Interactive Fly, Drosophila

See the embryonic expression pattern of Antp at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site

Antp is expressed in the nuclei of cells of the thoracic embryonic epidermis and several segments of the ventral and peripheral nervous systems. Strongest staining is in the posterior prothorax and anterior metathorax, corresponding to parasegment 4. Later staining extends to all of the mesothorax. The distributions of the ANTP and the Ultrabithorax proteins in doubly-labeled embryos suggest that the UBX protein may be one direct negative regulator of Antp gene expression. During neurogenesis, staining is seen in ventral thoracic and abdominal neural cells and in cells of the peripheral nervous system (Carroll, 1986).

The Drosophila visceral mesoderm (VM) is a favorite system for studying the regulation of target genes by Hox proteins. The VM is formed by cells from only the anterior subdivision of each mesodermal parasegment (PS). The VM itself acquires modular anterior-posterior subdivisions similar to those found in the ectoderm. Mesodemal cells located just under the engrailed-expressing cells in the posterior ectodermal compartment have been called the mesodermal "P domain." The dorsal-most cells of the mesodermal P domain in each PS express the homeobox gene bagpipe (bap); they detach from the mesodermal fold and move inward toward the center of the embryo. These bap-expressing cells form the VM progenitor groups. The VM subdivisions, and the metameric expression of Connectin, form in response to ectodermal production of secreted signals encoded by the segment polarity genes hedgehog and wingless and are independent of Hox gene activity. A cascade of induction from ectoderm to mesoderm to endoderm thus subdivides the gut tissues along the A-P axis. Induction of VM subdivisions may converge with Hox-mediated information to refine spatial patterning in the VM. Con patches align with ectodermal engrailed stripes, so the VM subdivisions correspond to PS 2-12 boundaries in the VM. The PS boundaries demarcated by Connectin in the VM can be used to map expression domains of Hox genes and their targets with high resolution. The resultant map suggests a model for the origins of VM-specific Hox expression in which Hox domains clonally inherited from blastoderm ancestors are modified by diffusible signals acting on VM-specific enhancers (Bilder, 1998).

Since Con expression marks the imprint of ectodermal PS boundaries on the VM, Con patches can be used to precisely map the domains of Hox gene transcription in relation to Con patches. teashirt is expressed in two domains. The anterior midgut domain extends from visceral mesoderm segment (VS) 4 to mid-VS 6, where it shares a posterior boundary with Antennapedia; the central midgut domain extends several cells to either side of the VS 8 boundary. dpp is also expressed in two domains: at the gastric caeca, it is found in the A domain of VS2 and the P domain of VS 3, while in the central midgut it extends from the A domain of VS 6 to terminate just anterior to the VS 8 boundary. wg is expressed just anterior to the VS 8 boundary, with some cells after stage 12 lying in VS 8. pnt is expressed throughout VS 8, although expression is not seen until early stage 13. At stage 13, the two domains of odd paired (opa) expression extend from the P domain of VS 4 to the VS 6 boundary and from VS 9 through VS 11 (Bilder, 1998).

To gain further insights into homeotic gene action during CNS development, the role of the homeotic genes was characterized in embryonic brain development of Drosophila. Neuroanatomical techniques were used to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS. This order is virtually identical in the CNS of Drosophila and mammals. All five genes of the Antennapedia Complex are expressed in specific domains of the developing brain. The labial gene has the smallest spatial expression domain; it is only expressed in the posterior part of the tritocerebral anlage. This contrasts with previous reports that lab is expressed throughout the tritocerebral (intercalary) neuromere. The proboscipedia gene has the largest anteroposterior extent of expression, however, in contrast to other homeotic genes, pb is only found in small segmentally repeated groups of 15-20 cells per neuromere. These groups of pb-expressing cells range from the posterior deutocerebrum toward the end of the VNC. Since pb-expressing cells are found anterior to the lab-expressing cells in the brain, this is an exception to the spatial colinearity rule. (Spatial colinearity is conserved in the epidermis, where pb expression is posterior to lab expression). The Deformed gene is expressed in the mandibular neuromere and the anterior half of the maxillary neuromere and the Sex combs reduced gene is expressed in the posterior half of the maxillary neuromere and the anterior half of the labial neuromere. The Antennapedia gene is expressed in a broad domain from the posterior half of the labial neuromere toward the end of the VNC. The three genes of the Bithorax Complex are expressed in the VNC. Ultrabithorax gene expression extends in a broad domain from the posterior half of the T2 neuromere to the anterior half of the A7 neuromere, with highest expression levels in the posterior T3/anterior A1 neuromeres. The abdominal-A gene is expressed from the posterior half of the A1 neuromere to the posterior half of the A7 neuromere. For the above mentioned genes, the anterior border of CNS expression remains stable from stage 11/12 until the end of embryogenesis. In contrast, the anterior border of CNS expression for the Abdominal-B gene shifts at stage 14. Before this stage Abd-B expression extends from the posterior half of neuromere A7 to the end of the VNC; afterwards, it extends from the posterior half of neuromere A5 to the end of the VNC with the most intense expression localized to the terminal neuromeres. With the exception of the Dfd gene, the anterior limit of homeotic gene expression in the CNS is always parasegmental (Hirth, 1998).

Sex combs reduced and Antp are expressed in the visceral mesoderm but not in the endoderm. The two genes are required for different aspects of the midgut morphogenesis. T he gastric caeca fail to form in Scr null mutant embryos. Scr is expressed in the visceral mesoderm cells posterior to the primordia of the gastric caeca and appears to be indirectly required for the formation of the caeca. Antp is expressed in visceral mesoderm cells that overlie a part of the midgut where a constriction will form, and Antp null mutant embryos fail to form this constriction. An ultrastructural analysis of the midgut reveals that the visceral mesoderm imposes the constriction on the endoderm and the yolk. The mesodermal tissue contracts within the constriction and thereby penetrates the layer of the midgut endoderm. Microtubules participate in the morphological changes of the visceral mesoderm cells (Reuter 1990).

The expression of Antp in leg discs is described, along with its effects on homothorax expression. In mature leg discs (about 120 hours after egg laying), strong Antp expression is restricted to the most proximal cells; in the rest of the disc, Antp expression is weak and limited to very few cells. However, earlier in disc development (72 hours after egg laying) strong Antp expression is present throughout the entire leg disc; it is gradually lost from the central and middle regions of the disc as development proceeds. Even earlier, during embryogenesis, Antp is expressed in the cells that give rise to all three leg discs. In embryogenesis, from stage 14 onward, hth expression and nuclear-localized Extradenticle are observed only in the most proximal cells of the leg primordia. In contrast, for wild-type antennal discs, in which Antp is never expressed, hth is expressed and Exd is nuclear throughout most of the disc (Casares, 1998).

