox complex directly competes with Senseless for enhancer binding, with the transcriptional outcome dependent upon their relative binding activities. Thus, Hox-Senseless antagonism forms a molecular switch that integrates neural and anterior-posterior positional information. As the vertebrate Senseless homolog is essential for neural development as well as hematopoiesis, it is proposed Hox-Senseless antagonism will broadly control cell fate decisions (Li-Kroeger, 2008).

Hox genes have long been known to specify distinct cell types along the body axes of both vertebrates and invertebrates. However, it has remained elusive how Hox factors regulate transcription in a tissue- or cell-specific manner. In this study, a Hox-regulated enhancer (Rho654) active within a subset of PNS cells was identified. Rho654 drives gene expression in abdominal C1-SOP cells to induce oenocytes, and an Exd/Hth/Abd-A complex stimulates gene expression by directly competing with Sens for this enhancer. These findings have three main implications: (1) They demonstrate how a Hox selector gene integrates A-P positional information with a PNS factor to differentially regulate gene expression along the body plan. (2) They uncover a permissive rather than instructive role for Hox factors in regulating transcription. (3) As Hox and Sens binding sites share a common core sequence, they suggest that additional target genes will be regulated through this mechanism. Moreover, genetic studies in mice have linked Gfi1 and Hox factors to both neural and blood cell development, and this study found that vertebrate Hox and Gfi1 factors compete for binding sites in blood cells (Li-Kroeger, 2008).

Sensory organs within the fly head, thorax, and abdomen require sens for their development. However, the type, location, and number of sensory organs that form in different body regions are regulated, at least in part, by Hox factors. The results provide new insight into how Hox factors provide positional information to modify gene expression in sensory cells. A series of point mutations was used to demonstrate that Hox-Sens competition forms a molecular switch whose outcome correlates with the binding activity of each factor. Intrinsic to this model is the following prediction: If Hox factors differ in their ability to interact with composite sites, then A-P differences in Hox-Sens target expression will be observed. Previous biochemical studies revealed that posterior Hox factors have higher affinity for DNA when bound with Pbx (Exd) than anterior Hox proteins (LaRonde-LeBlanc, 2003). Consistent with these results, this study found that a posterior Hox complex (Abd-A/Hth/Exd) that stimulates Rho654 binds 5-fold more RhoA than an anterior Hox complex (Antp/Hth/Exd) that fails to stimulate Rho654. Thus, differences in binding activities between Hox factors for Hox-Sens composite sites result in the differential regulation of gene expression along the A-P axis of the sensory system (Li-Kroeger, 2008).

Hox proteins instructively regulate gene expression by either activating and/or repressing transcription. In fact, the same Hox factor can perform both functions. Abd-A directly binds regulatory elements to activate wingless (wg) and repress decapentaplegic (dpp) in the same cells of the visceral mesoderm. So what determines if a Hox factor activates or represses transcription? Two recent studies revealed that the transcriptional outcome depends upon the binding of additional transcription factors (Gebelein, 2004; Walsh, 2007). The repression of Distal-less (Dll) by the Abd-A and Ultrabithorax (Ubx) Hox factors requires the binding of two transcription factors in addition to Exd and Hth. In posterior compartment cells, the Engrailed (En) protein collaborates with Abd-A/Exd/Hth to bind DNA and repress Dll. In anterior compartment cells, the Sloppy-paired (Slp) protein binds DNA near the Hox complex to repress Dll (Gebelein, 2004). As both En and Slp interact with the Groucho (Gro) corepressor, their recruitment by Hox factors suggests a mechanism to repress transcription. Similarly, Walsh and Carroll found that Ubx and Smad binding are required to repress spalt-major (salm) in the wing. In this case, the Smad proteins recruit the Schnurri corepressor to inhibit transcription. Thus, Hox factors collaborate with additional factors to determine the transcriptional outcome (Li-Kroeger, 2008).

Studies on Abd-A stimulation of a rho enhancer reveal an unexpected mechanism by which Hox factors control gene expression: through competition with the Sens repressor for DNA binding sites. Sens binds RhoA to repress thoracic gene expression, whereas in the abdomen Exd/Hth/Abd-A is permissive for activation by out-competing Sens. Importantly, mutations that disrupt both Sens and Hox binding to RhoA (SensM/HoxM) are expressed in the thorax and abdomen, revealing that Exd/Hth/Abd-A binding is not required to activate gene expression. In addition, coexpression of Exd, Hth, and Abd-A in cultured cells failed to stimulate Rho654- or RhoAAA-luciferase unless Abd-A is fused to a potent activation domain. Thus, unlike other Hox target genes, Hox complexes on RhoA are permissive rather than instructive and stimulate Rho654 by interfering with the binding of a transcriptional repressor (Li-Kroeger, 2008).

A comparison of consensus Sens, Hox/Exd, and Exd/Hth sites reveal a shared core sequence, suggesting that additional target genes will be regulated through Hox-Sens antagonism. In fact, bioinformatics reveals many Hox-Sens composite sites throughout the Drosophila and mammalian genomes. However, both the Sens and Hox sites extend beyond this core sequence, indicating that only a subset of target genes will comprise composite sites. Thus, three types of target genes for those factors are proposed: (1) those regulated by only Hox factors, (2) those regulated by only Sens/Gfi1, and (3) those regulated by both Hox and Sens/Gfi1. For example, many of the previously characterized Hox target genes in the Drosophila embryo are controlled in tissues that do not express Sens, suggesting they are only regulated by Hox genes. However, the Hox and Sens/Gfi1 factors are coexpressed in many neural cells of the developing PNS in both flies and vertebrates, indicating that similarly to rho regulation in abdominal SOP cells, additional targets will be coregulated by Hox and Sens (Li-Kroeger, 2008).

Like Hox genes, the Sens gene family is conserved in C. elegans (Pag-3), Drosophila, and vertebrates (Gfi1 and Gfi1b). These zinc finger transcription factors are essential for nervous system development in all three organisms. In addition, Gfi1 plays a critical role in hematopoiesis, where it participates in regulating stem cell renewal as well as specific blood cell lineages. Interestingly, Hox factors also regulate blood cell differentiation, proliferation, and stem cell renewal. HoxA9, for example, is required for normal hematopoiesis in mice, and alterations in HoxA9 expression have been implicated in acute myeloid leukemia (AML). In fact, a study analyzing the expression profile of 6817 genes in AML patients who either responded or did not respond to treatment found the highest correlated gene associated with poor prognosis is HoxA9. To determine if the Hox-Sens mechanism uncovered in Drosophila is conserved in mammals, in vitro DNA binding assays were used to show that HoxA9 forms a complex with Pbx and Meis that competes with Gfi1 for common binding sites. Moreover, mouse genetic studies support the hypothesis that Hox-Gfi1 factors antagonize each other to regulate gene expression and blood cell development. Thus, Hox-Sens/Gfi1 competition for composite binding sites is likely a conserved mechanism for the regulation of gene expression in organisms from flies to humans (Li-Kroeger, 2008).

