Deformed
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 cephalopharyngeal skeleton or pupal "mouth," is composed of four major cuticular structures: mouth hooks, median tooth, H-piece and cephalopharyngeal plates. These chitinous structures are secreted by cells of the atrum and pharynx of the developing embryo. In Deformed mutants these structures, specified by mandibular and maxillary epidermis, are absent, disrupted or duplicated. Antennal and maxillary sense organs are also disrupted (Regulski, 1987).
Dfd+ is required in three embryonic cephalic segments to form a normal head. Mutant embryos of Dfd display defects in derivatives of the maxillary segment, of the mandibular segment, and of some more anterior segments. In the adult fly, defects are seen in the posterior aspect of the head when the gene is mutant. A transformation from head to thoracic-like tissue is seen dorsally and a deletion of structures is seen ventrally. The gene product is necessary during at least two periods of development: during embryonic segmentation and head involution, and during the late larval and pupal stages (Merrill, 1987).
Only a few genes have been identified that participate in the developmental pathways that modulate homeotic (HOX) protein specificity or mediate HOX morphogenetic function. To identify more HOX pathway genes, a screen was carried out for mutations in loci on the Drosophila second chromosome that interact with the homeotic gene Deformed. Genetic and molecular tests on the eight genes isolated in the screen place them in three general categories. (1) Two genes appear to encode trithorax group functions, i.e. they are general activators of Hox gene expression or function. (2) Four genes encode abundant, widely expressed proteins that may be required to mediate Dfd morphogenetic functions in certain tissues, including two genes for collagen IV protein variants. (3) two of the genes are required for the development of a subset of embryonic Dfd-dependent structures, while leaving many other segmental structures intact. One of these two was cloned and characterized. The cloned gene, apontic (apt), is required for the elaboration of dorsal and ventral head structures. It encodes a 484-amino-acid protein with no significant similarity to known protein sequences. The apt transcript pattern is normal in Dfd and Scr mutants, and the Dfd and Scr transcript patterns are normal in apt mutants. It is proposed that apt acts in parallel to, or as a cofactor with, HOX proteins to regulate homeotic targets in the ventral gnathal region (Gellon, 1997).
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).
Deformed mutants dying during metamorphosis share defects of CNS reorganization, ventral adult head development, and adult salivary gland morphogenesis. Specifically, the shared phenotypes show failure to separate the subesophageal ganglion (SEG) from the thoracic ganglion (TG); structural and functional abnormalities of the proboscis and maxillary palps, innervated by the SEG; and failure of the adult salivary glands to extend into the thorax. Broad Complex/Deformed double mutants show synergistic enhancement of the ventral head defects. This genetic interaction suggests that the segment identity and steroid hormone-sensitive regulatory hierarchies intersect during postembryonic development (Restifo, 1994).
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). Also studied was the expression of the empty spiracles (ems) gene, which in the wild-type brain is expressed in a large domain anterior to lab . In lab loss-of-function mutants, the ems gene is expressed ectopically in the tritocerebral domain in which lab is normally expressed; this ectopic ems expression occurs with 100% penetrance and ranges from 5-7 cells per hemisegment. The expression of pb disappears in the deutocerebrum and tritocerebrum of lab loss-of-function mutants but not in more posterior neuromeres. In contrast, the expression patterns of Dfd and Scr remain unaltered. Thus, in the tritocerebral domain in which lab is normally expressed, two changes in regulatory gene expression occur: activation of ems and inactivation of pb. To determine if ubiquitous overexpression of labial also alters regulatory gene expression patterns in the tritocerebral domain, transgenic flies were used carrying the lab gene under control of a heat-inducible promoter. In these mutants, ubiquitous overexpression of lab following heat-shock results in ectopic expression of the posteriorly active Dfd gene in the posterior tritocerebrum; this occurred with 100% penetrance when the heat shock was given around embryonic stage 10/11 (Hirth, 1998).
Immunocytochemical analysis shows that a number of other molecular labels are present in the tritocerebral lab domain of the wild type, but are absent in the corresponding tritocerebral domain of lab mutants. Among these are the LIM-homeodomain gene islet, the neuron-specific NAC epitope recognized by the anti-HRP antibody, the axon-specific BP102 epitope and the segment polarity gene engrailed. Taken together, these findings imply that the lab mutant cells have not acquired a neuronal identity. This suggests that the lab mutant cells either fail to differentiate into neurons or adopt another cell fate such as that of glial cells. To determine if these cells do differentiate into glia, the expression of the glial-specific repo gene was studied in the lab mutant tritocerebral domain. The pattern and number of repo-expressing cells in the tritocerebral labial domain are similar in the wild type and in the labial null mutant. This indicates that the lab mutant cells have not acquired a glial identity. It also suggests that gliogenesis is not affected in the lab mutant domain. It is therefore postulated that the lab gene is necessary for the establishment of correct neuronal cell fate, but not glial cell fate, in the part of the developing brain in which it is normally expressed (Hirth, 1998).