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart (Lo, 2002).

Antp is strongly expressed in four consecutive pairs of cardioblasts in the anterior of the dorsal vessel. The three anterior cardioblast pairs of this domain of strong Antp expression are the posterior three tinman (tin) cardioblast pairs of segment A1, while the fourth pair corresponds to the anterior pair of the two seven up (svp) cardioblast pairs located between A1 and A2. There is also strong expression in at least six pericardial cells flanking the domain of strong cardioblast expression, all of which are non-Tin expressing pericardial cells. Weaker Antp expression is seen in a row of three or four consecutive cardioblast pairs in T3 immediately anterior to the domain of strong Antp, and also in the four tin cardioblast pairs of segment A2 (Lo, 2002).

Located posterior to the domain of Antp expression is a domain of Ubx expression in the midsection of the dorsal vessel. The highest levels of Ubx are observed in the tin cardioblasts of segments A3 and A4, while lower levels are seen in the svp cardioblasts at the A3/A4 border and in the cardioblasts of segments A2 and A5. In addition, the cardioblasts in the heart segments contain barely detectable levels of Ubx. There also appears to be Ubx expression in some of the pericardial cells within A2 to A5, but due to the low expression levels, it is difficult to determine their exact number and whether any of these are tin pericardial cells (Lo, 2002).

The spatially restricted expression of Antp and Ubx in portions of the aorta indicates that these two Hox genes function in the regulation of the A-P polarity of the dorsal vessel as well. Based upon loss- and gain-of-function experiments with Antp and Ubx, these two genes do not appear to be involved in the subdivision into aorta and heart. However, it is conceivable that Antp and Ubx are involved in the later subdivision of this anterior portion of the dorsal vessel into additional chambers that are seen in the adult stage after the remodeling of the dorsal vessel. In addition, Ubx has a role in the A-P patterning of the larval dorsal vessel that appears to be due to its expression in pericardial progenitors. It has been proposed that lymph glands and pericardial cells descend from a common type of progenitor cell, which form the lymph glands in T3/A1 and pericardial cells in more posterior segments. By contrast, in Ubx mutant embryos, the lymph gland is strongly expanded toward more posterior abdominal segments. Together, these observations suggest that during normal development the activity of Ubx within pericardial progenitors of the posterior portion of the aorta acts to suppress lymph gland formation from these cells (Lo, 2002).

Functional analyses of tiptop and Antennapedia in the embryonic development of Oncopeltus fasciatus suggests an evolutionary pathway from ground state to insect legs

In insects, selector genes are thought to modify the development of a default, or 'ground state', appendage into a tagma-specific appendage such as a mouthpart, antenna or leg. In the best described example, Drosophila melanogaster, the primary determination of leg identity is thought to result from regulatory interactions between the Hox genes and the antennal-specifying gene homothorax. Based on RNA-interference, a functional analysis of the Teashirt family selector gene tiptop (see Drosophila tiptop) and the Hox gene Antennapedia in Oncopeltus fasciatus embryogenesis is presented. It is shown that, in O. fasciatus, tiptop is required for the segmentation of distal leg segments and is required to specify the identity of the leg. The distal portions of legs with reduced tiptop develop like antennae. Thus, tiptop can act as a regulatory switch that chooses between antennal and leg identity. By contrast, Antennapedia does not act as a switch between leg and antennal identity. This observation suggests a significant difference in the mechanism of leg specification between O. fasciatus and D. melanogaster. These observations also suggest a significant plasticity in the mechanism of leg specification during insect evolution that is greater than would have been expected based on strictly morphological or molecular comparisons. Finally, it is proposed that a tiptop-like activity is a likely component of an ancestral leg specification mechanism. Incorporating a tiptop-like activity into a model of the leg-specification mechanism explains several mutant phenotypes, previously described in D. melanogaster, and suggests a mechanism for the evolution of legs from a ground state (Herke, 2005).

To investigate the variation and evolution of mechanisms of insect appendage formation, the role of selector genes in the formation of embryonic appendages of the milkweed bug O. fasciatus was examined. There are several advantages in using this species to study leg development and evolution. (1) As a hemimetabolous insect, O. fasciatus first instars have fully formed legs (i.e. all segments are present). Thus, the entire process of leg formation is readily apparent in the embryo and is not stretched out over the process of imaginal disc formation and metamorphosis as it is in holometabolous insects such as D. melanogaster. (2) O. fasciatus is positioned more basally on the insect phylogeny than any other insect for which the RNA-I technique has been successful in assaying gene function. (3) Because O. fasciatus is more distantly related to D. melanogaster than the more common genetically tractable model insects (e.g., the holometabolous insects Tribolium castaneum and Bombyx mori), there is greater potential for uncovering regulatory variation and perhaps gaining greater insight into the evolution of the leg specification mechanism. (4) Because O. fasciatus is generally less derived and has an ancestral leg composition, it may also have conserved the ancestral mechanisms of leg development allowing the direction of evolutionary change to be inferred (Herke, 2005).

From O. fasciatus embryos, partial cDNAs were cloned that represent a homolog of the D. melanogaster gene tiptop. tiptop is a member of the tsh-family that is typified by zinc-finger motifs and is presumed to be a transcription factor. Also, while present in the D. melanogaster genome, this gene was identified solely on the basis of molecular data. No mutations have been reported in tiptop, and its developmental function has not been previously reported for any arthropod (Herke, 2005).

tsh-family genes have been cloned by different methods from at least five insect species and, with the exception of D. melanogaster tsh, all appear to have greater similarity to tiptop. Through extensive PCR on genomic DNA and cDNA, both tiptop and tsh were recovered from D. melanogaster, but only a single gene was recovered from O. fasciatus or other insects. Also, a gene tree constructed using PAUP^* for the tsh-family genes shows that the two D. melanogaster genes cluster together. The gene tree and the absence of a tsh gene in the other insects surveyed suggest that the two tsh-family genes in D. melanogaster result from a recent duplication of an ancestral gene. This ancestral gene has been called tiptop because of its greater similarity to that gene and not to imply a closer evolutionary relationship of the ancestral gene to either the D. melanogaster tsh or tiptop (Herke, 2005).

tsh has a variety of developmental functions in the cuticle of the Drosophila larva and adult. Specifically, tsh is thought to specify trunk identity in the larva through interactions with Hox genes, is required for the formation of proximal regions of appendages in adults, and plays a role in restricting the development of the adult eye. There is little similarity between any of these activities of the Drosophila tsh gene and O. fasciatus tiptop. Thus, these activities appear to have been acquired by the tsh-family relatively recently. Significant to the discussion here is that the function tsh has in the formation of the proximal region of the leg in D. melanogaster cannot be provided by tsh in O. fasciatus because the gene is not present. These roles may be provided by other proximally expressed genes such as hth and exd (Herke, 2005).

