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 hhts2 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 hhts2 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).

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).

Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade

During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. This study found that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI> 0 switch is triggered by activation of Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS (Baumgardt, 2014).

Proliferation analysis of the developing Drosophila VNC reveals that most, if not all, lateral NBs initially divide in the type I proliferation mode, generating daughters that divide once. Three specific lineages, as well as many other NBs, subsequently switch to generating daughters that do not divide (type 0 mode). The full extent of the typeI>0 switch is currently difficult to precisely assess for several reasons. One such complicating issue pertains to possible developmental changes in daughter cell-cycle length over time. On this note, however, no obvious change was found in NB5-6T daughter divisions prior to the switch. In addition, if the accelerated decline in daughter division was indeed caused by a lengthening of the cell cycle rather than a typeI>0 switch, a long 'tail' of daughter divisions would be expected, perduring into St16-17. This is not the case; rather, daughter proliferation drops down to almost zero by St16-17. Similarly, no evidence was found for changes in NB cell-cycle length over time in the three specific NB lineages. Another complicating issue pertains to the fact that NBs differ in their time point of delamination, number of division rounds, and time point of switching, so that even if all NBs switched, only a fraction of the NBs would be in their type 0 window at the same time. However, these complications are more likely to lead to under- rather than overappreciation of the extent of the typeI>0 switch, and it is tempting to speculate that it may indeed involve the vast majority of NBs (Baumgardt, 2014).

The typeI>0 switch is triggered by the onset of Dap expression in NBs at precise stages of lineage progression. The mammalian Dap orthologs, p21CIP1/p27KIP1/p57Kip2, can act as inhibitors of the CycE/Cdk2 complex. By analogy, the mechanism behind the typeI>0 switch is, presumably, that type 0 daughters are prevented from entering the cell cycle by the presence of Dap at the G1/S checkpoint. The onset of Dap expression already in the NB suggests that Dap needs to be present at an early stage in newborn daughters to block their entry into S phase. These findings are also in line with the emerging role for the Cip/KIP family and cell-cycle exit in the mammalian CNS, although there has been no report of a connection to changes in daughter proliferation mode (Baumgardt, 2014).

No evidence was found for a role of pros in the type 0 mode, and, conversely, no evidence was found for a role of dap in the type I mode. The distinct roles of pros and dap in control of the type I versus 0 modes is further underscored by the expression of E2f, CycE, and Dap. In type I daughters (GMCs), E2f and CycE are rapidly repressed, by pros, and Dap is only weakly expressed at a later stage, around the time point of mitosis. The short window of E2f and CycE expression is still sufficient for the GMC to enter another cell cycle, since Dap expression is absent. As each GMC divides, the postmitotic cells (neurons/glia) are prevented from entering the cell cycle by the lack of E2f and CycE. In type 0 daughters, on the other hand, E2f and CycE expression is robust, but daughters still fail to enter the cell cycle due to the presence of high levels of Dap. These findings point to strikingly different strategies in daughter proliferation control: pros repression of E2f/CycE in type I, and Dap overriding E2f/CycE/Cdk2 in type 0 daughters (Baumgardt, 2014).

Changes in daughter cell proliferation could perhaps have been envisioned to merely result from a gradual loss of the proliferative potential of each progenitor, as a result of its undergoing many rapid cell cycles. If so, typeI>0 switches could have been predicted to occur somewhat stochastically toward the end stage of each lineage, perhaps loosely linked to the last NB division. In contrast to such simplified models, this study found that the typeI>0 switch can occur many divisions prior to NB exit and that it is programmed to occur at a precise stage during each lineage development. In the thorax, it was found that the precise timing of typeI>0 switches is controlled by the temporal gene cas and the Hox gene Antp, which are expressed at a late stage within NBs. Remarkably, in cas mutants, most, if not all, thoracic typeI>0 lineages fail to enter the type 0 mode. The primary mechanism by which cas and Antp control the switch appears to be by activating the expression of Dap, evident by the reduction of Dap in cas and Antp mutants; by the finding that cas-Antp co-misexpression triggers ectopic Dap expression; and by the finding that cas can be cross-rescued by elav>dap (Baumgardt, 2014).

The finding that the timing of the typeI>0 switch is scheduled by a temporal gene cascade points to an intriguing regulatory model where daughter cell proliferation mode switches are executed at stereotyped positions within the lineage tree by the activity of specific temporal genes. Since temporal genes also control the progression of NB competence, evident by their roles in cell fate specification, the temporal cascade can act to simultaneously control both cell fate and cell number, thereby ensuring that precise number of each neural cell subtype is produced (Baumgardt, 2014).

After a stereotyped number of divisions, each NB subtype stops proliferating. This study found that, for many NBs, this is a G1/S decision influenced by the activities of E2f, CycE, and dap. The nuclear localization of Pros was previously identified to be associated with cell-cycle exit in postembryonic NBs. However, previous studies of NB5-6T, and the current study on NB7-3A, do not indicate a general role for pros in NB cell-cycle exit in the embryonic CNS. Instead, in the thorax, the expression levels of E2f, CycE, and Dap are gradually modulated during lineage progression, by the temporal genes cas and grh as well as Antp. Because Cas, Grh, and Antp are progressively activated in thoracic NBs, this brings into view a logical model for timely NB cell-cycle exit where sequential activation of temporal and Hox genes act combinatorially to push E2f, CycE, and/or Dap to limiting levels after a determined number of divisions (Baumgardt, 2014).

For the majority of NBs in the thorax, cell-cycle exit is followed by quiescence until larval stages. In contrast, for the majority of abdominal NBs, cell-cycle exit is followed by apoptosis. However, for some NBs, such as NB7-3A, apoptosis is the functional exit mechanism. Thus, three general strategies for lineage stop are emerging: (1) cell-cycle exit > quiescence (most thoracic NBs), (2) cell-cycle exit > apoptosis (NB5-6T), and (3) lineage stop by apoptosis (NB7-3A). The balance of E2f, CycE, and Dap is involved in the first two strategies, while the balance of apoptosis gene expression presumably is at the core of the latter strategy (Baumgardt, 2014).

In addition to the type I and type 0 daughter proliferation modes described here in the embryo, recent studies of Drosophila larval CNS development have identified a third, more prolific, proliferation mode: the type II mode, identified in a small number of larval brain. Type II NBs divide asymmetrically, renewing themselves while budding of daughters that, in turn, undergo multiple rounds of proliferation before finally differentiating. This allows for the generation of very large lineages (some 500 cells) from each individual type II NB (Baumgardt, 2014).

In mammals, the most obvious equivalent of Drosophila NBs is the radial glia cell (RG), which divides asymmetrically to generate neurons and. During these RG asymmetric divisions, studies have identified several different division modes; RGs dividing asymmetrically to bud off a neuron, to bud off a daughter cell that divides once to generate two neurons, or to bud off daughter cells that themselves divide multiple times before generating neurons. Although mammalian CNS development likely will involve more complex and more elaborate lineage variations, there is, nevertheless, a striking similarity between these alternate mammalian daughter proliferation modes and the type 0, I and II modes now identified in Drosophila. Intriguingly, in line with these analogies between Drosophila and mammals, recent time-lapse studies on the developing primate cortex have revealed a global temporal switch in the proliferation profiles of daughter cells (Betizeau, 2013). It will be interesting to learn if such temporal proliferation changes are intrinsically controlled and if they are stereotypically linked to changes in neural subtype specification also in mammals (Baumgardt, 2014).