Comparable effects to those seen in labial mutants are seen in Deformed mutants but not in other homeotic gene mutants. In Dfd mutants the longitudinal pathways that normally project through the mandibular neuromere are missing or reduced. The mandibular commissure, which interconnects the mandibular hemineuromeres, is completely absent. The cells in the mandibular neuromere are generated in Dfd mutants. Homeotic gene expression patterns are altered in Dfd mutants; in the null mutant, lab is ectopically expressed in the mandibular segment in which Dfd is normally expressed. Ubiquitous overexpression of Dfd results in ectopic expression of the posteriorly active Scr gene in the Dfd domain. Cell-autonomous and cell-nonautonomous axogenesis defects occur in the Dfd mutants; mutant cells do not project axons, and descending and ascending axons from other parts of the brain do not project through the mutant domain. The Dfd mutant cells of the mandibular neuromere show a loss of neuronal markers such as ELAV, whereas the pattern and number of repo-expressing glial cells in the mutant domain is unaffected. These findings demonstrate that the action of the homeotic genes labial and Deformed are required for neuronal differentiation in the developing brain of Drosophila (Hirth, 1998).
Proteins produced by the homeotic genes of the Hox family assign different identities to cells on the
anterior/posterior axis. Relatively little is known about the signaling pathways that modulate their
activities or the factors with which they interact to assign specific segmental identities. To identify
genes that might encode such functions, a screen was carried out for second site mutations that reduce
the viability of animals carrying hypomorphic mutant alleles of the Drosophila homeotic locus,
Deformed. Genes mapping to six complementation groups on the third chromosome were isolated as
modifiers of Deformed function. Products of two of these genes, sallimus and moira, have been
previously proposed as homeotic activators since they suppress the dominant adult phenotype of
Polycomb mutants. Mutations in hedgehog, which encodes secreted signaling proteins, were also
isolated as Deformed loss-of-function enhancers. hedgehog mutant alleles also suppress the Polycomb
phenotype. Mutations were also isolated in a few genes that interact with Deformed but not with
Polycomb, indicating that the screen identified genes that are not general homeotic activators. Two of
these genes, cap 'n' collar and defaced, have defects in embryonic head development that are similar to
defects seen in loss of function Deformed mutants (Harding, 1995).
Insect metamorphosis provides a valuable model for studying mechanisms of steroid hormone action on the nervous system during a dynamic phase of
functional remodeling. The Drosophila Broad Complex (BRC) holds a pivotal position in the gene expression cascade triggered by the molting hormone
20-hydroxyecdysone (20E) at the onset of metamorphosis. BrC is essential for transducing 20E signals into the morphogenetic movements and cellular assembly that alter the CNS from juvenile to adult form and
function. The relationship of BR-C to two other genes was examined: Ecdysone-inducible gene E1 (IMP-E1), coding for an
EGF-like domain cysteine pattern protein, and Deformed (Dfd), involved in the metamorphic transition of the
CNS. Both BR-C and Dfd are required for maturation of the subeosphageal ganglion. Representatives of the whole family of BrC transcript isoforms accumulate in the CNS during the larval-to-pupal transition and respond directly to
20E in vitro. IMP-E1 is also directly regulated by 20E, but its induction is independent of BR-C, revealing that 20E works through at least two pathways in
the CNS. Dfd expression is also independent of BR-C function. Full induction of a number of other primary response genes (e. g., E74 and E75) requires BR-C function. Surprisingly, BR-C and Dfd proteins are expressed in distinct, nonoverlapping subsets of
neuronal nuclei of the subesophageal ganglion (SEG) even though both are required for SEG migration into the head capsule. The midline of the ventral ganglion contains many BR-C expressing cells, which may be neurons or glia. Expression in the optic lobes, which undergo BR-C dependent developmental events, is most extensive, with the vast majority of cells staining from BR-C. In contrast, the brain, SEG, and thoracic ganglia show clusters of stained cells, often surrounding a central zone devoid of staining. The pattern of "holes" is reminiscent of the positions of proliferating neuroblasts. This suggests that the segment identity (represented by Dfd expression)
and ecdysone cascades operate in separate but parallel pathways to control region-specific reorganization during metamorphosis (Restifo, 1998).
