Deformed
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).
date revised: 18 July 98
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