Abdominal-A mediated repression of Cyclin E expression during cell-fate specification in the Drosophila central nervous system

Homeotic/Hox genes are known to specify a given developmental pathway by regulating the expression of downstream effector genes. During embryonic CNS development of Drosophila, the Hox protein Abdominal-A (AbdA) is required for the specification of the abdominal NB6-4 lineage. It does so by down regulating the expression of the cell cycle regulator gene cyclin E (CycE). CycE is normally expressed in the thoracic NB6-4 lineage to give rise to mixed lineage of neurons and glia, while only glial cells are produced from the abdominal NB6-4 lineage due to the repression of CycE by AbdA. This study investigated how AbdA represses the expression of CycE to define the abdominal fate of a single NB6-4 precursor cell. Both in vitro and in vivo, the regulation was examined of a 1.9 kb CNS-specific CycE enhancer element in the abdominal NB6-4 lineage. CycE was shown to be a direct target of AbdA and it binds to the CNS specific enhancer of CycE to specifically repress the enhancer activity in vivo. These results suggest preferential involvement of a series of multiple AbdA binding sites to selectively enhance the repression of CycE transcription. Furthermore, the data suggest a complex network to regulate CycE expression where AbdA functions as a key regulator (Kannan, 2010).

All progenies of both thoracic and abdominal NB6-4 can be traced using Eagle (Eg) as a lineage marker, and Reversed polarity (Repo) for differentiating glial cells. Thus, Eg-only expression marks neuronal fate. The thoracic variant of NB6-4 (NB6-4t) gives rise to both neuronal and glial cells, whereas the abdominal variant (NB6-4a) gives rise to only glial cells. The Hox gene Antennapedia (Antp) is expressed in the NB6-4t lineage of thoracic segments (T1-T3) whereas abdominal A (abdA) and Abdominal B (AbdB) are expressed in the NB6-4a lineage of abdominal segments A1-A6 and A7-A8, respectively. However, loss of Antp function does not affect the lineage development in contrast to loss of abdA or AbdB, which results in NB6-4a to NB6-4t homeotic transformations. Thus, thoracic identity of NB6-4 lineage acts as a default state without the requirement of any Hox gene input, while abdominal identity of the lineage is imposed by the function of abdA and AbdB. AbdA and AbdB function by suppressing the expression of CycE, a cell cycle molecule necessary for G1-S phase transition. This study has focused on the mechanism by which AbdA regulates CycE expression (Kannan, 2010).

Exd and AbdA cooperatively bind as a heterodimer to a consensus DNA sequence. During development, nuclear localization of Exd is regulated by interaction with another homeodomain protein Homothorax. The expression pattern of the cofactors Exd and Hth was examined in NB6-4 lineages of wild-type embryos. Exd expression was detected in glial precursors of the NB6-4a lineage, Interestingly, it was not found in the NB6-4t lineage, although in the ectoderm expression levels of nuclear Exd are higher in thoracic segments than in the abdominal segments. In the case of Hth, the protein was detected in NB6-4a glial cells only after late stage 11, and also weakly in NB6-4t derived glia. Thus, consistent with their requirement to modulate the function of Hox proteins, Exd was found expressed in the NB6-4a lineage and not in NB6-4t lineage, although weak expression of Hth was detected in glial cells of NB6-4t lineage. Assuming that both are required together to modulate Hox function, modulation of Hox function is predicted only in NB6-4a lineage (where abdA and AbdB are expressed) and not in NB6-4t (where Antp is expressed). Indeed, loss of abdA and AbdB show NB6-4a to NB6-4t transformations, while loss of Antp has no phenotypic consequence (Kannan, 2010).

To gain additional evidence on the relevance of exd and hth expression in the NB6-4a lineage, their loss of function mutations were analyzed. exd mutant embryos showed an increase in the number of NB6-4a progeny. Some of these cells migrated medially in a pattern similar to glial cells of NB6-4t, while others migrated to the dorso-lateral cortex, suggesting neuronal identity. Abdominal hemisegments of hth mutant embryos did not show an increase in glial progeny, but generated ectopic neurons in the dorsal lateral cortex suggesting homeotic transformation of NB6-4a to NB6-4t. The fact that mutations in both the cofactors of AbdA independently induced homeotic transformations, although at much lesser degree (11% in exd mutants and 7% in hth mutants, compared to 100% in abdA mutants), suggests that this observation is a phenocopy of the abdA loss of function phenotype. The mildness of the phenotypes could be an indication of their role as cofactors to enhance the effect of AbdA rather than essential factors to regulate cell-fate specification (Kannan, 2010).

Next the expression pattern of CycE transcripts was investigated in the transformed abdominal NB6-4 lineage in exd mutant embryos. Consistent with the phenotype at the cellular level, CycE mRNA was observed exclusively in neuronal cells of transformed NB6-4a (Eg expressing cells) in exd mutant background as is the case for the thoracic NB6-4. In hemisegments that show no transformation and thus represent the wild-type NB6-4a, absence of CycE mRNA was found (Kannan, 2010).

The complex cis-regulatory region of zygotic CycE comprises of tissue and stage specific activator and repressor elements within an at least 10 kb genomic region including upstream and downstream elements. Based on the expression pattern of a 1.9 kb lacZ reporter gene (CycE-lacZ) in transgenic assays, it is evident that this region includes cis-acting sequences that drive zygotic CycE transcription both in epidermis (mitotic cycles 14-16) and CNS and regulatory elements responsible for CycE down regulation at the end of st11. Similar to CycE transcripts and CycE protein, CycE-lacZ is not expressed in NB6-4a and is activated in abdA, AbdB double mutant embryos. Thus, this 1.9 kb lacZ reporter reliably reflects the CycE expression in the abdominal NB6-4 lineage (Kannan, 2010).

To elucidate the mechanisms that AbdA specifies in collaboration with Exd/Hth, the 1.9 kb CNS-specific CycE regulatory element for known AbdA, Exd and Hth-binding sites. The element harbours at least 3 binding sites for AbdA, and one each for Exd and Hth in close association. Since strong repression of β-Gal expression from the 1.9 kb CycE-lacZ regulatory element was observed in the NB6-4a lineage, it was wondered whether this regulation could be due to the presence of AbdA and Exd/Hth-binding sites. Therefore, the 1.9 kb CycE-lacZ fragment in more detail both in vitro and in vivo (Kannan, 2010).

Binding of AbdA, Exd and Hth to the regulatory sequences of CycE was tested by electro-mobility shift assays (EMSAs) on three spatially separated AbdA binding sites named AbdA-1, -2 and -3. AbdA-2 is in close association with binding sites of cofactors Exd and Hth, named as 68 bp fragment. EMSA suggested physical association of AbdA protein with all the three putative binding sites (AbdA-1, -2 and -3) when tested independently with corresponding oligosequences. In addition, the association of AbdA, Exd and Hth complex was observed in the 68 bp fragment. To check whether the putative AbdA, Exd and Hth sites identified within the CycE enhancer are responsible for assembling the complex, the core sequences that make critical contact to each of these factors was mutated. Mutations in Hox binding sites resulted in the loss of association of AbdA, Exd and Hth to the core sequence. These results suggest cooperative binding of AbdA and its cofactors Exd and Hth to the CycE enhancer element (Kannan, 2010).