A model of the leg specification mechanisms in D. melanogaster and the proposed differences from O. fasciatus is presented. In contrast to D. melanogaster, where loss of Hox (Antp, Scr, Ubx) function produces dramatic transformations of leg to antenna, no transformation toward antennae of Antp-phenocopy legs is detected that could be interpreted as an expansion of hth activity. Thus, although it is possible that some residual Antp function remains in these animals, it is suggested that it is tiptop and not Antp that represses the activity of hth (or other antennal specifier) in the O. fasciatus leg. A role for Antp in the segmentation of the distal region is absent in D. melanogaster while its role in medial segmentation is conserved between the two insects. Also, given that neither tiptop nor the Hox genes act as specifiers of proximal identity (leg vs. antenna) in the leg specification mechanism of milkweed bugs, additional undetermined genes are implicated. This further distances the mechanism of appendage specification in milkweed bugs from the relatively simple two-gene (Hox, hth) system evident in D. melanogaster (Herke, 2005).

A tiptop-like activity is also evident in D. melanogaster. This is illustrated most convincingly by the persistent pretarsi formed on legs that are otherwise transformed to antennae in the absence of Antp activity. Also, the leg-like appendage (composed primarily of tarsi and pretarsi) produced by hth Antp null clones in D. melanogaster is what might be predicted if an independent tiptop-like activity for distal segmentation and specification remained active in these appendages. Genetic analysis of Drosophila tiptop has not revealed a role in distal specification or segmentation of the adult leg (Laurent Fasano, personal communication to Herke, 2005). However, due to the technical difficulties of determining the role embryonic gene activities have in adult structures in D. melanogaster, it has not been possible to rule out that embryonic activities of either tiptop or tsh affect the adult leg. Thus, it remains a possibility that a tiptop-like activity could be provided by tiptop or tsh, as well as by other genes in D. melanogaster (Herke, 2005).

Interestingly, the defects induced by reduced Antp activity in O. fasciatus are in striking contrast to the transformations of mouthparts to antennae seen when Scr and Dfd activity are reduced. These latter transformations have been used as evidence for a universal mechanism of Hox specification of insect appendages. However, in O. fasciatus, Scr and Dfd apparently repress the activity of antennal specification in gnathal appendages while Antp does not repress this activity in thoracic appendages. Thus, in O. fasciatus, two mechanisms (one Hox-dependent and one Hox-independent) exist for specifying the identity of appendages. Additional factors (including tiptop) must mediate the differences in the active mechanisms in these tagma (Herke, 2005).

It is possible to describe the genetic changes required for the O. fasciatus mechanism of leg development to evolve into that of D. melanogaster. (1) Antp acquired the ability to repress the antennal specifier (hth) in the distal leg and lost its role in distal segmentation. These changes might have been relatively simple. A mechanism for Hox genes (e.g. Scr and Dfd) to repress the antennal specifier already existed and the segmentation functions of Antp might be partially provided by tiptop. (2) The change in Antp function relaxed the constraints on tiptop, thereby allowing its function (including hth repression) to diverge. (3) Duplication and further divergence of the ancestral tiptop gene produced the tsh and tiptop genes of D. melanogaster (Herke, 2005).

A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors

The Drosophila lymph gland is a haematopoietic organ in which pluripotent blood cell progenitors proliferate and mature into differentiated haemocytes. Previous work (Jung, 2005) has defined three domains, the medullary zone, the cortical zone and the posterior signalling centre (PSC), within the developing third-instar lymph gland. The medullary zone is populated by a core of undifferentiated, slowly cycling progenitor cells, whereas mature haemocytes comprising plasmatocytes, crystal cells and lamellocytes are peripherally located in the cortical zone. The PSC comprises a third region that was first defined as a small group of cells expressing the Notch ligand Serrate. This study shows that the PSC is specified early in the embryo by the homeotic gene Antennapedia (Antp) and expresses the signalling molecule Hedgehog. In the absence of the PSC or the Hedgehog signal, the precursor population of the medullary zone is lost because cells differentiate prematurely. It is concluded that the PSC functions as a haematopoietic niche that is essential for the maintenance of blood cell precursors in Drosophila. Identification of this system allows the opportunity for genetic manipulation and direct in vivo imaging of a haematopoietic niche interacting with blood precursors (Mandal, 2007).

The Drosophila lymph gland primordium is formed by the coalescence of three paired clusters of cells that express Odd-skipped (Odd) and arise within segments T1-T3 of the embryonic cardiogenic mesoderm. At developmental stages 11-12, mesodermal expression of Antp is restricted to the T3 segment. A fraction of these Antp-expressing cells will contribute to the formation of the dorsal vessel, whereas the remainder, which also express Odd, give rise to the PSC. By stages 13-16, the clusters coalesce and Antp is observed in 5-6 cells at the posterior boundary of the lymph gland. The expression of Antp is subsequently maintained in the PSC through the third larval instar. The embryonic stage 16 PSC can also be distinguished by Fasciclin III expression and at stage 17 these are the only cells in the lymph gland that incorporate BrdU (Mandal, 2007).

Previous studies have identified the transcription factor Collier (Col) as an essential component regulating PSC function. The gene for this protein is initially expressed in the entire embryonic lymph gland anlagen and by stage 16 is refined to the PSC. In col mutants, the PSC is initially specified, but is entirely lost by the third larval instar. To address further the role of Antp and Col in embryonic lymph gland development, the expression of each gene was investigated in the loss-of-function mutant background of the other. It was found that loss of col does not affect embryonic Antp expression. In contrast, col expression is absent in the PSC of Antp mutant embryos, establishing that Antp functions genetically upstream of Col in the PSC (Mandal, 2007).

In imaginal discs, the expression of Antp is related to that of the homeodomain cofactor Homothorax (Hth). In the embryonic lymph gland, Hth is initially expressed ubiquitously but is subsequently downregulated in PSC cells, which become Antp-positive. In hth loss-of-function mutants, the lymph gland is largely missing, whereas misexpression of hth causes loss of PSC and the size of the embryonic lymph gland remains relatively normal. It is concluded that a mutually exclusive functional relationship exists between Antp and Hth in the lymph gland such that Antp specifies the PSC, whereas Hth specifies the rest of the lymph gland tissue. Interestingly, knocking out the mouse homologue of Hth, Meis1, eliminates definitive haematopoiesis (Hisa, 2004; Azcoitia, 2005). Meis1 is also required for the leukaemic transformation of myeloid precursors overexpressing HoxB9 (Mandal, 2007).