Neuroblast lineage identification and lineage-specific Hox gene action during postembryonic development of the subesophageal ganglion in the Drosophila central brain

The central brain of Drosophila consists of the supraesophageal ganglion (SPG) and the subesophageal ganglion (SEG), both of which are generated by neural stem cell-like neuroblasts during embryonic and postembryonic development. Considerable information has been obtained on postembryonic development of the neuroblasts and their lineages in the SPG. In contrast, very little is known about neuroblasts, neural lineages, or any other aspect of the postembryonic development in the SEG. This study characterized the neuroanatomy of the larval SEG in terms of tracts, commissures, and other landmark features as compared to a thoracic ganglion. Then clonal MARCM labeling was used to identify all adult-specific neuroblast lineages in the late larval SEG, and a surprisingly small number of neuroblast lineages, 13 paired and one unpaired, were found. The Hox genes iDfd, Scr, and Antp are expressed in a lineage-specific manner in these lineages during postembryonic development. Hox gene loss-of-function causes lineage-specific defects in axonal targeting and reduction in neural cell numbers. Moreover, it results in the formation of novel ectopic neuroblast lineages. Apoptosis block also results in ectopic lineages suggesting that Hox genes are required for lineage-specific termination of proliferation through programmed cell death. Taken together, these findings show that postembryonic development in the SEG is mediated by a surprisingly small set of identified lineages and requires lineage-specific Hox gene action to ensure the correct formation of adult-specific neurons in the Drosophila brain (Kuert, 2014).

A total of 14 identified postembryonic neuroblast lineages generate the adult-specific secondary neurons in the larval SEG. This is a surprisingly small number compared with the approximately 80 neuroblast lineages in the embryonic SEG. Cell counts indicate that only about one fourth of these ~80 neuroblasts are reactivated postembryonically. This is markedly different in the supraesophageal ganglion (SPG), where about 85 of the 100 embryonically active neuroblasts are reactivated and proliferate in larval stages. The experiments indicate that the fate of half of the embryonic SEG neuroblasts that are not present postembryonically is programmed cell death. This situation is comparable to that of embryonic neuroblasts in the abdominal ganglia where the majority of neuroblasts undergo apoptosis at the end of embryogenesis. The molecular cues that trigger cell death in these embryonic neuroblasts have not been studied. The fate of the other half of the embryonic SEG neuroblasts is unknown. They may terminate proliferation through other reaper/hid/grim-independent cell death mechanisms or through cell cycle exit at the end of embryogenesis. Further experiments will be necessary to elucidate this (Kuert, 2014).

The low number of postembryonic SEG lineages has interesting consequences for the relationship between primary neurons and secondary neurons in the mature SEG. Most neuroblasts generate 10-20 neural cells embryonically and 100-150 neural cells postembryonically. Thus, the ~80 embryonic SEG neuroblasts should generate 800-1600 primary neural cells per hemiganglion while the 14 postembryonic neuroblasts generate approximately 900 secondary neural cells (as estimated by cell counts) per hemiganglion. Assuming that most of the primary neurons survive metamorphosis, this suggests that a substantial fraction of the neurons in the adult SEG could be primary neurons that comprise the functional larval SEG before their integration into the adult brain (Kuert, 2014).

Previous work has shown that 75 neuroblast lineages generate the secondary neurons of the three thoracic neuromeres. This is in striking contrast to the 14 neuroblast lineages that generate secondary neurons in the three SEG neuromeres. This reduction is most evident in the SA region, where only one commissure (ISA) is present which is also formed by only one lineage, SA3. The labial neuromere is also reduced but not as dramatically. Moreover, it retains the two commissures (aI, pI) which are also characteristic of the thoracic neuromeres. This relatively small number of postembryonic neuroblast lineages in the SEG neuromeres is likely to reflect the marked reduction and fusion of segmental appendages in the three gnathal segments that are innervated by the SEG. From an evolutionary perspective, a loss/reduction of gnathal appendages in insects such as flies would eliminate or reduce the need for corresponding neural control circuitry at least in the adult. Interestingly, and in contrast to the VNC, no evidence was found for the presence of postembryonically generated motoneurons in the SEG, indicating that all secondary neurons in the SEG are interneurons. This notion is supported by the fact that none of the 14 SEG neuroblast lineages join the labial or pharyngeal nerves (which contain the motor axons from the proboscis), but instead they project their secondary axon tracts (SATs) to areas within the CNS (Kuert, 2014).

During embryonic and postembryonic brain development, the Hox genes Dfd, Scr, and Antp are regionally expressed in discrete and largely non-overlapping domains in the neuromeres of the SEG. In both cases Dfd is expressed in an anterior domain, Scr is expressed in a posteriorly adjacent domain, and Antp expression begins in a small labial domain adjacent to the prothoracic neuromere. Moreover, while the total number of neuroblast lineages that express a given Hox gene may be different embryonically and postembryonically, most of the postembryonic neuroblast lineages do express one of these genes suggesting that Hox gene expression is a stable developmental feature of SEG lineages. Indeed, most if not all of the Hox genes that are expressed in the embryonic CNS, are re-expressed in the neuroblast lineages of the postembryonic CNS (Kuert, 2014).

Hox genes are known to be expressed during CNS development in a number of bilaterian animal groups, including vertebrates, hemichordates, insects, and annelids, and in all of these animal groups the order of Hox gene expression domains in the developing CNS appears to be conserved. For example, the order of expression of orthologous Hox genes in the developing CNS of Drosophila, mouse, and human is virtually identical. Taken together, these findings suggest that a conserved pattern of Hox gene expression domains may be a common feature in the developing CNS of all bilaterians (Kuert, 2014).

This study reveals two types of lineage-specific requirement for Hox genes during postembryonic SEG development. The first is a requirement of the Hox genes Dfd, Scr and Antp for correct postembryonic development of a subset of those lineages that are normally present in the wildtype SEG. Hox genes are required for correct SAT projections in the lineages SA1 (Dfd), SA5 (Scr) and LB3 (Antp). Interestingly, in all three cases the lineage-specific loss-of-function of these Hox genes results in specific, reproducible SAT misprojections and not in randomized axonal misprojections. While this could, in principle, be the result of a homeotic transformation phenotype, no evidence was found for such a transformation, since in terms of their projection patterns mutant SATs of these three lineages do not resemble any of the wildtype SATs present in the larval SEG (Kuert, 2014).

Hox genes are also required for correct cell number in the lineages LB5 (Scr) and LB3 (Antp). While these Hox mutant lineages lose about half of their cells, which would suggest the involvement cell death in a hemilineage-dependent manner, no evidence was found for hemilineage-specific Hox gene expression in these lineages. Thus, further studies of Hox gene action in the lineages LB3 and LB5 are necessary to dissect the functional requirement of Scr and Antp in lineage-specific cell survival (Kuert, 2014).

The second type of lineage-specific requirement for Hox genes during postembryonic SEG development is the prevention of ectopic lineage formation. Thus, in addition to their requirement for correct development of normal wildtype lineages, the genes Dfd and Scr are also required for suppressing the appearance of aberrant ectopic lineages that are not normally present in the wildtype SEG. When Dfd or Scr mutant neuroblast clones are induced at early larval stages and recovered at late larval stages, five distinct types of ectopic neuroblast clones are found. Each of these is identifiable based on reproducible neuroanatomical features such as position, secondary axon tract projection and cell number. These ectopic lineages do not represent homeotic transformations of other wildtype neuroblast lineages, since all other SEG neuroblast lineages are present. Whether these ectopic lineages become functionally integrated into the adult brain of Drosophila is currently unknown. Evidence for an integration of ectopic neuron groups into a mature brain comes from mammalian studies, which show that Hoxa1 mutant hindbrain progenitors can establish supernumerary ectopic neural cell groups that escape apoptosis and give rise to a functional circuit in the postnatal brain (Kuert, 2014).