To test the functional relevance of binding of AbdA, Exd and Hth in vivo, reporter gene constructs were constructed, with presence or absence of either of three AbdA binding sites, upstream of a minimal promoter driving β-Gal expression. Transgenic flies were generated by P-element mediated transformation (Kannan, 2010).

Embryos homozygous for lacZ transgenes (two independent insertion lines for each transgene to rule out position variation effects) were stained for Eg, Repo and β-Gal to visualize the regulatory behaviour of the CNS-specific CycE element in the abdominal NB6-4 lineage. The enhancer elements deleted independently for putative binding sites AbdA-1 and AbdA-2 drive β-Gal expression in abdominal NB6-4 cells, suggesting the preferential requirement of both sites for transcriptional repression of CycE. In contrast, the transgene deleted for AbdA-3 showed the wild-type 1.9 kb CycE-lacZ expression pattern i.e. no expression in NB6-4a, suggesting that AbdA-3 may not be a preferred binding site for repressive activity. As expected, regulatory elements deleted for both AbdA-1 and AbdA-3 or AbdA-2 and AbdA-3 drive lacZ expression in abdominal NB6-4 progenies. However, deletion of both the repressor elements abdA1 and abdA2 (CycE-lacZAbdA-1&2) did not result in de-repression of lacZ in NB6-4a. Interestingly, deletion of all the three elements resulted in the activation of lacZ in NB6-4a. This suggests that AbdA-3 may act as a cryptic repressor, functional only in the absence of both AbdA-1 and AbdA-2. In addition, while deletion of AbdA-2 alone resulted in the activation of β-Gal expression in NB6-4a, CycE-lacZ68bp element deleted for Exd/Hth and AbdA-2 mimicked wild-type β-Gal expression pattern suggesting that in its absence, AbdA-1 and AbdA-3 may maintain repression of CycE in NB6-4a (Kannan, 2010).

These results do not rule out the possibility of other sequences in the regulatory region of CycE that contribute to the AbdA-mediated repression. The fact that lacZ is strongly repressed in CycE-lacZAbdA-1&2 and CycE-lacZ68bp embryos, but de-repressed in CycE-lacZAbdA-1,2&3, CycE-lacZAbdA-1&3 and CycE-lacZAbdA-2&3 embryos, suggest that other regulators may function together with this enhancer in vivo. There is a possibility that this regulatory region is between AbdA-1 and AbdA-2, which is required to assemble a repressor complex. Computational screening of the 1.9 kb enhancer fragments revealed the existence of at least 3 Engrailed (En)-binding sites. Two of the En sites are between AbdA-1 and AbdA-2. It is likely that a minimum of two AbdA binding sites along with this regulatory region is required to assemble a repressor complex that also involves Exd/Hth and probably En. When either AbdA-1 or AbdA-2 is deleted, this repressor complex fails to assemble and hence leads to activation of CycE-lacZ in NB6-4a. In the absence of both AbdA-1 and AbdA-2, the putative En-binding region may come closer to AbdA-3 and still be able to assemble a repressor complex. Interestingly, repression of lacZ was observed when the whole 68 bp region is deleted. This could also be due to the fact that AbdA-3 is now much closer to the putative En-binding sites. Unfortunately, this could not be tested in the background of loss of function of en since NB6-4 itself is not born in those embryos. Nevertheless, the above mentioned model appears to be identical to the way Dll expression is repressed in the epithelial cells, which is mediated by Ubx and En. Further investigation in this direction involves Chromatin immunoprecipitation for AbdA or En followed by Western blot analyses for the other protein under different conditions (Kannan, 2010).

To conclude, these results suggest the preferential involvement of a series of multiple AbdA binding sites for enhanced repression of CycE transcription. These data suggests a complex network to regulate CycE expression where AbdA functions as a key regulator. This may have evolved to ensure tight repression of CycE as it is a potent regulator of cell fate in NB6-4 and possibly other CNS lineages (Kannan, 2010).

Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo

Hox transcription factors control many aspects of animal morphogenetic diversity. The segmental pattern of Drosophila larval muscles shows stereotyped variations along the anteroposterior body axis. Each muscle is seeded by a founder cell and the properties specific to each muscle reflect the expression by each founder cell of a specific combination of 'identity' transcription factors. Founder cells originate from asymmetric division of progenitor cells specified at fixed positions. Using the dorsal DA3 muscle lineage as a paradigm, this study shows that Hox proteins play a decisive role in establishing the pattern of Drosophila muscles by controlling the expression of identity transcription factors, such as Nautilus and Collier (Col), at the progenitor stage. High-resolution analysis, using newly designed intron-containing reporter genes to detect primary transcripts, shows that the progenitor stage is the key step at which segment-specific information carried by Hox proteins is superimposed on intrasegmental positional information. Differential control of col transcription by the Antennapedia and Ultrabithorax/Abdominal-A paralogs is mediated by separate cis-regulatory modules (CRMs). Hox proteins also control the segment-specific number of myoblasts allocated to the DA3 muscle. It is concluded that Hox proteins both regulate and contribute to the combinatorial code of transcription factors that specify muscle identity and act at several steps during the muscle-specification process to generate muscle diversity (Enriquez, 2010).

Eve expression in the DA1 muscle lineage provided the first paradigm for studying the early steps of muscle specification. Detailed characterization of an eve muscle CRM showed that positional and tissue-specific information were directly integrated at the level of CRMs via the binding of multiple transcription factors, including dTCF, Mad, Pnt, Tin and Twi. Based on this transcription factor code and using the ModuleFinder computational approach, this study has identified a CRM, CRM276, that precisely reproduces the early phase of col transcription. This CRM also drove expression in cells of the lymph gland, another organ that is issued from the dorsal mesoderm where col is expressed. Parallel to this study, two col genomic fragments were selectively retrieved in chromatin immunoprecipitation (ChIP-on-chip) experiments designed to identify in vivo binding sites for Twi, Tin or Mef2 in early embryos. One fragment overlaps with CRM276. Based on this overlap and interspecies sequence conservation, a 1.4 kb subfragment of CRM276 that retained most of the transcription factor binding sites identified by ModuleFinder was tested, and it was found to specifically reproduced promuscular col expression. This in vivo validation shows that intersecting computational predictions and ChIP-on-chip data should provide a very efficient approach to identify functional CRMs on a genome-wide scale (Enriquez, 2010).

The eve and col early mesodermal CRMs are activated at distinct A/P and D/V positions. It is now possible to undertake a comparison of these two CRMs, in terms of the number and relative spacing of common activator and repressor sites and their expanded combinatorial code, in order to understand how different mesodermal cis elements perform a specific interpretation of positional information (Enriquez, 2010).