Although lymph gland development is initiated in the embryo, the establishment of zones and the majority of haemocyte maturation takes place in the third larval instar. At this stage, Antp continues to be expressed in the wild-type PSC. To investigate how the loss of PSC cells affects haematopoiesis, Antp expression was examined in third instar col mutant lymph glands. In this background, all Antp-positive PSC cells are missing, consistent with the previously described role for col in PSC maintenance. Overexpression of Antp within the PSC increases the size of PSC from the usual 30-45 cells to 100-200 cells. These PSC cells are scattered over a larger volume, often forming two or three large cell clusters rather than the single, dense population seen in wild type (Mandal, 2007).

To determine the role of PSC in haematopoiesis, the expression pattern of various markers was investigated in lymph glands of larvae of the above genotypes, which either lack a PSC or have an enlarged PSC. The status of blood cell progenitors was directly assessed using the medullary-zone-specific markers ZCL2897, DE-cadherin (Shotgun) and domeless-gal4. In col mutant lymph glands, expression of these markers is absent or severely reduced and when the PSC is expanded, the medullary zone is greatly enlarged. Previous work demonstrated that medullary zone precursors are relatively quiescent, a characteristic similar to the slowly cycling stem cell or progenitor populations in other systems. BrdU incorporation in the wild-type lymph gland is largely restricted to the cortical zone, but in third-instar col mutants incorporation of BrdU is increased relative to wild type and becomes distributed throughout the lymph gland, suggesting that the quiescence of the medullary zone haematopoietic precursors is no longer maintained in the absence of the PSC. Similarly, when the PSC domain is expanded, BrdU incorporation is significantly suppressed throughout the lymph gland (Mandal, 2007).

P1 and ProPO were used as markers for plasmatocytes and crystal cells, respectively, to assess the extent of haemocyte differentiation within lymph glands of the above genotypes. Loss of the PSC does not compromise haemocyte differentiation; rather, mature plasmatocytes and crystal cells are found abundantly within the lymph gland. Furthermore, the distribution of these differentiating cells is not restricted to the peripheral region that normally constitutes the cortical zone and many cells expressing ProPO and P1 can be observed medially throughout the region normally occupied by the medullary zone. Increasing the PSC domain causes a concomitant reduction in the differentiation of haemocytes (Mandal, 2007).

In summary, loss of the PSC causes a loss of medullary zone markers, a loss of the quiescence normally observed in the wild-type precursor population and an increase in cellular differentiation throughout the lymph gland. Similarly, increased PSC size leads to an increase in the medullary zone, a decrease in BrdU incorporation and a decrease in the expression of maturation markers. It is concluded that the PSC functions as a haematopoietic niche that maintains the population of multipotent blood cell progenitors within the lymph gland. The observed abundance of mature cells in the absence of the PSC suggests that the early blood cell precursors generated during the normal course of development will differentiate in the absence of a PSC-dependent mechanism that normally maintains progenitors as a population. This situation is reminiscent of the Drosophila and C. elegans germ lines in which disruption of the niche does not block differentiation per se, but lesser numbers of differentiated cells are generated as a result of the failure to maintain stem cells. It is also interesting to note that col mutant larvae are unable to mount a lamellocyte response to immune challenge. It is speculated that this could be because of the loss of precursor cells that are necessary as a reserve to differentiate during infestation (Mandal, 2007).

Recent work on several vertebrate and invertebrate developmental systems has highlighted the importance of niches as unique microenvironments in the maintenance of precursor cell populations. Examples include haematopoietic, germline and epidermal stem cell niches that provide, through complex signalling interactions, stem cells with the ability to self-renew and persist in a non-differentiated state. The work presented in this report demonstrates that the PSC is required for the maintenance of medullary zone haematopoietic progenitors. The medullary zone represents a group of cells within the lymph gland that are compactly arranged and express the homotypic cell-adhesion molecule, DE-cadherin. These cells are pluripotent, slowly cycling and undifferentiated and are capable of self-renewal. It is presently uncertain whether Drosophila has blood stem cells capable of long-term repopulation as haematopoietic stem cells are in vertebrates. Nevertheless, it is clear that the maintenance of medullary zone cells as precursors is niche dependent (Mandal, 2007).

In order for the PSC to function as a haematopoietic niche there should exist a means by which the PSC can communicate with precursors. As such, a signal emanating from the PSC and sensed by the medullary zone represents an attractive model of how this might occur. Although it has been reported that Ser and Upd3 are expressed in the PSC, preliminary analysis suggests that elimination of either of these ligands alone will not cause the phenotype seen for Antp and col mutants. Therefore the haematopoietic role of several signalling pathways was investigated and the hedgehog (hh) signalling pathway was identified as a putative regulator in the maintenance of blood cell progenitors. The hh^ts2 lymph gland is remarkably similar in its phenotype to that seen for Antp hypomorphic or col loss-of-function mutants. Blocking Hh signalling in the lymph gland through the expression of a dominant-negative form of the downstream activator Cubitus interruptus (Ci, the Drosophila homologue of Gli) also causes a phenotype similar to that observed in Antp and col loss-of-function backgrounds. This is true when expressed either specifically in the medullary zone or throughout the lymph gland (Mandal, 2007).

Consistent with the above functional results, Hh protein is expressed in the second instar PSC and continues to be expressed in third instar PSC cells. In the hh^ts2 mutant background, the PSC cells continue to express Antp at the restrictive temperature indicating that, unlike col and Antp, Hh is not essential for the specification of the PSC. Rather, Hh constitutes a component of the signalling network that allows the PSC to maintain the precursor population of the medullary zone. Consistent with this notion, downstream components of the Hh pathway, the receptor Patched (Ptc) and activated Ci, are found in the medullary zone. On the basis of both functional and expression data, it is proposed that Hh in the PSC signals through activated Ci in medullary zone cells, thereby keeping them in a quiescent precursor state (Mandal, 2007).

The Hh pathway has been studied extensively in the context of animal development. Although the Hh signal does not disperse widely on secretion, many studies have shown that this signal can be transmitted over long distances. The mechanism by which this occurs is not fully clear and this is also true of how the PSC delivers Hh to medullary zone progenitors. However, when labelled with green fluorescent protein (GFP), it was found that PSC cells extend numerous thin processes over many cell diameters. The morphology of the PSC cells, taken together with the long-range function of Hh revealed by the mutant phenotype, indicates that the long cellular extensions may deliver Hh to receiving cells not immediately adjacent to the PSC. In this respect, the Drosophila haematopoietic system shows remarkable similarity to the C. elegans germline. In both cases, precursors are maintained as a population over some distance from the niche and in both instances, the niche cells extend long processes when interacting with the precursors (Mandal, 2007).