The Drosophila X chromosome has been screened for genes whose dosage affects the function of the
homeotic gene Deformed. One of these genes, extradenticle, encodes a homeodomain transcription
factor that heterodimerizes with Deformed and other homeotic Hox proteins. Mutations in the nejire
gene, which encodes a transcriptional adaptor protein belonging to the CBP/p300 family, also interact
with Deformed. The other previously characterized gene identified as a Deformed interactor is Notch,
which encodes a transmembrane receptor. These three genes underscore the importance of
transcriptional regulation and cell-cell signaling in Hox function. Four novel genes were also identified in
the screen. One of these, rancor, is required for appropriate embryonic expression of Deformed and
another homeotic gene, labial. Both Notch and nejire affect the function of another Hox gene,
Ultrabithorax, indicating they may be required for homeotic activity in general (Florence, 1998).
Dfd is required for the formation of the mouth hook, ectostomal sclerite, H-piece bar, anterior lateralgräten, cirri, ventral organ, and maxillary sense organ. Ablation experiments have provided a rough fate map of the embryonic head for the larval cuticular structures, and they show that these Dfd-dependent structures are derived from the maxillary and mandibular segments where Dfd is expressed. The Dfd targets Distalless (Dll), paired (prd), and Serrate (Ser) are all expressed in subsets of Dfd-expressing cells. The Dll, prd, and Ser mutant phenotypes in the maxillary segment are strongly correlated with the maxillary subregions in which they are expressed (Florence, 1998 and references).
The effects of the Dfd interactors on Dfd-dependent structures and target genes are discussed below:
Notch: The classical dominant Notch phenotype includes notched wing margins. Although all three Dfd-interacting alleles show notched wings in adults, their phenotypes are less severe than a Notch null, suggesting that all three alleles are hypomorphic. Mutant embryos lacking Notch have a neurogenic phenotype where the ventral epidermis is transformed into nervous tissue and, therefore, only the dorsal cuticle is produced. However, approximately half the NS177 cuticles have laterally derived maxillary cirri, whereas NUB53 animals do not. This suggests that NS177 is a weaker mutation than NUB53 even though NS177 shows the strongest interaction with Dfd (Florence, 1998).
nejire: The extant nej3 allele has been molecularly characterized as a null. However, in the Dfd interaction test, the viability of Dfd hypomorphs is not affected by heterozygous nej3. The failure of a nej null to interact with Dfd suggests that the Dfd-interacting nej alleles are not amorphic. Consistent with this interpretation, the lethal phases of the nej alleles in the current study differ from that of the nej3. Only ~15% of nej3 males die as embryos, demonstrating that the nej maternal component is sufficient for embryogenesis. However, 100% of both nejQ7 males and nejTA57j/nejQ7 females, as well as ~35% of nejS342/nejQ7 females, die as embryos. The premature lethality of the Dfd-interacting alleles indicates that they provide a less functional maternal component, perhaps because of an antimorphic Nej protein. In nejTA57/nejQ7 or nejQ7/Y cuticles, the maxillary and antennal sense organs often show a slight disruption in patterning; the mouth hooks and median tooth are reduced, and the proventriculus is sclerotized. No other phenotypes are consistently observed in cuticles of any nej genotype (Florence, 1998).
rancor: Cuticles of rncTA54, rncTA54/Df(1)DesiS3, and Df(1)DesiS3 embryos show defects in derivatives of the antennal and ventral gnathal lobes, including Dfd-dependent structures. Specifically, the hypostomal sclerite and the H-piece arm are present but malformed; the base of the mouth hook is reduced; the mouth hooks are multiply serrated, and head involution fails. The involution defect causes the maxillary and antennal sense organs to develop on the lateral aspect of the head instead of at the normal anterior-dorsal location. Less often, the H-piece cross-bar is reduced or fails to fuse medially; the dorsal-lateral or medial papillus is missing or misplaced; the ventral organ is disrupted, and the median tooth is translocated dorsally. The head involution defect is similar to that caused by mutations in another homeotic gene, labial (lab). In addition, both lab and rnc mutations affect many of the same head structures, such as mouth hooks, hypostomal sclerite, and H-piece (Florence, 1998).