A progenitor is selected from the Col promuscular cluster in T2 and T3 but not T1. One cell issued from the Col-expressing promuscular cluster in T1 nevertheless shows transiently enhanced Col expression, suggesting that the generic process of progenitor selection is correctly initiated in T1. This process aborts, however, in the absence of a Hox input, as shown by the loss of progenitor Col expression and DA3 muscle in specific segments in Hox mutants. The similar changes in Nau and Col expression observed under Hox gain-of-function conditions leads to the conclusion that the expression of 'identity' transcription factor iTFs is regulated by Hox factors at the progenitor stage. The superimposition of Hox information onto the intrasegmental information thereby implements the iTF code in a segment-specific manner and establishes the final muscle pattern. Unlike DA3, a number of specific muscles are found in both T1 and T2-A7, such as the Eve-expressing DA1 muscle; other muscles form in either abdominal or thoracic segments, as illustrated by the pattern of Nau expression in stage 16 embryos. This diversity in segment-specific patterns indicates that Hox regulation of iTF expression is iTF and/or progenitor specific (Enriquez, 2010).

As early as 1994, Hox proteins were proposed to regulate the segment-specific expression of iTFs. Seven years later, an apterous mesodermal enhancer (apME680) active in the LT1-4 muscles was characterized and itwas proposed that regulation by Antp was direct. However, mutation of the predicted Antp binding sites present in apME680 abolished its activity also in A segments, suggesting that some of the same sites were bound by Ubx/AbdA. Evidence is now available that the regulation of col expression by Ubx/AbdA in muscle progenitors is direct and involves a single Hox binding site. However, regulation by Antp does require other cis elements. It remains to be seen whether regulation by Antp is also direct. Since Antp, Ubx and AbdA display indistinguishable DNA-binding preferences in vitro, the modular regulation of col expression by different Hox paralogs suggests that other cis elements and/or Hox collaborators contribute to Hox specificity. Direct regulation of col by Ubx has previously been documented in another cellular context, that of the larval imaginal haltere disc, via a wing-specific enhancer. In this case, Ubx directly represses col expression by binding to several sites, contrasting with col-positive regulation via a single site in muscle progenitors. This is the second example, in addition to CG13222 regulation in the haltere disc, of direct positive regulation by Ubx via a single binding site. Hox 'selector' proteins collaborate on some cis elements with 'effector' transcription factors that are downstream of cell-cell signaling pathways. In the DA3 lineage, it seems that Dpp, Wg and Ras signaling act on one col cis element and the Hox proteins on others. The regulation of col expression by Hox proteins in different tissues via different CRMs provides a new paradigm to decipher how different Hox paralogs cooperate and/or collaborate with tissue- and lineage-specific factors to specify cellular identity (Enriquez, 2010).

The DA3 muscle displays fewer nuclei in T2 and T3 than in A1-A7, an opposite situation to that described for an aggregate of the four LT1-4 muscles. It has been proposed that the variation in the number of LT1-4 nuclei was controlled by Hox proteins. These studies of the DA3 muscle extend this conclusion by showing that the variations due to Hox control are specific to each muscle and are exerted at the level of FCs. Since the number of nuclei is both muscle- and segment-specific, Hox proteins must cooperate and/or collaborate with various iTFs to differentially regulate the nucleus-counting process. As such, Hox proteins contribute to the combinatorial code of muscle identity. Identifying the nature of the cellular events and genes that act downstream of the iTF/Hox combinatorial code and that are involved in the nucleus-counting process represents a new challenge (Enriquez, 2010).

Drosophila castor is regulated negatively by the Ubx and abdA genes, but positively by the AbdB gene

The ventral nerve cord (VNC) of Drosophila exhibits significant segmental-specific characteristics during embryonic development. Homeotic genes are expressed over long periods of time and confer identity to the different segments. castor (cas) is one of the genes which are expressed in neuroblasts along the VNC. However, at late embryonic stages, cas transcripts are found only in head and thoracic segments and terminal abdominal segments, while Cas protein lasts longer in all segments. This study investigated the regulation of temporal and spatial expression of cas by the bithorax complex genes. In the loss-of-function mutants of Ultrabithorax (Ubx) and abdominal-A (abdA), cas transcripts were ectopically expressed in abdominal segments at late embryonic stage. However, unlike in Ubx and abdA mutants, in Abdominal-B (AbdB) loss-of-function mutant embryos, cas disappeared in the terminal region. Ectopic Ubx and abdA suppressed cas expression, but ectopic AbdB activated cas expression in most abdominal segments. Moreover, cas was co-expressed in the cells in which AbdB was normally expressed, and overexpressed in the ectopically expressed AbdB embryos. These results suggest that the expression of cas is segment-specifically regulated negatively by Ubx and abdA genes, but positively by the AbdB gene (Ahn, 2010).

cas is transiently expressed in a subset of neuroblasts in their cell lineage. Its transcripts are present with homologous patterns in thorax and abdomen at early embryonic stages. At late embryonic stages, cas transcripts are found in only a few cells per hemisegment in thoracic and posterior abdominal segments, but not in other abdominal segments. This indicates that cas is expressed in segment-specific mode during late embryonic stages. This study investigated how this regional diversity was produced (Ahn, 2010).

The segment-specific expression of cas was regulated by the homeotic genes. Ubx or abdA mutation caused the homeotic transformation of abdominal cuticle belts to the more anterior ones. These transformation patterns were also observed in cas expression. Mutations in Ubx or abdA genes caused ectopic cas expression in abdominal segments, which was normally observed in the thoracic segments at stage 15 of wild-type embryo, suggesting transformation of the thoracic pattern to an abdominal pattern at that stage. This transformation was synergistically enhanced in the Ubx and abdA double mutants (Ahn, 2010).

cas was ectopically expressed in A1 segment in Ubx mutant embryos. This result is coincident with the function of Ubx to specify the posterior thorax and a portion of A1 segment. cas was also ectopically expressed in A1 to A4 segments in abdA mutant embryos, which coincide with the function of abdA. In Ubx abdA double mutant embryos, cas was expressed in virtually all abdominal segments and in more cells than in each single mutant embryo. The roles of Ubx and abdA on cas expression in the abdominal segments were confirmed in the ectopically expressed Ubx and abdA mutant embryos. For this experiment, proper embryonic stages were very important because cas expression changed dramatically in the abdominal segments between stages 14 and 15. Whether ectopic Ubx or abdA repressed cas expression in the abdominal segments was tested at this stage. The GAL4-mediated induction of Ubx or abdA suppressed cas expression in the abdominal segments (Ahn, 2010).

However, in contrast to the Ubx and abdA mutations, AbdB mutation caused reverse effects on cas expression in the abdominal segments, which have never been reported. Loss-of AbdB function caused lack of cas expression, while ectopic ABDB activated cas expression in the abdominal segments. This phenotype was also observed in Polycomb mutant embryos. Although Polycomb mutation induced ectopic expression of Ubx, abdA and AbdB at the same time, cas was ectopically expressed in the abdominal segments of stage15 embryos, suggesting that ABDB dominated the effects of ectopic UBX or ABDA. The co-localization of cas and ABDB is found in a few cells in the posterior abdominal segments, supporting the positive regulation of cas by ABDB (Ahn, 2010).