The molecular regulators through which the Hox genes Dfd, Scr and Antp exert their diverse roles in lineage-specific SEG development are currently not known. In terms of the Hox gene requirement for correct development of wildtype lineages, only 4 of the 14 SEG lineages (11 of which express Hox genes) show misprojection or cell number mutant phenotypes. However, in these 4 lineages, the Hox gene mutant phenotypes are highly penetrant and reproducible. The lineage-restricted nature of these mutant phenotypes suggests that Hox genes interact with other lineally acting control elements to determine the developmental features of the affected lineages. While the ensemble of these control elements is currently unknown, there is increasing evidence for the importance of transcription factor codes in controlling the expression of axonal guidance molecules. In terms of the Hox gene requirement for preventing the formation of ectopic lineages, the data suggest that this involves lineage-specific programmed cell death of the corresponding postembryonic neuroblasts. Indeed, all Hox genes studied to date have been implicated in some aspect of programmed cell death in postembryonic neuroblasts. The lab gene is required for the termination of specific tritocerebral neuroblasts, Dfd and Scr are required for lineage-specific neuroblast termination in the SEG, Antp und Ubx can trigger neuroblast death if misexpressed in thoracic lineages, and abd-A induces programmed cell death in neuroblasts of the central abdomen. It is therefore concluded that a general function of Hox genes in postembryonic neural development is in the regionalized termination of progenitor proliferation as a key mechanism for neuromere-specific differentiation and specialization of the adult brain (Kuert, 2014).


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 AntpScx mutation isolated in 1953 (Southworth, 2002).

The AntpScx 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 AntpScx 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 AntpScx mutant chromosome fails to complement Antp alleles that specifically lack P1 function, such as AntpB, Antp73b, AntpCB, and Antp17. There is no difference in the average number of sex comb teeth per first leg in AntpScx heterozygous males compared to homozygous wild type or in AntpScx/Scr4 males compared to +/Scr4 males. Males heterozygous for AntpScx, 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 AntpScx (ScrE2 AntpScx/+ and ScrE3 AntpScx/+) have fewer sex comb teeth per second leg (an average of 0.8); males with Scr mutations on the homolog (AntpScx/Scr2 and AntpScx/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 AntpScx [ScrE2 AntpScx/ 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 AntpScx 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 AntpScx mutant chromosome. It is believed that the repetitive DNA inserted near the Antp P1 promoter on the AntpScx 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 AntpScx insertion disrupts silencing of the Scr promoter in cis or in trans, respectively. In this respect, the AntpScx 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 AntpScx mutant chromosome and the endogenous regulatory elements in the Scr gene. Interactions between the AntpScx 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 AntpScx insertion and the endogenous elements in the Scr gene in trans should not silence the Antp P2 promoter. Since the AntpScx 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 AntpScx 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, AntpScx interferes significantly with function of the P2 promoter; AntpScx 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 AntpScx 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 AntpScx mutant chromosome that were not detected in the molecular analyses. However, it should be emphasized that the AntpScx 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).

Hox genes regulate the same character by different strategies in each segment

Hox genes control regional identity along the anterior-posterior axis in various animals. Each region contains morphological characteristics specific to that region as well as some that are shared by several different regions. The mechanism by which one Hox gene regulates region-specific characteristics has been extensively analyzed. However, little attention has been paid to the mechanism by which different Hox genes regulate the same characteristics in different regions. This study shows that two Hox genes in Drosophila, Sex combs reduced and Ultrabithorax, employ different mechanisms to achieve the same out-put, the absence of sternopleural bristles, in the prothorax and metathorax, respectively. Sternopleural bristles are characteristics of the mesothorax, and it was found that spineless is involved in their development. Analysis of the regulatory relationship between Hox genes and spineless indicated that ss expression is repressed by Sex combs reduced in the prothorax. Since sole misexpression of ss could induce ectopic sternopleural bristle formation in the prothorax irrespective of the expression of Sex combs reduced, spineless repression appears to be critical for inhibition of sternopleural bristles by Sex combs reduced. In contrast, spineless is expressed in the metathorax independently of Ultrabithorax activity, indicating that Ultrabithorax blocks sternopleural bristle formation through mechanisms other than spineless repression. This finding indicates that the same characteristics can be achieved in different segments by different Hox genes acting in different ways (Tsubota, 2008).

This study found that three genes, Antp, ss and al, are involved in sternopleural bristle formation. In the al mutant, no appreciable Ac expression in the T2 leg disc is detected and sternopleural bristles are not formed, indicating that the requirement of al is absolute. In contrast, Ac expression is detectable in the ss mutant T2 leg disc and in the Antp mutant clones, indicating that the requirement of both ss and Antp for ac expression is not absolute. However, sternopleural bristles were never found in the ss mutant, despite the fact that Antp expression was unaffected in the ss mutant clone in the T2 leg disc. In contrast, Antp mutant cells, in which ss is expressed normally, formed sternopleural bristles. In addition, sole misexpression of ss in the T1 segment produces sternopleural bristles ectopically, while that of Antp did not. Therefore, ss appears to be necessary and sufficient for sternopleural bristle formation, while Antp appears to be insufficient and not necessarily required. Moreover, Ac expression is ectopically induced in the T1 leg disc by misexpression of ss but not of Antp and in the ss mutant T2 leg disc is very weak, highly restricted, and only transient. This indicates that ss but not Antp appears to be one of the major activators of ac expression. Taken together, ss appears to be much more fundamental for sternopleural bristle formation than Antp (Tsubota, 2008).

The initiation of ac expression coincides with the initiation of ss expression. Since al and Antp are already expressed before ac induction in the early third instar stage, the timing of ac induction may be determined by the regulation of ss expression. Interestingly, the residual Ac expression seen in the ss mutant leg disc is first observed in the mid third instar as in the wild-type leg disc. This implies that at least one additional gene (referred to as X hereafter), whose expression or function is activated at the same stage as the initiation of ss expression, may be involved in ac induction. One possibility may be a gene functioning in hormonal regulation. Nonetheless, the ability of the sole misexpression of ss to induce ectopic ac expression and sternopleural bristle formation strongly indicates that ss is much more fundamental than X (Tsubota, 2008).

The restriction of ac expression to the overlap between the ss and al expression domains indicates the importance of determining the distal limit of ss expression and the proximal limit of al expression. Analysis of clones lacking ss activity or misexpressing ss indicates that ss has a repressive activity on al expression. How can al be expressed in the overlap domain? In the overlap domain, ss represses al expression when misexpressed at high levels but does not when misexpressed at approximately endogenous levels. The level of ectopic Al expression in the ss mutant clone located in a region proximal to the normal al expression domain is lower than that of endogenous Al expression. Moreover, Al expression in the wild-type leg disc gradually decays at its proximal edges. Considering all of these observations, the following hypothesis is suggested: al expression is activated according to the proximodistal information and the proximal limit of the al expression domain may be determined by a balance between activation according to the proximodistal information and repression by ss. The activation force may dominate the repressive activity of ss in the overlapping region but may gradually decay towards the proximal edges of the al expression domain. In contrast, ss expression does not appear to be regulated by al. As with the case of al activation, it may be possible that ss is repressed according to the proximodistal information (Tsubota, 2008).

The morphological identities of the T1 and T3 segments, including the absence of sternopleural bristles, are determined by Scr and Ubx, respectively. Analyses of the T1 leg disc with Scr mutant clones and the T2 leg disc with ectopic Scr activity indicate that both ss and Antp are repressed by Scr in the T1 leg disc. In addition, there is a possibility that the expression or function of gene X is repressed by Scr. Weak Ac expression is transiently observed in the ss mutant T2 leg disc, indicating that ac expression can be weakly activated without ss activity in the presence of gene X and Antp activity. In addition, Scr does not appear to repress ac expression directly, since ectopic induction of ac by ss misexpression in the T1 leg disc was not associated with an alteration in Scr expression. If gene X is active in the T1 leg disc, sole misexpression of Antp is expected to activate ac expression at least weakly and transiently. However, no ectopic Ac expression was found upon sole misexpression of Antp. Therefore, the activity of gene X is likely to be repressed in the T1 leg disc. For evaluating the significance of these three genes on Scr-dependent inhibition of sternopleural bristle formation, the ability of ss misexpression to induce ectopic ac expression and sternopleural bristle formation without affecting Scr expression is of crucial importance. At present, whether ac expression and sternopleural bristle formation can be induced solely by ss or only in a combination of ss and Antp and/or gene X is unclear. However, ss misexpression induced Antp expression and, thus, at least ss and Antp were coexpressed upon sole misexpression of ss. As for gene X, if it is not activated by ss misexpression, the results indicate that ac expression and sternopleural bristle formation can be induced without gene X activity at least in the presence of both ss and Antp expression. In contrast, if ac expression and sternopleural bristle formation require gene X activity, ss misexpression must activate gene X. After all, the results indicate that sole misexpression of ss can fulfill at least a minimum requirement for ac expression and sternopleural bristle formation. In other words, if Scr could not repress ss expression, ac expression would be activated and sternopleural bristles would be formed irrespective of the expression and function of Antp and gene X. Therefore, Scr must repress ss expression and this appears to be a key step to block sternopleural bristle formation in the T1 segment (Tsubota, 2008).