Several studies have highlighted the importance of homeodomain proteins in stem cell development and leukaemias. Likewise, the role of Hh in vertebrate and invertebrate stem cell maintenance has recently received much attention. This study describes direct roles for Antp in the specification and Hh in the functioning of a haematopoietic niche. The medullary zone cells are blood progenitors that are maintained in the lymph gland at later larval stages by Hh, a signal that originates in the PSC. The maintenance of these progenitors provides the ability to respond to additional developmental or immune-based haematopoietic signals. On the basis of these findings, understanding the specific roles of Hh signalling and Hox genes in the establishment and function of vertebrate haematopoietic niches warrants further investigation. The identification of a haematopoietic niche in Drosophila will allow future investigation of in vivo niche/precursor interactions in a haematopoietic system that allows direct observation, histological studies and extensive genetic analysis (Mandal, 2007).

EFFECTS OF MUTATION OR DELETION

The absence of Antp+ function during embryogenesis results in the larval mesothorax exhibiting characteristics of the prothorax and an ensuing lethality; the loss of Antp+ function in the development of the adult thorax causes specific portions of the leg, wing and humeral imaginal discs to develop abnormally. Every Antp mutation, however, does not cause all of these developmental defects. Certain mutant alleles disrupt humeral and wing disc development without affecting leg development, and they are not deficient for the wild-type function required during embryogenesis. Other Antp mutations result in abnormal legs, but do not alter dorsal thoracic development. Mutations of each type can complement to produce a normal adult fly, which suggests that there are at least two discrete functional units within the locus (Abbott, 1986).

Ectopic expression of homeotic genes, Dfd, Scr and Antp, results in the disruption of the developing PNS in the abdomen. Thus homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression (Heuer, 1992).

In Drosophila, segment-specific muscle pattern is thought to be determined by the autonomous function of homeotic selector genes in the mesoderm in combination with inductive cues from the developing epidermis and nervous system. The expression patterns of homeoproteins were determined in the mesoderm of the thoracic segments during embryonic and adult development. Unlike the mesoderm of the first and third thoracic segments which express Sex combs reduced and Antennapedia, respectively, the mesoderm of the second thoracic segment does not express any known homeotic selector gene of the Antp or bithorax complex. In animals homozygous for Antp null mutations, the muscles of the second thoracic segment were affected in the embryo, probably as an indirect consequence of its requirement in the ectoderm. Animals that specifically lack Antp function in the mesoderm, but expressed the gene in the epidermis, developed with a normal muscle pattern in the second thoracic segment. Specific ectopic expression of Antp and other homeotic selector genes in the mesoderm of the second thoracic segment respecifies its muscle pattern, indicating that these genes are not required autonomously during muscle development in this segment. Antp continues to be expressed in the mesoderm of the homeotically transformed third thoracic segment in the "four-winged fly." This is a likely reason for the failure of flight muscle development in the transformed segment. A model for muscle development in the second thoracic segment is presented whereby mesodermal properties are specified entirely by induction, in contrast to muscle development in other segments, where autonomous function for homeotic selector genes is also required (Roy, 1997).

Segment-specific differences are evident in the number of neuroblasts (NBs) that persist beyond the end of embryogenesis and proliferate during larval stages. At stage 17 of embryogenesis, all NBs have stopped dividing but can still be monitored by NB-specific expression of grainyhead. Analyses of Grh expression pattern in the CNSs of wild type embryos and of mutant embryos where cell death is suppressed, strongly suggest that a number of NBs normally die towards the end of embryogenesis. The degree of cell death shows segment-specific differences: many more NBs die in the central abdomen than in the thorax and anterior abdomen. As a consequence, when NBs resume proliferation as postembryonic NBs in the larval stages, 47 NBs are detected in each thoracic segment; about 12 are detected in the two anterior abdominal neuromeres, but only six in central abdominal segments. Furthermore, postembryonic NBs in the thorax and anterior abdomen produce hundreds of daughter cells each, whereas those in abdominal neuromeres 3-A7 give rise to only five to 15 cells. In summary there are three major factors regulating the segment-specific proliferation of NBs: (1) the period and frequency of embryonic NB proliferation; (2) the number of NBs eliminated at the end of embryogenesis, and (3) the frequency and period of postembryonic proliferation (Prokop, 1998 and references).

The number and pattern of neuroblasts that initially segregate from the neuroectoderm in the early Drosophila embryo are identical in thoracic and abdominal segments. However, during late embryogenesis, differences in the numbers of NBs and in the extent of neuroblast proliferation arise between these regions. The homeotic genes Ultrabithorax and abdominal-A regulate these late differences. Abdominal NBs in Ubx and abd-A mutants continue replicating DNA, and consequently the number of NBs in these mutants resembles that of thoracic neuroblasts. In embryos lacking the Antp gene, DNA synthesis in ventrolateral/lateral NBs is normal, however, additional cells are detected in ventral positions resembling the ventral patterns of the subesophageal ganglion. Therefore abd-A function is needed to repress DNA replication in some lateral NBs of abdominal neuromeres, and Antp function is required to repress DNA replication in ventral NBs of the thorax. Misexpression of either Ubx or abd-A in thoracic neuroblasts, after segregation, is sufficient to induce abdominal behaviour in lateral neurons and subesophageal characteristics in ventral neurons. The ventral pattern appears to be due to the ability of Ubx to repress Antp expression, since the pattern of ventral neurons resembles the phenotype found in Antp mutant embryos. In wild type embryos, Abdominal-A and Ultrabithorax proteins are only detected in early neuroblasts. In stage 15 embryos no cells are found which co-express Ubx and Grh. This suggests that neither Abd-A nor Ubx are present in the NBs shortly before segment-specific differences in the numbers of cells and Grh patterns occur. Asense is expressed in NBs shortly after their segregation from the neuroectoderm and so can be used as an early marker for NBs. Ubx is detected in many NBs at stages 8-12 although there is wide variation between levels of Ubx present in different NBs and a subset of NBs contain no detectable Ubx. Similarly, Abd-A is present in many NBs at early stages. Thus both Ubx and Abd-A are present in embryonic NBs, but their expression fades before segment-specific differences become detectable (Prokop, 1998).