This idea was further intensified by the appearance of the ectopic cas mRNA in the numerous abdominal cells with the ectopic AbdB expression. Real-time PCR experiment showed the overexpession of cas mRNA in the ectopically expressed AbdB embryos, also supporting the positive regulation of cas by ABDB. Furthermore, seven AbdB DNA binding sites were found within 5kb upstream from the cas transcription start site enhancing the possibility that ABDB directly regulates the cas expression. ABDB binds preferentially to a sequence with an unusual 5'-TTAT-3' core (Ahn, 2010).

One of questions was why all the cells with the AbdB expression does not show cas mRNA expression. In wild-type embryos, all AbdB-expressing cells does not show cas mRNA. Only a few cells among AbdB-expressing cells could maintain the expression of cas and the other cells lost it. This might be that the homoetic proteins carry out their function by interacting with other cofactors to regulate distinct sets of downstream genes (Ahn, 2010).

Accumulating evidence shows that the bithorax complex genes are involved at different steps in the segment-specific divergence of the CNS. Ubx or abdA activity is required for the abdominal pathway of the NB1-1 lineage. Both ectopic induction of Ubx- or abdA expression until several hours after gastrulation and homeotic de-repression in Polycomb mutants, override thoracic determination of NB1-1. The abdominal NB6-4 lineage is also specified by the abdA and AbdB. abdA is expressed in the NB6-4 lineage of abdominal segments A1-A6, whereas AbdB is expressed in the NB6-4 lineage of segments A7-A8. They specify the abdominal NB6-4 lineage by down-regulating levels of G1 Cyclin (CycE) (Ahn, 2010).

In summary, UBX and ABDA suppress cas expression in abdominal segments, so that mutation in both genes causes ectopic expression of cas in abdominal segments at late embryonic stage. However, ABDB activates cas expression, which is supported by co-localization of cas and ABDB in cells ectopically expressing AbdB, and real-time PCR in ectopically expressed AbdB embryos (Ahn, 2010).

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

The effect were analyzed of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulate the castor expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5'-TTAT-3' canonical core motifs than the probe containing the 5'-TTAC-3' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes (Kim 2013).

Proneural and abdominal Hox inputs synergize to promote sensory organ formation in the Drosophila abdomen

The atonal (ato) proneural gene specifies a stereotypic number of sensory organ precursors (SOP) within each body segment of the Drosophila ectoderm. Surprisingly, the broad expression of Ato within the ectoderm results in only a modest increase in SOP formation, suggesting many cells are incompetent to become SOPs. This study shows that the SOP promoting activity of Ato can be greatly enhanced by three factors: the Senseless (Sens) zinc finger protein, the Abdominal-A (Abd-A) Hox factor, and the epidermal growth factor (EGF) pathway. First, it was shown that expression of either Ato alone or with Sens induces twice as many SOPs in the abdomen as in the thorax, and does so at the expense of an abdomen-specific cell fate: the larval oenocytes. Second, Ato was shown to stimulate abdominal SOP formation by synergizing with Abd-A to promote EGF ligand (Spitz) secretion and secondary SOP recruitment. However, it was also found that Ato and Sens selectively enhance abdominal SOP development in a Spitz-independent manner, suggesting additional genetic interactions between this proneural pathway and Abd-A. Altogether, these experiments reveal that genetic interactions between EGF-signaling, Abd-A, and Sens enhance the SOP-promoting activity of Ato to stimulate region-specific neurogenesis in the Drosophila abdomen (Gutzwiller, 2010).

How proneural pathways that specify sensory precursor cells throughout the body are integrated with region-specific patterning genes to yield the correct type and number of sensory organs is not well understood. This study shows that three factors enhance the ability of Ato to promote ch organ SOP cell fate in the Drosophila abdomen; the EGF pathway mediated by the Spi ligand, the Abd-A Hox factor, and the Sens zinc finger transcription factor (Gutzwiller, 2010).

EGF signaling is used reiteratively throughout development to specify the formation of distinct cell types along the body plan. In the embryonic Drosophila abdomen, EGF signaling initiated by the activation of rhomboid (rho) in a set of ch organ SOP cells induces the formation of both a cluster of abdomen-specific oenocytes as well as a set of 2° ch organ SOP cells. But how does the EGF-receiving cell know whether to become a larval oenocyte that is specialized to process lipids or a ch organ SOP cell that forms part of the peripheral nervous system? Previous studies have shown that oenocyte specification requires at least two inputs: (1) the reception of relatively high levels of EGF signaling and (2) the expression of the Spalt transcription factors. Hence, oenocytes develop in close proximity to the abdominal C1 SOP cells that lie within a Spalt expression domain and express high levels of rho. In contrast, 2° SOP cells require less EGF signaling and form if the receiving cells lack Spalt. Consistent with this model, genetic studies have shown that oenocytes fail to develop and one to two additional ch organ SOP cells are specified in Spalt mutant embryos, whereas ectopic Spalt expression in the ventral ectoderm inhibits the recruitment of 2° SOP cells. Thus, Spalt promotes oenocyte development and antagonizes 2° ch organ specification in the Drosophila embryo (Gutzwiller, 2010).

Evidence that ato has the opposite effect as Spalt: it promotes ch organ SOP cells at the expense of oenocyte specification. Witt (2010) showed that ato loss-of-function results in decreased expression of activity of the rho enhancer, RhoBAD (Witt, 2010), in C1 SOP cells and induces fewer oenocytes. These data are consistent with EGF signaling being compromised in ato mutant embryos and oenocyte specification being dependent upon the reception of high levels of Spi. This study shows that Ato gain-of-function stimulates RhoBAD expression yet results in the inhibition of oenocyte formation. Importantly, the loss of oenocytes is not due to decreased EGF signaling as similar whorls of phospho-ERK-positive cells and even extra phospho-ERK staining are observed in Ato-expressing segments compared with non-expressing segments. In addition, no difference was detected in cell death between Ato-expressing and non-Ato-expressing segments (using an anti-cleaved Caspase3 marker), indicating the oenocyte loss is not due to apoptosis. Instead, Ato promotes the formation of additional ch organ SOP cells in abdominal segments that normally form oenocytes. Moreover, while the broad activation of EGF signaling (PrdG4;UAS-Rho) induces many extra oenocytes and a few scolopodia, the co-expression of Ato and Rho induces many scolopodia and few oenocytes. These data suggest that if the Spi-receiving cell expresses high Ato relative to Salm then ch organ development occurs whereas if the Spi-receiving cell expresses high Salm relative to Ato then oenocytes are formed. Thus, Ato plays a role in both the Spi-secreting (induction of rho expression) and Spi-receiving cell to dictate the choice of cell fate (Gutzwiller, 2010).