In contrast to the T1 leg disc, strong Ss expression was observed in the wild-type T3 leg disc and it is unaltered in Ubx mutant clones. Therefore, Ubx appears to act through a mechanism unrelated to ss expression. How does Ubx function? Simultaneous expression of both ss and Antp seemed insufficient for ac expression and sternopleural bristle formation in the T3 segment, since Antp misexpression failed to induce Ac expression in the T3 leg disc, in which ss is prominently expressed. It may be possible that Ubx represses ac expression directly. Alternatively, Ubx may compromise the function of the Ss protein directly or indirectly through regulation of its downstream gene products. Another possibility is that Ubx acts through repression of gene X activity. These possibilities are not mutually exclusive with each other (Tsubota, 2008).

The occurrence of ac expression and sternopleural bristle formation in the absence of Antp activity indicates that the absence of sternopleural bristles is not the ground state. However, the number of sternopleural bristles is variable in that condition, indicating that the complete formation of sternopleural bristles is not also the ground state. Since ss misexpression experiment suggests that sternopleural bristles can be formed as long as ss is expressed, one possible aspect of the ground state may be the expression of ss and the production of at least some kind of bristles. Antp may have acquired the ability to modify this state to produce the current-type of sternopleural bristles. On the other hand, Scr may have evolved the ability to block sternopleural bristle formation by acquiring the activity to repress ss expression and Ubx by acquiring another, yet unknown function. Taken together, the current state of sternopleural bristles in all three thoracic segments appears to be the derived state (Tsubota, 2008).

Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors.

Neural stem cell quiescence is an important feature in invertebrate and mammalian central nervous system development, yet little is known about the mechanisms regulating entry into quiescence, maintenance of cell fate during quiescence, and exit from quiescence. Drosophila neural stem cells (neuroblasts or NBs) provide an excellent model system for investigating these issues. Drosophila NBs enter quiescence at the end of embryogenesis and resume proliferation during larval stages; however, no single neuroblast lineage has been traced from embryo into larval stages. This study establishes a model NB lineage, NB3-3, which allows reproducibly observation of lineage development from NB formation in the embryo, through quiescence, to the resumption of proliferation in larval stages. Using this new model lineage, a continuous sequence of temporal changes is shown in the NB, defined by known and novel temporal identity factors, running from embryonic through larval stages; quiescence suspends but does not alter the order of neuroblast temporal gene expression. NB entry into quiescence is regulated intrinsically by two independent controls: spatial control by the Hox proteins Antp and Abd-A, and temporal control by previously identified temporal transcription factors and the transcription co-factor Nab (Tsuji, 2008).

This study has revealed for the first time the temporal changes in a Drosophila NB lineage from embryonic NB formation, through entry into quiescence, to resumption of proliferation in larval stages. Using a model NB system with which the complete lineage formation can be reproducibly traced at the resolution of individual cell divisions, it was shown that despite considerable differences in extracellular environment the temporal changes (as defined by the switching of transcription factor/co-factor expression) proceeded continuously in each NB throughout the embryonic and larval stages. Moreover, mutual regulation was found between quiescence and the series of the temporal transcription factors/co-factor; the temporal transcription factors/co-factor endogenously control the timing of triggering NB quiescence, which in turn suspends the switching of late temporal transcription factor expression (Tsuji, 2008).

In the Antp mutant and following ectopic expression of Abd-A there was a lack of NB quiescence, and consequently what appeared to be a precocious generation of larval neurons during embryogenesis was observed. This strongly supports the notion that temporal changes in NBs actually continue in sequence before and after quiescence, i.e., through embryogenesis and larval stages, and in the absence of quiescence the changes occur precociously. In addition, this indicates that spatial and temporal factors control NB quiescence through independent routes (Tsuji, 2008).

Antp mutants did not exhibit NB3-3T quiescence in all thoracic T1-T3 segments. In Antp mutants, epidermis in T2 and T3 segments transform into that in the T1 segment, and some thoracic NB lineages retain thoracic-specific features. These facts indicate that the inhibition of NB3-3T quiescence by Antp mutation is not just a consequence of global transformation of thoracic-to-abdominal segments but rather results from specific effects on individual NBs. NB-specific misexpression of Abd-A also repressed Antp and inhibited NB3-3T quiescence. This also provides evidence that the effect is specific to NBs. Furthermore, because the effect could be observed even when Abd-A was induced after several divisions of the NB, thoracic NBs probably maintain plasticity of their identities during lineage formation (Tsuji, 2008).

It was shown that the temporal transcription factors/co-factor Pdm, Cas, Sqz and Nab play a role in triggering NB quiescence intrinsically in NBs. All of these factors also controlled temporal specification within late lineages of embryonic NBs in both thoracic and abdominal segments. This was confirmed by further examining the relationships of the temporal factors. For example, the loss of Pdm function in NB3-3T resulted in precocious transcription factor switching and precocious quiescence. Conversely, in cas mutant embryos, in which Pdm expression was de-repressed, quiescence was inhibited and expression of late-stage-specific temporal factors disappeared. Similar to Pdm upregulation, loss of nab function resulted in loss of both transcription factor switching and quiescence (Tsuji, 2008).

Although Nab and Sqz can form a complex, nab and sqz mutants displayed very different phenotypes. Both mutants showed de-repression of Kr expression; however, sqz mutants showed no other abnormality in transcription factor switching, whereas nab mutants showed the above-mentioned defects in transcription factor switching and timing of quiescence. These mutant phenotypes revealed that regulation of late temporal events is distributed into multiple pathways. Pdm probably coordinately regulates all factors involved in the timing of NB quiescence, because the loss of Pdm alone is sufficient to cause precocious entry into quiescence (Tsuji, 2008).

Nab and Sqz were shown to work for NB quiescence in NBs. The Nab/Sqz-mediated repression of Kr may be controlled in NBs due to changes in NB temporal identity, or in both NBs and their neurons. Nab might inhibit transcription by recruiting the nucleosome remodeling and deacetylase chromatin remodeling complex as does mammalian Nab (Srinivasan, 2006). Mammalian Nab acts with EGR-1, EGR-2 to determine the fate of cells in hematopoiesis (Laslo, 2006; Svaren, 1996), but whether it can act with the mammalian homolog of LIN-29/Sqz has not been reported. Loss of lin-29, a C. elegans homolog of sqz, causes a heterochronic phenotype in which adulthood is not reached and molting is repeated (Ambros, 1984; Rougvie, 1995). C. elegans has a nab homolog gene, mab-10, that acts with lin-29 in a heterochronic genetic cascade (Tsuji, 2008).

It is unclear what molecular mechanisms enable NBs to suspend the switching of transcription factor expression and maintain temporal identity during quiescence. It is known that the mechanisms for switching expression of early temporal transcription factors can be either cell division dependent or independent. Irrespective of the mechanism used, a NB can 'memorize' its temporal state before quiescence and resume the intrinsic temporal changes once cell cycle progression is reactivated. Embryonic stem cells may maintain multipotency during a slow proliferation state by staying in S phase. When quiescent NBs re-entered the cell cycle, their initial progeny incorporated BrdU fed since hatching, indicating that quiescent NBs stay either prior to S phase or early in S phase. It will be important to identify the point in the cell cycle at which NB enters quiescence (Tsuji, 2008).