Transplantation experiments reveal that segment-specific behaviour is determined even prior to neuroblast segregation, that is, prior to expression of Ubx or Abd-A. When cells are heterotopically transplanted from thoracic to abdominal sites of the early gastrula neuroectoderm, 67% give rise to a large nest of postembryonic cells with postembryonic NB (pNB), consistent with the characteristics of thoracic NBs. Conversely, when cells are transplanted from abdominal to thoracic sites, all clones fail to express thoracic features and contain only embryonic cells. It is concluded that segment-specific differences in neuroblast behaviour seem to be determined in the early embryo, mediated through the expression of homeotic genes in early neuroblasts, and executed in later programs controlling neuroblast numbers and proliferation. Two models are presented for the action of the homeotic genes. They could act as transcriptional repressors that initiate a repressed state for their target genes, which can be maintained after the proteins have disappeared, or alternatively, they may activate target genes that have the capacity for autoregulation, so that the targets maintain their own expression in the absence of homeotic proteins (Prokop, 1998).

Null mutations in Antp result in a transformation of T2 and T3 towards T1 in the embryonic body plan. In addition, Antp mutant embryos develop ectopic head-like sclerites in the dorsal thorax (between T1 and T2), similar in kind but not in position to the ectopic sclerite phenotype seen in split ends (spen) mutants. To test whether spen and Antp function in an additive or synergistic manner in the repression of head-like sclerites in the thorax, spen^-; Antp^- cuticle phenotypes were examined. Embryos mutant for both spen and Antp have more sclerotic material in dorsal T2 than do Antp mutants alone. In addition, the ectopic head-like sclerites in the ventral thorax of spen^-;Antp^- mutants are more sclerotized and extensive than in spen mutants alone. The sclerotic material in spen^-;Antp^- mutants frequently appears in two distinct bands, one in the center of the segment similar to the position in spen mutants, and at another position in the posterior of T1 and T2. These posterior ectopic sclerites do not develop in T3. The enhanced formation of head-like sclerites in spen^-;Antp^- mutants suggests that spen and Antp function in a common or interacting pathway(s) in subregions of T1 and T2 (Wiellette, 1999).

The synergistic effect of Antp and spen might be due to a regulatory effect of Antp on spen transcription pattern, or to Spen effects on Antp transcript pattern or translation. However, Antp transcript and protein expression patterns are unchanged in spen mutant embryos, and spen transcript expression is unchanged in Antp mutant embryos. Therefore, spen and Antp appear to be acting in parallel, presumably due to direct or indirect regulation of common downstream genes (Wiellette, 1999).

If spen and Antp regulate common targets, then induction of high levels of exogenous Antp expression might result in suppression of the spen mutant phentoype. The ability of excess Antp protein to suppress the spen mutant phenotype was examined. Overexpression of Antp under heat shock promoter control (hsAntp) causes a transformation of head regions to thoracic identity, but leaves T2 and T3 nearly unchanged. When Antp is overexpressed in a spen mutant background, the ectopic head-like sclerites are strongly suppressed. The number of hsAntp; spen^- embryos that exhibit any detectable ectopic sclerites is less than half the expected number compared to spen^- mutant siblings from the same cross, or compared to spen^-; hsAntp embryos that were not subjected to heat shock. In addition, the sclerites which do occasionally appear in heat shocked hsAntp; spen^- embryos are smaller than those in their spen^- siblings. The ability of excess Antp to suppress the spen^- homeotic transformation indicates that the two genes interact to repress ectopic head-like sclerites (Wiellette, 1999).

Introgression of homeotic mutations into wild-type genetic backgrounds results in a wide variety of phenotypes and implies that major effect modifiers of extreme phenotypes are not uncommon in natural populations of Drosophila. A composite interval mapping procedure was used to demonstrate that one major effect locus accounts for three-quarters of the variance for haltere to wing margin transformation in Ultrabithorax flies, yet has no obvious effect on wild-type development. Several other genetic backgrounds result in a pronounced enlargement of the haltere, significantly beyond the normal range of haploinsufficient phenotypes, suggesting genetic variation in cofactors that mediate homeotic protein function. Introgression of Antennapedia produces lines with heritable phenotypes ranging from almost complete suppression to perfect antennal leg formation, as well as transformations that are restricted to either the distal or proximal portion of the appendage. It is argued that the existence of potential variance, which is genetic variation whose effects are not observable in wild-type individuals, is a prerequisite for the uncoupling of genetic from phenotypic divergence (Gibson, 1999).

The closely related Hox transcription factors Ultrabithorax (Ubx) and Antennapedia (Antp) respectively direct first abdominal (A1) and second thoracic (T2) segment identities in Drosophila. It has been proposed that their functional differences derive from their differential occupancy of DNA target sites. A hybrid version of Ubx (Ubx-VP16), which possesses a potent activation domain from the VP16 viral protein, no longer directs A1 denticle pattern in embryonic epidermal cells. Instead, it mimics Antp in directing T2 denticle pattern, and it can rescue the cuticular loss-of-function phenotype of Antp mutants. In cells that do not produce denticles, Ubx-VP16 appears to have largely retained its normal repressive regulatory functions. These results suggest that the modulation of Hox activation and repression functions can account for segment-specific morphological differences that are controlled by different members of the Hox family. These results also are consistent with the idea that activity regulation underlies the phenotypic suppression phenomenon in which a more posterior Hox protein suppresses the function of a more anterior member of the Hox cluster. The acquisition of novel activation and repression potentials in Hox proteins may be an important mechanism underlying the generation of subtle morphological differences during evolution (Li, 1999).

Interestingly, although Ubx-VP16 acquires an Antp-like ability in denticle patterning, it preserves the Ubx ability to repress Keilin's organ development in thoracic segments. Therefore, Ubx-VP16 displays a mix of Antp-like and Ubx-like functions, dependent on tissue types and cell positions. Since development of Keilin's organs requires the appendage-promoting gene Distalless (Dll), the regulation of Dll by Ubx-VP16 was examined. The expression of Dll in thoracic appendage primordia cells is repressed by Ubx by means of the Dll304 element, presumably by eliciting the Ubx repression function on the element. In ectopic Ubx-VP16 embryos, both Dll expression and the activity of the Dll304 element are partially repressed. However, unlike ectopic Ubx, Ubx-VP16 is capable of activating Dll304 in other cells outside the appendage primordia. Thus, the Ubx-like function of Ubx-VP16 in repressing Keilin's organ development stems from retaining the Ubx repressive function upon Dll transcription. Since this repression appears specific for appendage primordia cells, the repression function of Ubx-VP16 is not constitutive but rather generated in a regulated manner. Taken together, the above results suggest that Ubx-VP16 functions are due to normal Ubx repressive effects on some targets (e.g., Dll), despite the attached VP16 activation domain, as well as a novel activation function on other targets (e.g., Antp) caused by the VP16 domain. The mix of functions that Ubx-VP16 exhibits is also often observed for natural Hox proteins (Li, 1999).