The broad expression of Ato within the ectoderm revealed differences in sensory organ competency between the thorax and abdomen. In particular, it was found that Ato induced approximately twice as many ch organ SOP cells in the abdomen as in the thorax. Moreover, the co-expression of Ato with the Abd-A Hox factor induced significantly more ch organ cell formation than expression of either factor alone (none by Abd-A, four by Ato, and eight by Ato/Abd-A). These data suggest that Ato and Abd-A synergize to enhance ch organ SOP formation in the abdomen, an prompted an examination whethere these SOP cells are predominantly 1° or 2° cells. This problem was first addressed by first showing that the co-expression of Ato and Abd-A stimulates Rho enhancer activity (RhoAAA) within additional cells and results in enhanced phospho-ERK staining. Second, it was shown that Ato and Abd-A require the EGF pathway to enhance ch organ development as co-expression of both factors in a spi mutant embryo failed to promote more ch organs than expression of Ato alone. These data indicate that the co-expression of Ato and Abd-A enhances the ability of 1° ch organ SOP cells to activate rho, stimulates Spi secretion and, since the receiving cell expresses Ato, 2° SOPs form instead of oenocytes. The net result is that Ato and Abd-A synergize to activate the EGF pathway to promote region-specific neurogenesis within the Drosophila abdomen (Gutzwiller, 2010).

The Sens transcription factor is essential for the formation of much of the peripheral nervous system in Drosophila and previous studies revealed that Sens can stimulate the sensory bristle-forming activity of the Scute and Achaete proneural factors in the wing disc. Similarly, it was found that Sens stimulates the ability of Ato to generate internal stretch receptors in the embryo and that Ato and Sens promote more sensory organ development in the abdomen than in the thorax. In addition, while the overall number of ch organs formed by Ato and Sens co-expression is decreased in spi mutant embryos, significantly more ch organ SOP cells in the abdomen than in the thorax are observed in this EGF-compromised genetic background. Thus, Ato and Sens can stimulate abdominal ch organ SOP cell development in the presence or absence of Spi-mediated cell signaling (Gutzwiller, 2010).

So, what is the relationship between Ato, Sens, and Abd-A in regulating both EGF signaling and region-specific sensory organ formation? It was previously found that Ato, Sens, and Abd-A control EGF signaling through the regulation of a cis-regulatory element within the rhomboid (rho) locus (RhoBAD) (Li-Kroeger, 2008; Witt, 2010). RhoBAD acts in abdominal C1 SOP cells to induce oenocyte formation, and Ato and Abd-A both stimulate RhoBAD expression, at least in part, by limiting the ability of Sens to repress RhoBAD activity. Moreover, they do so using different mechanisms. An Abd-A Hox complex containing Extradenticle and Homothorax directly competes with the Sens repressor for overlapping binding sites in RhoBAD (Li-Kroeger, 2008). In contrast, Ato does not directly bind RhoBAD but does directly interact with Sens to limit its ability to bind and repress Rho enhancer activity (Witt, 2010). Consequently, SOPs that co-express Ato and Abd-A are likely to limit the ability of Sens to repress Rho and thereby increase the number of ch organ SOP cells that secrete Spi. Consistent with this prediction, the co-expression of Ato and Sens preferentially stimulates Rho enhancer activity within abdominal segments compared to thoracic segments. Each SOP cell that expresses rho would further enhance sensory organ development through the recruitment of 2° SOP cells via Spi-mediated signaling. Hence, the genetic removal of spi results in a significant decrease in the number of ch organ SOP cells that develop in response to Ato and Sens. Thus, the ato-sens genetic pathway, which is used throughout the body to promote SOP formation, interacts with an abdominal Hox factor to stimulate EGF signaling and promote additional cell fate specification in the abdomen (Gutzwiller, 2010).

While the above model fits well with most of the data, two unexpected findings were observed when comparing the ability of Ato-Sens co-expression to induce ch organ development in the presence and absence of spi function: First, it was predicted that Ato-Sens co-expression in the thoracic regions, which lack Abd-A, should predominantly induce the formation of 1° ch organ SOP cells that do not require EGF signaling for their development. However, it was found that significantly fewer ch organs form in the thorax of spi mutants, indicating that EGF signaling can enhance 2° sensory organ formation within thoracic segments that co-express Ato and Sens. Interestingly, previous studies have shown that both rho and the Rho enhancers are weakly active within thoracic C1 SOP cells, but their levels do not reach a high enough threshold to induce oenocyte formation. However, it is possible that the co-expression of Ato and Sens sufficiently sensitizes the receiving cells to respond to low levels of EGF signaling and become ch organ SOP cells. The second unanticipated finding is that Ato and Sens co-expression still induced significantly more ch organ development within the abdomen (5-6 extra SOP cells) relative to the thorax (1-2 extra SOP cells) in the absence of Spi-mediated signaling. This finding suggests that Ato and Sens can genetically interact with the Abd-A Hox factor to promote sensory organ development in an Spi-independent manner. Currently, it is not understood how Abd-A enhances the proneural activity of the Ato-Sens factors in the absence of Spi signaling. One possibility is that Abd-A and Ato use similar mechanisms to limit Sens-mediated repression of additional target genes besides rho to stimulate ch organ development. Alternatively, Abd-A could independently regulate other factors such as those involved in the Notch-Delta pathway to enhance the competency of the ectoderm to respond to the Ato-Sens pathway. Intriguingly, a Hox factor (lin-39) in C. elegans has been shown to directly regulate Notch signaling during vulval development, and the vertebrate Hoxb1 factor regulates neural stem cell progenitor proliferation and maintenance by modulating Notch signaling. Since differential Notch-Delta signaling is a key pathway in deciding neural versus non-neural cell fates, the ability of Hox factors to modify this pathway could result in segmental differences in neurogenesis (Gutzwiller, 2010).

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

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

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

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

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

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

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

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

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

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

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

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

Hox proteins mediate developmental and environmental control of autophagy

Hox genes encode evolutionarily conserved transcription factors, providing positional information used for differential morphogenesis along the anteroposterior axis. This study shows that Drosophila Hox proteins are potent repressors of the autophagic process. In inhibiting autophagy, Hox proteins display no apparent paralog specificity and do not provide positional information. Instead, they impose temporality on developmental autophagy and act as effectors of environmental signals in starvation-induced autophagy. Further characterization establishes that temporality is controlled by Pontin, a facultative component of the Brahma chromatin remodeling complex, and that Hox proteins impact on autophagy by repressing the expression of core components of the autophagy machinery. Finally, the potential of central and posterior mouse Hox proteins to inhibit autophagy in Drosophila and in vertebrate COS-7 cells indicates that regulation of autophagy is an evolutionary conserved feature of Hox proteins (Banreti, 2013).

Autophagy is a cellular process whose induction or inhibition involves multiple levels of regulation, including developmental signals conveyed by the steroid hormone ecdysone, and environmental signals, sensed in the case of amino acid starvation by the InR/dTOR pathways. These regulatory paths do not act independently but seem rather to be interconnected as illustrated by developmentally induced ecdysone-mediated autophagy that acts by repressing the inhibitory function of the InR pathway. This indicates that whereas upstream control is distinct, downstream control may be common (Banreti, 2013).

This study shows that Drosophila Hox proteins are potent inhibitors of autophagy, with a potent and equivalent impact on both developmental and starvation-induced autophagy, and establish that both converge in the regulation of Hox gene expression. This highlights Hox genes as central regulators of autophagy, acting as a node for mediating autophagy inhibition. In regulating autophagy, Hox proteins act at least through regulation of Atg genes and other autophagy genes. Consistent with a direct transcriptional effect of Hox proteins in controlling Atg genes, Ubx DNA binding was found to be essential for autophagy inhibition, whereas have previously shown that Ubx associates to genomic regions immediately adjacent to Atg5 and Atg7 genes (Banreti, 2013).