Another well-established mechanism that governs temporal aspects of lineage formation is the heterochronic gene cascade in C. elegans. This cascade contains one each of the hunchback homolog and lin-29 genes and generates five distinct temporal cell identities within a single cell lineage. Drosophila NB lineage formation uses two types of Zn-finger proteins, namely the Hb/Cas class [Cas shares DNA-binding characteristics with Hb and the Kr/LIN-29 class. These pairs are expressed three times in NB lineages in the following order: (1) Hb and Kr-> (2) Cas, Kr and Sqz--> Cas and DmLin-29-->end of lineage. This sequence seems to produce at least ten distinct temporal identities within an NB lineage. The repetitive use of these temporal transcription factors in three distinct phases appears to have made the NB lineage longer and more diverse. Lack of Hb also generates NB lineage variety; the NB3-3 and NB2-1 lineages lack Hb and initiate their lineage with Kr. Although the model NB employed in this study lacks Hb, the sequence and entry into quiescence described in this study are common to many typical NB lineages that start with Hb. Interesting questions from the perspective of evolution are how do the three phases combine to form a single lineage and how has NB quiescence evolved in the middle of the NB lineages (Tsuji, 2008)?

Neural stem cells in the mouse cerebral cortex go through ~11 divisions and some enter quiescence in late embryo. The possibility has to be considered that mammalian neural stem cell and Drosophila NB share a similar intrinsic mechanism that induces quiescence (Tsuji, 2008).

Antagonistic roles for Ultrabithorax and Antennapedia in regulating segment-specific apoptosis of differentiated motoneurons in the Drosophila embryonic central nervous system

The generation of morphological diversity among segmental units of the nervous system is crucial for correct matching of neurons with their targets and for formation of functional neuromuscular networks. However, the mechanisms leading to segment diversity remain largely unknown. This paper reports that the Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp) regulate segment-specific survival of differentiated motoneurons in the ventral nerve cord of Drosophila embryos. Ubx is required to activate segment-specific apoptosis in these cells, and their survival depends on Antp. Expression of the Ubx protein is strongly upregulated in the motoneurons shortly before they undergo apoptosis, and these results indicate that this late upregulation is required to activate reaper-dependent cell death. Ubx executes this role by counteracting the function of Antp in promoting cell survival. Thus, two Hox genes contribute to segment patterning and diversity in the embryonic CNS by carrying out opposing roles in the survival of specific differentiated motoneurons (Rogulja-Ortmann, 2008).

Over the course of development, clear differences emerge between thoracic and abdominal segments in the form of specialized functional networks that support regional locomotion and sensory requirements. These differences are the result of region-specific division patterns of the neural progenitors (neuroblasts) and of the different numbers and types of neural cells that the neuroblasts generate. Regulation of CNS cell number is achieved through control of cell proliferation and death, and both of these processes have been shown to be orchestrated by Hox genes, in vertebrates and invertebrates. It has recently also been reported that mutations in Hox genes affect locomotor behavior in larval crawling, suggesting that they provide positional information for neurons and thus regulate the formation of neuromuscular networks that control region-specific peristaltic locomotion. The Drosophila Hox genes Ubx and Antp regulate segment-specific survival of two differentiated motoneurons, and may thus pattern neuromuscular networks by regulating the segment-specific presence of individual motoneurons. This study shows that Ubx is necessary and sufficient to induce apoptosis, and that it does so by impeding the positive effect of Antp on the survival of these cells (Rogulja-Ortmann, 2008).

The pattern of Ubx expression in the GW and NB2-4t motoneurons and their respective lineages is interesting. In early stages, the Ubx expression pattern conforms to its role as a homeotic gene: it is expressed from the posterior half of segment T3 to the anterior half of A7. The levels of Ubx protein in NB7-3 and NB2-4 and their progeny are not particularly high at these stages, but removal of both Ubx and abdA, for example, results in an NB7-3 lineage that is typical for T1 and T2, suggesting that Ubx determines the segmental identity of this neuroblast. At a later developmental stage, Ubx expression is strongly upregulated in the NB7-3 and NB2-4 progeny in a segment-specific manner. This dynamic pattern of expression implies two roles of Ubx in these lineages: (1) an early role in establishing tagma-specific identity of the neuroblast, such as has already been shown for NB1-1; and (2) a late role in inducing apoptosis of the motoneurons. Heat-shock experiments confirm that, at least in NB7-3, it is this late upregulation of Ubx that leads to apoptosis. NB2-4t could not be tested for the proapoptotic function of Ubx in the heat-shock experiments. However, it is believed that Ubx plays a late role in this lineage as well: since Ubx does not play an early role in specifying the T3 identity of the NB2-4t neuroblast, this suggests that the proapoptotic function of Ubx must be executed at a later point in development. Also, in poxN-Gal4,UAS-GFP/UAS-Ubx embryos, the NB2-4t lineage does not appear to be transformed into its abdominal counterpart, since two closely positioned dorsal motoneurons were observed in thoracic segments. In these embryos, Ubx is still capable of inducing apoptosis of the anterior motoneuron in all thoracic segments. Taken together, these data indicate that, also in this lineage, Ubx is needed at a late developmental stage to induce apoptosis. Dual requirements for Hox genes have also been described in determining thoracic bristle patterns and in cardiac tube organogenesis. It is not clear how the late expression of Ubx is regulated. It has been suggested that this might depend on genes that define the differences between cell types, and it will be interesting to see whether this is also the case for the NB7-3 neurons (Rogulja-Ortmann, 2008).

Interestingly, the late Ubx expression in NB7-3 extends to the second thoracic segment, but in a cell-specific manner: the GW motoneuron does not activate Ubx expression, whereas the EW interneurons do. These findings evoke the following questions: (1) why is it that the EWs in T2 to A7 do not undergo apoptosis although they also upregulate Ubx? and (2) what represses Ubx expression in the T2 GW (Rogulja-Ortmann, 2008)?

Regarding the first question, it is likely that the differentiation program of the EWs creates a different cellular context in which Ubx is unable to induce apoptosis. The results of ectopic Ubx expression experiments support this assumption: whereas GW undergoes apoptosis, the EWs do so very rarely, although they express Ubx at equally high levels. In addition, GW seems to acquire the competence to undergo Ubx-dependent apoptosis rather late, as en-Gal drives expression strongly from earlier stages but apoptosis does not occur until stage 15. This would suggest that the susceptibility to apoptosis of at least some motoneurons is coupled to differentiation. The context-dependent ability of Ubx to activate apoptosis also holds true for the NB2-4t lineage: the anterior motoneuron is susceptible to Ubx-induced apoptosis, whereas the posterior one is not, even when Ubx is overexpressed (Rogulja-Ortmann, 2008).

Preliminary attempts to determine at least some of the factors contributing to apoptosis-susceptibility were not successful. For the NB7-3 lineage, abdA expression was examined and it was found that, at the onset of GW apoptosis, it is weakly expressed only in the EWs from A1 to A7, but not in GW. However, the survival of the EWs is not impaired in abdA mutants. The cofactors homothorax (hth) and extradenticle (exd), which are known to be required for some functions of homeotic genes, were tested, but compelling evidence was obtained for their involvement in the apoptosis of these cells. Mutants of other factors that are differentially expressed in GW and EWs (numb, zfh1) were also examined, and no indication was found that any of these is involved in the differential effect of Ubx on cell survival (Rogulja-Ortmann, 2008).