strung out: Cuticles of stout mutants are similar to one another. Most striking are the defects surrounding the dorsal sac. The dorsal bridge is diffuse and occasionally unfused. Strands of sclerotic material are 'strung out' from the anterior vertical plate and ventral side of the dorsal bridge in the area between the dorsal sac and lateralgräten. Also, the H-piece lateral arm is bifurcated or widened, and the H-piece crossbar is widened or broken. The mouth hooks are of normal length, but thin, and the mouth hook bases are often reduced. The trunk of a stout mutant also shows narrow third thoracic denticle belts. A similar denticle phenotype is seen in mutants of another Dfd interactor, Ecdysone receptor. The stout gene appears to correspond to a previously identified lethal complementation group. stout adults heterogyous for certain alleles have thick aristae and arced wings, and many have one or more kinked macrochaete, phenotypes that are not seen in deficiency heterozygotes for the stout locus. In addition, males carrying these alleles over two duplications that cover the stout locus are sterile. The presence of these dominant phenotypes indicates that these stout alleles are either neomorphic or antimorphic. Another extant allele of stout does not exhibit these dominant phenotypes, nor does this mutant allele exhibit an interaction with Dfd (Florence, 1998).
extradenticle:
The exdS136 allele is embryonic-larval lethal (~40% die as embryos), while exd nulls are embryonic lethal. In addition, homeotic transformations of abdominal and thoracic cuticle are absent in exdS136 animals at 25° and only slightly apparent at 29°. The different lethal phase and milder cuticular phenotype indicates that exdS136 is hypomorphic and slightly temperature sensitive. Hemizygous exd1 cuticles show perturbations in structures derived from all head segments, except the antennal and hypopharyngeal lobes. All cuticular structures derived from the maxillary segment are malformed, with the exception of the cirri. The lateralgräten are short and often thick, and the hypostomal sclerite was elongated, perhaps fused to the ectostomal sclerite. Also, the structures surrounding the dorsal sac, such as the median tooth, epistomal sclerite, and dorsal bridge, are disrupted or fused. These dorsal sac region phenotypes may be caused by defects in dorsal head involution (Florence, 1998).
Wild-type Dfd expression is initiated in the blastoderm embryo in the primordia of the mandibular and maxillary segments. In early germ band-extended embryos, the Dfd expression domain includes the hypopharyngeal, mandibular, and maxillary lobes. During stages 11 and 12, Dfd expression is repressed in the hypopharyngeal and anterior mandibular epidermis. This refinement in the Dfd pattern requires the repressive function of an isoform from the cap'n'collar (cnc) gene. Soon after the initiation of Dfd transcription, Dfd protein is required in nearly all maxillary epidermal cells for persistent activation of Dfd transcription. Therefore, mutations that affect either Dfd transcription or protein levels would be expected to have an effect on Dfd transcript pattern in the maxillary epidermis. To determine if the X-linked, Dfd-interacting genes are required for the normal Dfd transcript pattern, hemizygous mutants were assayed for Dfd expression by RNA in situ hybridization. Only Notch and rnc mutant animals show perturbations in Dfd transcription during any stage of embryogenesis. In rnc mutants, ectopic Dfd expression is often seen in the anterior mandibular lobe. Because cnc is required for the repression of anterior mandibular Dfd expression, one possiblity is that the ectopic Dfd seen in rnc mutants is indirectly caused by a loss of cnc expression. cnc expression is severely reduced in the hypopharyngeal and mandibular lobes of rnc mutant embryos, suggesting that the aberrant mandibular Dfd expression is caused by reduced cnc levels. Expression of cnc in the clypeolabrum is unaffected in these mutants (Florence, 1998).
Although it was not obvious from the cuticular phenotype, the altered expression patterns
of Dfd and cnc suggests that the loss of rnc function may result in a partial homeotic transformation of cells in the mandibular segment to maxillary identity. If this hypothesis is correct, maxillary-specific target genes of Dfd should be inappropriately regulated in the mandibular lobe of rnc mutants. The expression of the Dfd targets prd, Ser, and Dll were examined in rnc mutants. In most rnc mutant embryos, two of these genes are expressed in maxillary-like patterns in the mandibular segment: prd being ectopically activated, and Ser inappropriately absent. Dll expression is unaffected in rnc mutants. These data are consistent with a partial transformation of the mandibular lobe toward maxillary identity. Since the rnc cuticular phenotype also have similarities to the lab phenotype, the lab transcription pattern was examined in rnc mutants. Early phases of lab expression are normal in the rnc mutant embryos. However, by stage 13/14, lab transcript abundance in the intercalary and tritocerebral primordia of the procephalic region is weaker or absent in rnc mutants than in wild-type embryos. The pattern and amounts of lab transcript in the midgut of rnc mutants is comparable to wild type. However, after stage 15, the second midgut chamber, as marked by lab expression, is smaller and of variable diameter along its length, whereas the other chambers appear relatively normal. Therefore, even though both procephalic and midgut lab-expressing tissues are disrupted in rnc mutants, lab expression is affected only in the procephalic region (Florence, 1998).
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).
date revised: 18 July 98
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