A key aspect underlying Hox-mediated autophagy control is the regulation of Hox gene expression, where Hox downregulation induces autophagy. This aspect is true for both developmental- and starvation-induced autophagy, where the dynamics of Hox proteins respond to ecdysone (developmental autophagy) and to InR/dTOR (starvation) signaling. Signals mediating changes in Hox gene expression result from changes in the expression of Pont, a facultative component of the Brm complex known to act as a global and positive regulator of Hox genes. Although not establishing changes in Brm complex composition at the L3 feeding/L3 wandering transition, the dynamics of Pont expression suggest that a Pont-depleted Brm complex loses its ability to maintain the expression of Hox genes, resulting in the release of Hox-mediated inhibition of autophagy (Banreti, 2013).

Hox proteins are widely described as providing spatial information required for differential morphogenesis along the A-P axis, within which they largely display paralog-specific activities. However, in regulating autophagy, Hox function is distinct. First, it appears to be generic, with all Hox proteins tested providing inhibitory activity. The need to alleviate global Hox gene function (achieved in this study by impairing the activity of the Brm complex) in order to induce autophagy, further supports their redundant function in inhibiting autophagy. Second, they provide temporal, instead of spatial, information, mediating the temporality of developmental autophagy downstream of ecdysone signaling. Third, in the case of starvation-induced autophagy, Hox genes respond to the InR/dTOR pathways, acting as environmental effectors (Banreti, 2013).

Investigating the evolutionary conservation of Hox-mediated inhibition of autophagy by exploring the activity of mouse Hox proteins in Drosophila fat body cells as well as in vertebrate COS-7 cells indicates that vertebrate Hox proteins also act as potent autophagy inhibitors. Further studies in vertebrate cells should frame their activity to the multiple physiological and pathological situations that involve autophagy and allow for deciphering the molecular modalities of their regulatory roles (Banreti, 2013).

In summary, these findings broaden the framework of Hox protein functions, showing that besides providing spatial information during development, they also coordinate temporal processes and, more surprisingly, act as mediators of environmental signals for autophagy regulation (Banreti, 2013).

The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans

The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. This study investigated how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. The data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-a-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, this study discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages (Camino, 2015).

This study has traced the evolutionary history of two CREs required for a novel trait, and show that they have recently evolved similar expression patterns through remarkably different architectures in a common trans-regulatory landscape. The data indicates that the tergite-wide activities of the yBE and t_MSE did not exist in the monomorphic ancestor for Sophophora, but evolved in the lineage leading to the common ancestor of the melanogaster species group. The results support a scenario where the subsequent expansion and contraction of male pigmentation pattern was driven primarily by alteration of the trans-regulators, whereas repeated losses involved both cis- and trans-evolution with respect to these CREs. Though the t_MSE and yBE drive coordinated patterns of gene expression, striking differences were found in their upstream regulators and direct regulatory linkages. These results bear on the understanding of how new gene regulatory networks form, diversify, and how coordinated regulatory activities can arise through the independent evolution of unique regulatory codes (Camino, 2015).

Hox transcription factors play a prominent role in generating the differences in serially homologous animal body parts, and the origin of novelties. The diversification of homologous parts can be driven by changes in the spatial domains of Hox protein expression, as has been shown for crustacean appendage morphology, snake limblessness, and for the water strider appendage ground plan. Changes in the downstream Hox targets are evident in cases such as the hindwings of insects, and for fruit fly tergite pigmentation. The origin of novel structures can also be traced to the co-option of Hox proteins, as exemplified by cases such as the Photuris firefly lantern and the sex combs residing on the forelegs of certain Drosophila species. For many of these evolved traits, the molecular mechanisms by which Hox expression patterns and target genes evolve remain unknown (Camino, 2015).

While mechanistic studies on the evolution of Hox-regulated CREs remain limited, several target gene CREs have been thoroughly characterized and serve as exemplars of Hox-regulation during development. Hox proteins can interact with CRE binding sites as monomers or through cooperative interactions with Hox-cofactors. The activity of these bound complexes can be further modulated through interactions with collaborating transcription factors. However, to date, few direct Hox target linkages have been traced to their evolutionary beginnings. Expression of yellow in the male A5 and A6 segments required the gain of two binding sites for Abd-B, but it remains uncertain whether these binding events require cooperative interactions with Hox cofactors and which transcription factors are acting as collaborators (Camino, 2015).

The t_MSE presented an opportunity to study how a second Hox-responsive CRE evolved in parallel to the activity at yellow. This study shows that Abd-A and Abd-B respectively are necessary and sufficient for t_MSE regulatory activity. However, the ablation of the resident Hox sites had little effect on this CRE's activity in the A5 and A6 segments, though mutations to nearby CRE sequences resulted in dramatically reduced activity. This result strongly implies that both Abd-A and Abd-B indirectly activate the t_MSE through a downstream factor or factors. While it can't be entirely ruled out that these factors are operating directly through other non-canonical Hox sites, the gel shift assays did not provide convincing evidence that such sites exist. While the Hox sites were not necessary for activation in the A5 and A6 segments, their ablation resulted in a drastic gain of regulatory activity in the A4 and A3 segments, a setting in which Abd-A is the only Hox protein present. This indicates that Abd-A is a direct repressor of t_MSE function in these anterior abdomen segments. The observed dichotomy in Abd-A function can be explained by at least two-not necessarily mutually exclusive-scenarios. First, in the A5 and A6 segments Abd-B may not act as a direct activator of the t_MSE but its occupancy of Hox sites might preclude the direct repressive effects of Abd-A. Secondly, Abd-A may interact cooperatively or collaboratively with other transcription factors in the more anterior segments to impart repression. The results with Hth support this second scenario (Camino, 2015).

The Hox co-factors Hth and Exd were prime candidates to mediate the context-dependent modulation of Abd-A activity. First, RNAi suppression of hth and exd expression each resulted in ectopic pigmentation (Rogers, 2014) and t_MSE activity in the male A4 and A3 segments. Furthermore, inspection of the t_MSE sequence revealed sites characteristic of Hth (AGACAG) and Exd (GATCAT) binding that reside in close proximity to Hox sites. This site content and arrangement is strikingly similar to that found in an abdominal-repressive module for the CRE controlling thoracic Distalless expression. Along a similar vein, this study shows that the ablation of the Hth-like site led to an anterior expansion in t_MSE activity similar to that induced by the Hox site mutations. This outcome supports the interpretation that the more recent origin of the t_MSE involved the formation of novel regulatory linkages with Hox proteins and Hox cofactors (Camino, 2015).

Morphological traits result from the activities of gene regulatory networks, in which each network is governed by a trans-regulatory tier of transcription factors and cell signaling components that ultimately regulate the expression of a set of differentiation genes. For animals, the trans-regulatory genes are remarkably conserved. It is plausible that the origin of new morphologies occurs through the formulation of new gene regulatory networks, while diversification and losses in traits would likely occur through the modification and dismantling of extant networks. The empirical evaluation of such trends of network evolution necessitates the study of trait evolution at the level of networks, CREs, and their encoded binding sites for multiple animal lineages, traits, and evolutionary time frames. The Drosophila pigmentation system is particularly well poised to make pioneering contributions to this growing body of knowledge (Camino, 2015).