Regarding the question of differential Ubx expression in NB7-3 cells of T2, this is an intriguing observation that might prove to be key in determining the developmental signal that upregulates Ubx late in development. One candidate for repressing Ubx expression in GW is the gap gene hb. It is known to repress Ubx in early embryonic development, and hb overexpression can suppress Ubx in the NB7-3 lineage. However, hb is also necessary to activate Antp expression and thereby specify the second thoracic segment. In hb mutants, T2 is not present and it was thus impossible to test whether this is the factor repressing Ubx expression in GW. Other obvious candidates are the Polycomb group (PcG) proteins, well-known repressors of Hox genes. It has recently been shown that, contrary to what had been believed for a long time, target gene repression by these proteins is not necessarily maintained throughout development, but can be reversed in certain developmental contexts. It is conceivable that repression by PcG proteins could be lifted in some cells (e.g. EWs in T2) and not in others (e.g. GW). However, the question would still remain as to how the difference between the GW and EW neurons is established specifically in this segment. Alternatively, differential Ubx regulation might be effected via micro RNAs or non-coding RNAs (Rogulja-Ortmann, 2008).

It was also shown that Antp is required for GW survival in all segments, and that Ubx counteracts Antp in T3 to A7 to induce apoptosis. Although the lower percentage of dying abdominal GWs in Antp mutants (69%) as compared with wild type (81.2%) might indicate a proapoptotic function of Antp in the abdomen, it is believed that this is not the case because Antp overexpression actually reduces the amount of abdominal GW apoptosis more than twofold. Moreover, removing Antp function in a Ubx heterozygous background increases GW apoptosis (both in T3 and in abdomen) in a dose-dependent manner, and removing both Ubx and Antp results in a recurrence of GW apoptosis, albeit with low penetrance, lending further support to a pro-survival function of Antp (Rogulja-Ortmann, 2008).

It is not clear at which level these two factors interact. GW apoptosis is inhibited both in T3 and in abdominal segments of Ubx mutants. However, Antp expression in Ubx mutants is upregulated only in T3, and remains low in abdominal segments, suggesting that here Ubx does not induce apoptosis through Antp repression. In addition, the pattern and levels of Ubx expression do not change at all in Antp mutants, indicating that in this context Antp does not promote survival via Ubx regulation. It is therefore proposed that in the wild type, Antp and Ubx might compete for a co-factor or for a target enhancer, rather than cross-regulating each other. The proapoptotic gene rpr, which is transcriptionally activated in both GW and MNa motoneurons, is a candidate target. In fact, several binding sites for both Ubx and Antp were found in the enhancer of the rpr gene. It will be interesting to see whether Antp can prevent activation of the apoptotic machinery by affecting an upstream factor or through direct repression of rpr. The presence of several Antp binding sites in the rpr enhancer permits such speculation, and although it awaits experimental validation, this does suggest a model for the antagonistic effects of Antp and Ubx on cell survival. According to this model, Ubx and Antp compete for sites in the rpr enhancer. In cells that express Antp at high level, this would repress rpr transcription. Ubx would overcome repression by Antp and activate rpr transcription to induce apoptosis. Such positive Hox regulation of rpr has already been demonstrated in shaping segment borders in Drosophila embryos, where Deformed directly activates rpr transcription. In addition, antagonistic transcriptional regulation of the P2 Antp promoter in the embryonic ventral nerve cord has been demonstrated for Antp and Ubx. In this case, Antp positively autoregulates its own P2 promoter in the thoracic segments, and Ubx competes with Antp for the same binding sites and thus prevents high-level expression of Antp in the more posterior segments (Rogulja-Ortmann, 2008).

A requirement for Hox genes in segment-specific cell survival has already been shown for the MP2 and MP1 pioneer neurons, where AbdB expression is necessary for survival of these neurons in the three most-posterior abdominal segments. In the more anterior segments, the dMP2 and MP1 motoneurons undergo apoptosis at the end of embryonic development, after they have completed their role in pioneering axonal tracts. The surviving dMP2 neurons innervate the hindgut and differentiate into insulinergic neurons. The exact function and targets of the GW and the anterior NB2-4t motoneurons in the first and second thoracic segment are unclear, as is the reason for their removal in the relevant segments. The surviving GW and MNa might exert a region-specific neurosecretory function and thus modulate neuronal or muscle activity, as has been described for neurosecretory cells in the larval brain, the processes of which arborize on the wall of the anterior aorta adjacent to the ring gland. Alternatively, the elimination of certain outward-projecting neurons in the posterior thoracic and/or abdominal segments might be related to the pattern of muscle fibers, which differs considerably between the thorax and the abdomen. It is suggested that Hox-regulated segment-specific motoneuron survival is a part of the patterning process that enables formation of region-specific functional neuromuscular networks (Rogulja-Ortmann, 2008).

Segment-specific generation of Drosophila Capability neuropeptide neurons by multi-faceted Hox cues

In the Drosophila ventral nerve cord, the three pairs of Capability neuropeptide-expressing Va neurons are exclusively found in the second, third and fourth abdominal segments (A2-A4). To address the underlying mechanisms behind such segment-specific cell specification, the developmental specification of these neurons was followed. Va neurons are initially generated in all ventral nerve cord segments and progress along a common differentiation path. However, their terminal differentiation only manifests itself in A2-A4, due to two distinct mechanisms: segment-specific programmed cell death (PCD) in posterior segments, and differentiation to an alternative identity in segments anterior to A2. Genetic analyses reveal that the Hox homeotic genes are involved in the segment-specific appearance of Va neurons. In posterior segments, the Hox gene Abdominal-B exerts a pro-apoptotic role on Va neurons, which involves the function of several RHG genes. Strikingly, this role of Abd-B is completely opposite to its role in the segment-specific apoptosis of other classes of neuropeptide neurons, the dMP2 and MP1 neurons, where Abd-B acts in an anti-apoptotic manner. In segments A2-A4 abdominal A was found to be important for the terminal differentiation of Va cell fate. In the A1 segment, Ultrabithorax acts to specify an alternate Va neuron fate. In contrast, in thoracic segments, Antennapedia suppresses the Va cell fate. Thus, Hox genes act in a multi-faceted manner to control the segment-specific appearance of the Va neuropeptide neurons in the ventral nerve cord (Suska, 2011).

Addressed here is the segment-specific appearance of one peptidergic neuronal subtype, the Capa-expressing Va neurons. One pair of Va neurons is initially generated in each segment of the VNC. At embryonic stage 14, differentiation begins and the cells commence the expression of the transcription factors Dac and Dimm. Only after this process is initiated, at stage 16, the posteriorly expressed Hox gene Abd-B triggers PCD in segments A5 to A8. This PCD involves the RHG motif genes, and mutant analysis indicates that grim, or grim and hid play the most important roles. As development progresses, the Va neurons in abdominal segments A2-A4 are further specialized under the influence of abd-A, which results in expression of the Capa neuropeptide at stage 17. The single pair of Dimm/Dac-expressing Va neurons in the first abdominal segment is present into larval stages, but does not express Capa. These alternate Va neurons depend upon Ubx for their Dimm expression, but it is unclear if they differentiate into peptidergic neurons, and if so, which neuropeptide gene they express. In thoracic segments, Antp is involved in the down-regulation of Dac and Dimm. These studies unravel a complex interplay of Hox gene input critical for the segment-specific survival and differentiation of the Va neurons and thereby highlight the involvement of Hox genes during the process of shaping the segment-specific structures of the nervous system (Suska, 2011).

Ectopic appearance of Capa expression through ectopic expression of abd-A indicates that abd-A is an important partner in the combinatorial code of transcription factors necessary for initiating the expression of Capa. The roles of Ubx and Antp are not as straightforward to assess. Ubx showed a participation in the specification of the Va neurons in more anterior segments of the VNC, mainly the thoracic area. Ectopic Ubx expression resulted in maintained Dac/Dimm expression in thoracic Va cells into late embryonic stages (18hAEL). Its endogenous role seems to be confined to segment A1, which is characterized by co-expression of Dac/Dimm and a lack of Capa. The role this pair of neurons plays is unknown, as they are not known to express any neuropeptide. The mutant analysis indicates a possible role of Antp in the down-regulation of Dac/Dimm in thoracic Va neurons. The ectopic expression of Antp however could not override specification signals provided by the other factors (Suska, 2011).