In these experiments the strength of activation function in Ubx is artificially varied. However, the partial change in segmental identity conferred by the Ubx-VP16 protein suggests that regulating the activity state of Ubx may modulate its functional specificity in denticle patterning. The fact that the Ubx-VP16 denticle patterning function is Antp-like suggests that the functional difference between the Ubx and Antp proteins in diversifying denticle patterns may reside in differences in activation and repression strengths on similar target genes rather than in differences in target occupancy. This suggestion is consistent with results indicating that Ubx and Antp recognize identical DNA sequences in vitro and regulate several common target genes in embryos. This evidence indicates that the segment identity functions of Ubx and Ubx-VP16 are distinct, but it does not eliminate the possibility that the VP16 domain increases activation function by altering the binding selectivity of the hybrid protein in developing embryos. This is thought to be unlikely because the specific Ubx targets such as dpp, Antp, and Dll are all regulated, and thus presumably occupied at similar Hox sites, by both Ubx and Ubx-VP16 (Li, 1999 and references).

Regulation of activation and repression functions may also be the mechanism that underlies the phenomenon of phenotypic suppression, in which one Hox protein can dominantly suppress the function of other coexpressed Hox proteins. It has been proposed that competition of Hox proteins for DNA binding sites is responsible for this phenomenon. A well studied example of phenotypic suppression is the parasegment-specific transcription of the decapentaplegic gene in the visceral mesoderm (VM). dpp is directly activated by Ubx protein in PS7 but is repressed by Abdominal-A (Abd-A) protein in PS8-12 of the VM, even when Ubx protein is ectopically expressed in PS8-12. The repression conferred by Abd-A and the activation conferred by Ubx involves separate clusters of Hox binding sites within the dpp674 element (34). This suggests that Abd-A does not compete with Ubx for binding to the same DNA sites to antagonize Ubx activation on dpp. Instead, Abd-A and Ubx proteins can occupy many sites on the dpp674 element in PS8-12, but only Abd-A is capable of conferring repression from one of the clusters of Hox sites. The Abd-A repression function can then override the Ubx activation function that is produced from another cluster of Hox binding sites on dpp674 (Li, 1999 and references).

The role of activity regulation in Hox segmental specificity may provide new insight into understanding how the Hox patterning system evolved. At an early point in metazoan evolution, prototypes of Hox genes such as Ubx and Antp were generated by the duplication of a common ancestral gene. After the duplication event, one or both of the two copies accumulated mutations and evolved distinct functions. One evolutionary event that altered function was the change in regulatory sequence that altered expression patterns, when compared with the ancestral copy. From the study of extant Hox genes, it is known that changes in the coding sequence during evolution have generated functional distinctions between adjacent Hox genes. It is proposed that coding region changes that resulted in different functional specificities did so by altering activation/repression strengths on a largely common set of downstream genes. One reason for this proposal is that mutations in coding sequences that altered binding specificity would presumably influence target occupancy on all or most downstream genes. Therefore, the newly evolved Hox protein T would no longer regulate many of the genes under the control of Hox protein S, and protein T would also immediately acquire a novel battery of downstream genes. It is imagined that these events would result in striking morphological changes in the body plan that would have a low chance of surviving and being selected. However, differences in coding sequences controlling activation/repression strengths could subtly or drastically vary the amount of gene expression from one or a few members of a common set of downstream genes (x and y for example). The difference that evolves in Hox T could be depicted as an adoption of a novel repression ability on gene y. This mode of Hox protein evolution would be more likely to result in subtle changes in metameric morphology compatible with survival. Occasionally some of these subtle morphological changes would result in slight advantages in natural selection for certain niches. This model also requires changes (either preexisting or acquired) in downstream gene regulatory sequences that are near Hox binding sites, so that factors that regulate the repression strength of Hox T could switch it into a repressive mode. This model of evolving diverse Hox functions by subtle changes in activation/repression strengths is not meant to discount the importance of evolutionary variation in downstream genes in morphological variation (Li, 1999 and references).

How can it be examined whether this process has occurred in evolution? In the embryo of the crustacean Artemia, the Antp, Ubx, and abd-A homologs are coexpressed in a trunk region that is composed wholly of appendage-bearing segments. In contrast to Drosophila, the Artemia Ubx and Abd-A homologs do not repress Dll transcription and do not repress appendage development. There are a variety of reasons why the Artemia Ubx and Abd-A proteins might be incapable of repressing appendages, but one possibility is that sequence motifs within the proteins that would allow them to repress the appendage enhancer of Dll are missing. This possibility may be testable by placing the Artemia versions of Ubx and Abd-A proteins in the context of Drosophila early embryonic cells and assaying their effects on appendage development (Li, 1999 and references).

The Sex combs reduced gene specifies the identities of the labial and first thoracic segments in Drosophila. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, a model is proposed in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element that is located physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome (Southworth, 2002).

The two putative negative regulatory elements are located distal and proximal to the 60–70 kb region that includes the chromosome rearrangements that cause the appearance of ectopic sex comb teeth. Although the distal and proximal elements may be different, both putative regulatory elements are referred to as maintenance elements for silencing (MES). In this model, when the Scr gene is active, flanking MESs fail to interact. When the Scr gene is silenced, the flanking MESs preferentially interact in cis to stabilize silencing of genes in between. The interaction of MESs may occur through the binding of different proteins to these elements when silencing is specified, or it may occur by the modification of proteins already bound even when the gene is active. Maintenance of silencing, however, affects only genes that lie between two elements; i.e., silencing requires the ability to form a physical loop of DNA between the two elements. Interaction of the elements on the wild-type homolog would preferentially occur in cis, maintaining silencing in most cells. However, because the silencing elements on the broken chromosome are no longer in cis, they could compete for interactions with the silencing elements on the wild-type homolog. If both elements on the aberration chromosome interact with the elements on the homolog, one configuration might be stable enough to prevent interaction of the two elements in cis on the wild-type chromosome. This would disrupt silencing of the Scr promoter between these two elements, allowing derepression of the wild-type Scr gene. It is believed that deletion chromosomes that contain only one MES are not able to effectively compete with the cis interactions on the wild-type homolog. This model can account for all of the data described so far, and it can also explain the behavior of an old mutation with very anomalous properties. This is the Antp^Scx mutation isolated in 1953 (Southworth, 2002).