The most recent common ancestor of monomorphic and dimorphic Sophophora lineages was inferred to have possessed monomorphic tergite pigmentation, in the context of an otherwise invariant morphological landscape, in which segment number and form has remained conserved at the genus level. Hence, the origin of this novel pigmentation trait may be expected to have co-opted spatial and sex-specific patterning mechanisms that shape the conserved abdomen features. Comparative analysis of orthologous yellow and tan non-coding sequences indicate that these co-option events involved the origination of novel CRE activities that connected a trans-regulatory tier of Hox, Hox-cofactors, and the Bab proteins to these key differentiation genes that encoded pigmentation enzymes (Camino, 2015).

The patterns of regulatory activity for the orthologous tan and yellow sequences support some additional inferences about the early events in this dimorphic trait's origin. While the t_MSE abdominal activity was strikingly lower in D. pseudoobscura and D. willistoni, the D. pseudoobscura yellow body element was active (albeit with expanded activity). These outcomes support at least two evolutionary scenarios. One scenario is a sequence of events where the origination of the t_MSE and y_BE in the lineage of D. pseudoobscura was followed by a secondary loss of the t_MSE. This scenario is supported by a previous observation of dimorphic Bab expression in the D. pseudoobscura abdomen, backing the notion that this species' broad pattern of monomorphic abdominal pigmentation evolved from a dimorphic ancestral state. For the other scenario, the body element-like regulatory activity of D. pseudoobscura could be due to this CRE's origin preceding that of the t_MSE. Distinguishing between these two scenarios will require a more rigorous comparison of the pigmentation phenotypes and networks within the melanogaster and obscura species groups. The outcomes would provide a more nuanced understanding of the early evolutionary history for the derived sexually dimorphic pigmentation network (Camino, 2015).

Tergite pigmentation evolution in the Sophophora subgenus has been relatively well-studied, and the accumulated results frame an extended perspective of trait evolution within a common network. Trans-evolution at the bric-à-brac (bab) locus has been found to be a major driver for the diversification of female tergite pigmentation. This study, in addition to previous studies, indicates that trans-evolution at as of yet unidentified loci may have played prominent roles in the diversification of male-limited tergite pigmentation. Regarding the repeated losses in male pigmentation, the current results are consistent with a scenario where both trans- and cis-evolution occurred, though the targets of cis-evolution have alternated between tan and yellow. While cis-evolution has been identified for a case of monomorphic gain (ebony) in tergite pigmentation, and for a case of monomorphic loss (ebony and tan), the full wealth of case studies portend to a more prominent role for evolutionary changes in the trans-regulatory tier of the pigmentation gene network. However, it is important to note that many of these case studies only assessed the activities of transgenes in D. melanogaster. While similarities in CRE activity might be indicative that expression divergence occurred through trans-evolution, it does not rule out the possibility that cis-changes occurred at other regions in the pigmentation enzyme gene loci, or that expression divergence results from combined cis- and trans-changes. In the future, it will be important to validate or reject the prominent role for trans-regulatory evolution by the reciprocal tests of CREs in species with the contrasting patterns of pigmentation. Two studies where CREs were tested in species with contrasting pigmentation phenotypes, showed that trans-regulatory evolution was a major driver for diversification of fruit fly wing spot patterns by modifying Distalless and wingless expression (Arnoult, 2013; Werner, 2010). Thus it appears the notion of a “conserved trans-landscape” requires more scrutiny (Camino, 2015).

In this study, and elsewhere, experiments indicate that pigmentation losses are associated with and perhaps result from both changes in the trans-regulatory tier and in the cis-regulatory regions of the yellow and tan genes. Interestingly, some instances of trans-regulatory modifications that cause loss of gene expression appear to leave perfectly good CREs intact. The current data provides a second instance in which loss of expression occurred without the loss of the encoded CRE. The yBE was found to be conserved in D. santomea, which diverged from D. yakuba ~400,000 years ago. The activity for this CRE has also remained for D. ananassae since its divergence from a pigmented ancestor. In contrast, D. kikkawai has lost pigmentation while still expressing tan in the abdomen through a perfectly active t_MSE. These results suggest that these CREs were maintained within the population for long periods of time, perhaps indicating additional functions that promote the preservation of these CREs' ancestral potential. Furthermore, the observed heterogeneity of changes in cis and trans to yellow and tan were at first surprising. However, study of the binding site architecture at the yBE and t_MSE provided key clues as to why their evolution may often be uncoupled (Camino, 2015).

The coordinated expression of genes is a ubiquitous theme in developmental biology. Gene expression is finely regulated during development through the activities of CREs that are individually encoded as evolved combinations of transcription factor binding sites (regulatory logic). A compelling question is whether such synchronized expression results from the independent evolution of CREs with similar logics. This question was previously pursued for CREs of regulatory genes coordinately expressed in the developing fruit fly neurogenic ectoderm. In this case, the coordinately activated CREs are encoded by a common regulatory logic, or a so called 'cis-regulatory module equivalence class'. However, the neurogenic ectoderm CREs are deeply conserved, and arose in the distant past (over 230 million years ago) (Camino, 2015).

The recently evolved male-specific expression patterns for tan and yellow present a case in which the evolutionary formation of coordinated regulation can be observed over shorter time-scales. Though both the t_MSE and yBE0.6 drive reporter expression in the dorsal A5 and A6 segment epidermis of males during late pupal development, this study found their regulatory logic to be surprisingly dissimilar. Whereas the yBE0.6 is directly activated by Abd-B, the results indicate that the t_MSE is indirectly activated by Abd-B and Abd-A, and is directly repressed in more anterior body segments by Abd-A and seemingly Hth. Thus, this study provides an example that illustrates how coordinated expression evolved through the evolution of very different binding site architectures and logic (Camino, 2015).

The disparity of regulatory logic governing the yBE0.6 and t_MSE sheds light on the evolutionary tendencies of gene regulatory networks. The incipient stages of the dimorphic pigmentation network's origin involved the derivation of CREs that generate similar patterns through distinct combinations of binding sites. This evolutionary history establishes a 'branched' network in which several of the possible trans-regulatory alterations are incapable of generating coordinated shifts in the expression patterns for co-expressed genes. Hence, an emerging theme from the work in this system is that the differences in regulatory logic of yBE and t_MSE may necessitate changes in one CRE or the other, but is unable to be altered through a common trans regulator that influences both CRE's patterning. Future studies are needed to substantiate the occurrence and identity of the trans changes altering this network's structure. As other recently derived morphological traits are resolved to the level of binding sites within their networks, it will be instructive to see whether similar branched networks and paths of cis and trans evolution permeate their origin and diversification. The net results may reveal general principles of gene regulatory network evolution (Camino, 2015).

abdominal-A: Biological Overview | Evolutionary Homologs | Transcriptional Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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