Several studies have identified roles for Hox genes in specifying neuronal subtypes. Of particular interest for the current study are previous findings that Antp acts at a late stage to specify two other neuropeptide cells; the thoracic Nplp1 and FMRFa neurons of the Apterous (Ap) cluster. In this study, Antp first acts together with the temporal gene castor to activate expression of the collier gene, an EBF family member, thus triggering specification of a transient 'generic' Ap cluster neurons identity. Subsequently, Antp acts in a feedforward manner with collier to activate late cell fate determinants, such as dimm, and ultimately the Nplp1 and FMRFa neuropeptide genes. Currently, the neuroblast origin of the Va neurons is unclear. Double-labeling with the neuroblast row 5-6 marker GooseberryNeuro indicates that Va neurons originate from a row 5 neuroblast. As the neuroblast origin of the Va neurons is established, and this lineage mapped, it will be possible to place the generation of Va neurons within a lineage tree. This will furthermore allow identification of the temporal window that generates Va neurons (Suska, 2011).

Programmed cell death plays a critical role in the generation of segmental diversity. Studies in the Drosophila embryo have revealed that this can act both at the level of progenitor and postmitotic, even differentiated cells. In progenitors, PCD acts to remove many abdominal neuroblasts after they have completed their lineages and become quiescent. This ensures that as neuroblasts re-enter proliferative states in the larvae, the abdomen has very few quiescent neuroblasts that can enter the cell cycle. Thus, in the adult CNS, the abdomen will end up containing substantially fewer neurons and glia. In postmitotic cells, PCD acts in two apparently different ways: (1) to remove certain postmitotic cells immediately after mitosis, or (2) to remove differentiated neurons. A particularly relevant case to the studies presented is the removal of the peptidergic dMP2 and MP1 neurons. These cells are generated in all VNC segments, extend axons to pioneer critical axon tracts, and subsequently undergo PCD in all segments but the A6-A8 segments. Strikingly, here Abd-B has an anti-apoptotic and promotes peptidergic identity role, while in the Va neurons it has a pro-apoptotic role. Moreover, the cell death of both MP1 and Va neurons also depends upon the RHG genes. These results suggest that Abd-B acts in an opposing manner, pro- versus anti-apoptotic, by differentially controlling the same PCD pathway in related neurons. An attractive and simple model for this dual role of Abd-B would be that MP1 and Va neurons express different regulatory genes, which can act with Abd-B to trigger either survival or death. Further studies of PCD in the dMP2, MP1 and Va neurons may help shed light on the molecular genetic mechanisms behind these dual roles of Abd-B (Suska, 2011).

Cell-autonomous and non-cell-autonomous function of Hox genes specify segmental neuroblast identity in the gnathal region of the embryonic CNS in Drosophila

In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of neuroblasts (NB) prior to their delamination from the neuroectoderm. This study identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. It was shown that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. The secreted molecule Amalgam (Ama) was identified as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dad pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, the study provides genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental patterning in the developing CNS (Becker, 2016).

The Drosophila head consists of seven segments (4 pregnathal and 3 gnathal) all of which contribute neuromeres to the CNS. The brain is formed by approximately 100 NBs per hemisphere, which have been individually identified and assigned to specific pregnathal segments [The anterior pregnathal region (procephalon) is composed of the labral, ocular, antennal, intercalary segments, see Segment polarity and DV patterning gene expression reveals segmental organization of the Drosophila brain]. As judged from comparison of the combinatorial codes of marker gene expression only few brain NBs appear to be serially homologous to NBs in the thoracic/abdominal ventral nerve cord, reflecting the highly derived character of the brain neuromeres. The connecting tissue between brain and the thoracic VNC consists of three neuromeres formed by the gnathal head segments named mandibular (mad), maxillary (max) and labial (lab) segment, but the number and identity of the neural stem cells and their lineage composition in these segments is still unknown. Compared to the thoracic ground state the segmental sets of gnathal NBs might be reduced to different degrees, but are thought to be less derived compared to the brain NBs. Therefore, to fully understand segmental specification during central nervous system development, it is important to identify the neuroblasts and their lineages in these interconnecting segments (Becker, 2016).

Assuming that most NBs in the gnathal segments still share similarities to thoracic and abdominal NBs, this study sought serially homologous NB-lineages, which are suitable for genetic analyses. Using the molecular marker eagle (eg), which specifically labels four NB-lineages in thoracic/abdominal hemisegments this study identified three serial homologs (NB3-3, NB6-4 and NB7-3) in the gnathal region. To investigate the mechanisms conferring segmental identities, focus was placed on one of them, the NB6-4 lineage, which shows the most significant segment-specific modifications. The analysis reveals a primary role of the Antennapedia-Complex (Antp-C) Hox gene Deformed (Dfd) in cell-autonomously specifying the maxillary fate of NB6-4 (NB6-4max). Surprisingly, an additional, non-cell-autonomous function was uncovered of the Antp-C Hox genes labial (lab, expressed anterior to Dfd) and Antennapedia (Antp, expressed posterior to Dfd) in specifying NB6-4max. In a mini-screen for downstream effectors the secreted protein Amalgam (Ama) was identified as being positively regulated by lab, Dfd and Antp and negatively regulated by the Antp-C Hox gene Sex combs reduced (Scr). Loss of function of Ama and its receptor Neurotactin (Nrt) as well as the downstream effector kinase Abelson tyrosine kinase (Abl) lead to a transformation of NB6-4max similar to Dfd single mutants. Thus, in parallel to the cell-autonomous role of Dfd, a non-cell-autonomous function of Hox genes lab and Antp, mediated via the Ama-Nrt-Abl pathway, is necessary to specify NB6-4max identity. Disruption of either of these pathways leads to a partial misspecification of NB6-4max (approx. 40%), whereas simultaneous disruption of both pathways leads to a complete transformation (approx. 100%) of NB6-4max to a labial/thoracic identity. It was further shown that both pathways regulate the expression of the cell cycle gene CyclinE, which is necessary and sufficient to generate labial/thoracic NB6-4 identity. Whereas Dfd seems to directly repress CyclinE transcription (similar to AbdA/AbdB in the trunk), indications are provided that the Ama-Nrt-Abl pathway prevents CyclinE expression by altering the activity of the Hippo/Salvador/Warts pathway effector Yorkie (Yki) (Becker, 2016).

Along the anterior-posterior axis the CNS consists of segmental units (neuromeres) the composition of which is adapted to the functional requirements of the respective body parts. In Drosophila the CNS comprises 10 abdominal, three thoracic, three gnathal and four pregnathal (brain) neuromeres that are generated by stereotyped populations of neural stem cells (neuroblasts, NBs). The pattern of NBs in thoracic segments resembles the ground state while NB patterns in the other segments are derived to various degrees. Within each segment individual NBs are specified by positional information in the neuroectoderm. NBs delaminating from corresponding positions in different segments express similar sets of molecular markers, generate similar lineages, and are called serial homologs. However, for thoracic and abdominal neuromeres it has been shown that the composition of a number of serially homologous NB-lineages shows segment-specific differences. In the more derived gnathal and pregnathal head segments embryonic NB-lineages and the mechanisms of their segmental specification have not been analyzed so far (Becker, 2016).