The Antp^Scx mutant was isolated originally on the basis of a dominant extra sex combs phenotype. It is lethal when heterozygous to Antp mutant alleles, but is viable when heterozygous to Scr mutant alleles. The Antp^Scx mutant chromosome is cytologically normal and the only molecular lesion identified in the ANTC was the insertion of repetitive DNA very close to the Antp P1 promoter. Given the physical location of the insertion, it is not surprising that the Antp^Scx mutant chromosome fails to complement Antp alleles that specifically lack P1 function, such as Antp^B, Antp^73b, Antp^CB, and Antp¹⁷. There is no difference in the average number of sex comb teeth per first leg in Antp^Scx heterozygous males compared to homozygous wild type or in Antp^Scx/Scr⁴ males compared to +/Scr⁴ males. Males heterozygous for Antp^Scx, however, do have a considerable number of ectopic sex comb teeth (an average of 2.7 per second leg). The ectopic sex comb teeth result from misexpression of Scr in cis and in trans. Males with Scr mutations in cis to Antp^Scx (Scr^E2 Antp^Scx/+ and Scr^E3 Antp^Scx/+) have fewer sex comb teeth per second leg (an average of 0.8); males with Scr mutations on the homolog (Antp^Scx/Scr² and Antp^Scx/Scr⁴) also have fewer sex comb teeth per second leg (an average of 1.3–1.6). Scr mutations both in cis and in trans to Antp^Scx [Scr^E2 Antp^Scx/ Scr⁴;Dp(3;Y)77ab] almost completely eliminate the ectopic expression of Scr (an average of only 0.02 sex comb teeth per second leg). Comparison of the effects of Scr mutations in cis and in trans also suggest that Antp^Scx derepresses the Scr promoter in cis about twice as much as the Scr promoter on the homolog. A molecular mechanism through which the insertion of repetitive DNA ~150 kb upstream of the Scr promoter might be responsible for transcriptional derepression of both the cis promoter and the Scr promoter on the homolog has not been previously suggested (Southworth, 2002).

This model is the first attempt to explain the unusual properties of the Antp^Scx mutant chromosome. It is believed that the repetitive DNA inserted near the Antp P1 promoter on the Antp^Scx mutant chromosome mimics the endogenous regulatory elements involved in the maintenance of silencing (the MES elements). By competing for interactions with the endogenous elements either on the same chromosome or on the homolog, the Antp^Scx insertion disrupts silencing of the Scr promoter in cis or in trans, respectively. In this respect, the Antp^Scx insertion appears to be more effective than a wild-type MES, since deletion chromosomes with a single MES do not interfere with silencing on the homolog. Not only does this model explain the existing data, but it also makes a prediction. The Antp P2 promoter is between the repetitive insertion on the Antp^Scx mutant chromosome and the endogenous regulatory elements in the Scr gene. Interactions between the Antp^Scx insertion and the endogenous elements in the Scr gene in cis should not only derepress the Scr promoter, but should also silence the Antp P2 promoter. Interactions between the Antp^Scx insertion and the endogenous elements in the Scr gene in trans should not silence the Antp P2 promoter. Since the Antp^Scx mutation appears to derepress Scr in cis about twice as much as in trans, about two-thirds of the cells are expected to lack Antp P2 function from the Antp^Scx chromosome. Two mutations (Antp¹ and Antp²³) have been characterized that inactivate the Antp P2 promoter but appear to have normal function for the Antp P1 promoter. These two mutations can be used to examine Antp P2 function on the homologous chromosome in heterozygotes. As expected from this model, Antp^Scx interferes significantly with function of the P2 promoter; Antp^Scx fails to complement both Antp¹ and Antp²³ for viability (no surviving adults were found among several hundred expected). In contrast, deletions that remove the Antp P1 promoter and chromosome aberrations that physically separate the P1 and P2 promoters are all viable when heterozygous to either Antp¹ or Antp²³. With these results, four genetic properties are now associated with the Antp^Scx mutant chromosome: (1) loss of Antp P1 function, (2) loss of Antp P2 function, (3) derepression of the Scr promoter on the mutant chromosome, and (4) derepression of the Scr promoter on the homolog (Southworth, 2002).

It is possible that there are multiple molecular lesions on the Antp^Scx mutant chromosome that were not detected in the molecular analyses. However, it should be emphasized that the Antp^Scx mutant chromosome is cytologically normal, is wild type for Scr function, and has the ability to derepress the Scr gene in trans. Only Scr mutations that have chromosome aberration breakpoints within the Scr locus have the ability to derepress Scr in trans. The model explains how the identified molecular lesion could lead to all of the mutant phenotypes observed (Southworth, 2002).

In the model, trans interactions between MESs occur when the cis interactions are disrupted. Although PREs are believed to normally act in cis to maintain silencing, they are also able to act in trans when included within transgenes. These trans interactions of PREs are enhanced when cis interactions are blocked. In addition, while single PREs appear to partially silence transgenes, silencing is often greater when multiple PREs can interact. A pair of major PREs has also been characterized in about the same position in the Ultrabithorax (Ubx) homeotic gene as the MESs in the Scr gene; i.e., one PRE is ~25 kb upstream of the Ubx promoter and a second PRE is within an intron in the middle of the transcription unit. Therefore, an important question is whether MESs are the same as PREs. They are likely to be distinct elements, but are often in close proximity. Many DNA fragments that contain PREs may also contain MES elements, but these activities may be separable. For example, a 2.9-kb DNA fragment from the Mcp region of the bithorax complex appears to contain at least two different types of regulatory elements. An 800-bp DNA fragment from the central region of the larger fragment is not sufficient for silencing, but it is sufficient for mediating pairing-sensitive interactions between transgenes on different chromosomes. It is also sufficient for mediating long-range interactions between enhancers and promoters in transgenes. Two XbaI restriction fragments from Scr (an 8.2-kb fragment from the second intron and a 10.0-kb fragment 35–45 kb upstream of the promoter) that have been tested in transgenes for PRE activity overlap with the putative MESs. Both fragments appear to partially silence the reporter gene in a transgene assay. This silencing is sensitive to some Polycomb group mutations; however, the two tested fragments differ as to which Polycomb group mutations had effects. Interestingly, only the 8.2-kb fragment exhibited pairing-sensitive silencing, while only the 10.0-kb fragment functioned as a PRE in embryos. The apparent independence of MES function and Polycomb group repression also suggests that MESs may be separate elements that are in close proximity to PREs. It is possible that MESs act to maintain interactions between nearby PREs, thus facilitating the maintenance of silencing. In this respect, MESs may be similar to the pairing-sensitive regulatory elements identified upstream of the engrailed promoter (Southworth, 2002).

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