Using the well-established molecular marker Eagle (Eg) which labels four embryonic NB-lineages (NB2-4, NB3-3, NB6-4, NB7-3) in all thoracic and most of the abdominal segments this study identified serially homologous lineages of NB3-3, NB6-4 and NB7-3 in gnathal segments. The embryonic NB7-3 lineage shows segmental differences as it comprises increasing cell numbers from mandibular (2 cells), maxillary (3 cells) to labial (3-5 cells) segments, while cell numbers are decreasing from T1-T2 (4 cells), T3-A7 (3 cells) to A8 (2-3 cells). Reduced cell numbers in the mandibular and maxillary NB7-3 lineages depend on Dfd and Scr function, respectively . While NB7-3 appeared in all three gnathal segments, NB3-3 and NB6-4 was only found in labial and maxillary segments, and NB2-4 was not found in any of them. Preliminary data suggest that the missing NBs are not generated in these segments, instead of being eliminated by apoptosis. For the terminal abdominal neuromeres (A9, A10) it has recently been shown that the formation of a set of NBs (including NB7-3) is inhibited by the Hox gene Abdominal-B. Similarly, in Dfd mutants the formation was observed of a NB with NB6-4 characteristics in mandibular segments (10%), in which it is never found in wild type (Becker, 2016).

Similar to the thoracic and abdominal segments NB6-4 showed dramatic differences between maxillary and labial segments. NB6-4max produces glial cells only (like abdominal NB6-4), whereas the labial homolog produces neurons in addition to glial cells (like thoracic NB6-4). The number of glial cells produced by the glioblasts NB6-4max (4 cells) and abdominal NB6-4 (2 cells) and by the neuroglioblasts NB6-4lab (3 glia) and thoracic NB6-4 (3 glia) is segment-specific(Becker, 2016).

Thus segment-specific differences among serially homologous lineages may concern types and/or numbers of specific progeny cells and may result from differential specification of NBs and their progeny, differential proliferation and/or differential cell death of particular progeny cells. It has been shown that the segment-specific modification of serially homologous lineages is under the control of Hox genes and that during neurogenesis Hox genes act on different levels, i.e. they act in a context-specific manner at different developmental stages and in different cells. In the thoracic/abdominal region segmental identity is conferred to NBs early in the neuroectoderm by cell-autonomous function of Hox genes of the Bithorax-Complex. This study used the NB6-4 lineage to clarify mechanisms of segmental specification in the gnathal segments (Becker, 2016).

In segments of the trunk, the action of Hox genes strictly follows the rule of the posterior prevalence concept: More posterior expressed Hox genes repress anterior Hox genes and thereby determine the segmental identities. In the gnathal segments this phenomenon was not observed on the level of the nervous system. Removing Hox genes of the Antp-C had no or only minor impact on the expression domain of other Antp-C Hox genes. Similar results were also obtained in a study that analyzed cross-regulation of Hox genes upon ectopic expression (Becker, 2016).

Moreover, it seems that at least in the case of the differences monitored between labial and maxillary segments Hox gene function has to be added to realize the more anterior fate. Antennapedia has no impact on NB6-4 identity in the labial segment, but specification of the maxillary NB6-4 requires the function of Deformed and Sex combs reduced. These two Hox genes are not repressed or activated by Antp. Also, cross-regulation between Dfd and Scr seems to be unlikely or is very weak since only mild effects were observed on the protein level and on the phenotypic penetrance. In principle Scr can repress Dfd, but it was suggested that this occurs only when products are in sufficient amounts. In NB6-4 Dfd and Scr are co-expressed, but Scr levels appear to be insufficient to repress Dfd. Dfd seems to be the major Hox gene that cell-autonomously confers the maxillary NB6-4 fate, since the loss of Dfd showed the highest transformation rate and, more importantly, ectopic expression of Dfd in thoracic segments leads to a robust transformation towards maxillary fate. Scr does not act redundantly since in double mutants Dfd/Scr no synergistic effect was observed. It might have a fine-tuning effect, as it was shown that Scr influences Ama by repressing its transcription, whereas all other Antp-C Hox genes seem to activate Ama. However, since only minor changes were found in cell identities and numbers in Scr LoF background, the role of Scr in NB6-4max stays enigmatic (Becker, 2016).

Surprisingly cell-autonomous Hox gene function was not the only mechanism that confers segmental identity in NB6-4max. Loss of Dfd showed an effect in approx. 43% of all segments. Moreover, mutations of the adjacently expressed Hox genes labial and Antennapedia in combination with Dfd LoF showed a dramatic increase in the transformation rate of NB6-4max. Their expression patterns on the mRNA and protein level were carefully studied in wild type and Hox mutant background. In no case were these genes found to be expressed in NB6-4max or in the neuroectodermal region from which NB6-4max delaminates. This indicates that labial and Antennapedia influence NB6-4max fate in a non-cell-autonomous manner. That Hox genes can act non-cell-autonomously on stem cells was recently shown in the male germ-line, were AbdB influences centrosome orientation and the proliferation rate through regulation of the ligand Boss in the Sevenless-pathway. In this study Antp-C Hox genes controled the expression of the secreted molecule Amalgam, which spreads to adjacent segments and ensures segmental specification of NB6-4max in a parallel mechanism to the cell-autonomous function of Dfd. Thus, this study provides first evidence for parallel non-cell-autonomous and cell-autonomous functions of Antp-C genes during neural stem cell specification in the developing CNS (Becker, 2016).

Abelson kinase (Abl) was shown to be required for proper development of the Drosophila embryonic nervous system. In neurons Abl interacts with proteins like Robo or Chickadee and influences the actin cytoskeleton in the growth cone to regulate axonogenesis and pathfinding. In this system it was also demonstrated that Ama and Nrt are dominant modifiers of the Abl phenotype. It is proposed that the interaction of secreted Ama and the membrane-bound Nrt regulates Abl function in NBs. This leads to the correct segmental specification of NB6-4max. Antp-C Hox genes lab, Antp and Dfd regulate the expression of Ama and in mutants for theses Hox genes expression of Ama is severely reduced, which leads to the transformation of NB6-4max due to missing Abl function and de-repression of the cell cycle gene CyclinE. That Abl can influence the expression of CyclinE was also demonstrated in a modifier-screen in the Drosophila eye, but the mechanism remained unclear. Genetic analysis now suggests that in NBs this might occur via the regulation of the highly conserved Hippo-Salvador-Warts pathway and its downstream transcriptional co-activator Yki, which is known to regulate CyclinE expression. The Hippo-Salvador-Warts pathway controls organ growth and cell proliferation in Drosophila and vertebrates but so far has not been implicated in embryonic NB development. This study observed Yki cytoplasmic localization in wild type NB6-4max prior to division suggesting the active Hippo pathway. Nuclear localization of Yki could not be detected in Abl mutants, the loss of Yki activity in the Abl mutant background leads to a significant reduction in the strength of the Abl single mutant phenotype showing their genetic interaction and therefore supporting the proposed model in which Abl influences Yki activity. Moreover, expression of constitutive active Yki also lead to the transformation of NB6-4max and phenotypes that were similar to those observed in Abl mutants. Attempts were made to assess how Abl might influence Yki activity. Work in vertebrates suggests that this could be at least on two levels: first, c-Abl was shown to directly phosphorylate and activate the vertebrate MST1 and MST2 (Hpo homologue) and the Drosophila Hpo on a conserved residue (Y81) and second, c-Abl can also phosphorylate YAP1, which changes its function to become pro-apoptotic. This analysis suggests that in NBs Abl might regulate Hpo, since changes were found in the stability of Salvador, which is used as a Hpo activity readout, but a parallel direct regulation of Yki could not be ruled out, since it was recently shown that other pathways like the AMPK/LKB1 pathway can directly influence Yki activity. Since severe over-proliferation was observed in Abl or lab/Dfd mutants, that have an impaired Ama-Nrt-Abl pathway, or upon overexpression of YkiCA, future studies need to elucidate whether and how the proto-oncogene Abl kinase and Hox genes act on growth and proliferation or even tumor initiation through regulation of the Hippo/Salvador/Warts pathway (Becker, 2016).

Antennapedia: Biological Overview | Evolutionary Homologs | Regulation | Targets of activity | Protein Interactions | Developmental Biology | References

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