Motor axons make synaptic connections to specific muscles. This specificity is determined during development as motorneuron growth cones choose specific pathways and ultimately recognize and then synapse on their specific muscle targets. The abrupt gene is required for the embryonic formation of specific synaptic connections at the neuromuscular junction between a subset of motorneurons and a corresponding subset of muscles. abrupt also has a role in establishing and maintaining muscle attachments, adult sensory cell formation and morphogenesis of adult appendages. abrupt is a transcription factor expressed in muscle cells and not in neurons, suggesting that abrupt controls the muscle expression of molecules required for correct motorneuron targeting, as well as molecules required for correct muscle attachments (Hu, 1995).

The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, the mechanism has been examined by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. A border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations (Cook, 2004).

The ab gene, which encodes a zinc finger protein containing a BTB/POZ domain, is required for L5 development as revealed by viable alleles such as ab¹, which bypass the early embryonic requirement for this gene in motor neuron axon guidance and result in distal truncation of the L5 vein (Hu, 1995). Four additional viable ab alleles have been recovered in a genome-wide screen for new wing vein mutants, one of which results in a somewhat stronger phenotype in which the L5 vein is consistently truncated proximal to the posterior cross-vein. Expression of ab in the wing disc was examined; it is expressed as a single stripe in the posterior compartment. The viable ab¹ allele is likely to be a regulatory mutation, since ab expression is greatly reduced in ab¹ mutant wing discs. ab expression is similarly reduced or undetectable in the other four independently isolated viable ab alleles. Double-label experiments with the vein marker Delta (Dl), which is expressed in L1 and L3-L5, reveal that ab is co-expressed with Dl in the L5 primordium (Cook, 2004).

Extension of a previous analysis of ab in initiating L5 development (Biehs, 1998; Sturtevant and Bier, 1995) has shown that ab functions early in L5 specification. Activation of all known vein genes, including rho, Dl, the caupolican and araucan genes of the Iroquois Complex (IroC), and argos, and repression of the intervein genes bs (also known as DSRF) and net, is lost in cells corresponding to the L5 primordium in ab¹ mutant wing discs. A determination was also made whether it is critical that ab expression is confined to a narrow stripe for regulating expression of vein or intervein genes. ab was ubiquitously misexpressed in the wing disc using the MS1096-GAL4 driver; such global activation of ab suppresses expression of vein genes, such as rho and Dl. This ab misexpression also caused vein-specific downregulation of the intervein gene bs, in the wing disc, but did not repress expression of other genes, including hh, ptc and dpp. This phenotype may result from unregulated production of a lateral inhibitory signal normally produced by vein cells to suppress vein development in adjacent intervein cells (Cook, 2004).

Whether restricted expression of ab in small clones is sufficient to induce vein development was also investigated. The flip-out misexpression system was used to generate clones of cells ectopically expressing ab in the wing disc; these cells (identified by Ab or ß-Gal expression) ectopically express the vein marker Dl and downregulate expression of the intervein marker Bs in a cell-autonomous fashion when located anywhere within the wing pouch. Adult wings containing small ab-expressing clones marked with forked also produce ectopic vein material cell autonomously. These results demonstrate that ab is necessary to control known gene expression in the L5 primordium, and is sufficient to induce vein development when expressed in a restricted number of cells. These data are consistent with ab acting in a vein-organizing capacity to direct L5 development (Cook, 2004).

The L2 primordium forms along the anterior boundary of the sal expression domain, in cells expressing low levels of sal and facing those expressing high levels of sal. The symmetrical disposition of the L2 and L5 veins, and the positioning of both of these veins by Dpp rather than Hh signaling, suggests that the L5 vein might form along the posterior border of the sal expression domain in much the same way that L2 is induced along its anterior border. However, two lines of evidence indicate that sal is not likely to be directly involved in determining the position of L5. (1) The posterior border of the sal expression domain is located several cells anterior to the L5 primordium (Sturtevant, 1997). (2) Although salm^- clones do occasionally result in the formation of ectopic posterior veins, they do so non-autonomously at a distance of several cell diameters from the clone border (Sturtevant, 1997). This phenotype is entirely different from the ectopic L2 veins that form at high penetrance immediately within the borders of anterior sal^- clones, located between the L2 and L3 veins (Sturtevant, 1997). Clones of a deficiency removing both salm and the related salr gene also result in the production of an ectopic vein, but this vein forms within the interior of such clones between L4 and L5, in a position corresponding to a cryptic vein, or paravein, which has a latent tendency to form along the posterior border of the sal domain (Cook, 2004).

Since the L5 primordium forms approximately four to six cell diameters posterior to the sal expression domain (Sturtevant, 1997), the expression was examined of other BMP target genes, omb and brk, relative to the L5 primordium. The borders of these gene expression domains are known to form posterior to that of the sal domain. Previous studies revealed that the domains of cells expressing high levels of omb and brk are largely reciprocal, although these genes are co-expressed at lower levels in cells along the border. Therefore the relative positions of the border of high level omb/brk expression was determined with respect to vein primordia marked by Dl (L1, and L3-L5) and Kni (L2). These experiments revealed that the L5 stripe of Dl expression forms inside and along the posterior border of the domain expressing high levels of omb, whereas the anterior border of the omb domain extends well beyond the L2 primordium. A complementary pattern was observed in wing discs of brk-lacZ flies double stained for ß-Gal and Dl, in which the L5 Dl stripe runs outside and along the border of the high level brk expression domain. Similar results were obtained using ab as a marker for the L5 primordium, in which the stripe of ab-expressing cells was found to lie within the omb domain, adjacent to high level brk-expressing cells. These expression studies reveal that omb and brk are expressed in the right location to play a role in positioning the L5 primordium (Cook, 2004).

As a first step in determining whether omb or brk play a role in L5 development, genetic interactions between these genes and ab were tested. Several viable or lethal ab alleles were crossed to stocks carrying the brk^m68 allele or a deficiency of brk, and trans-heterozygous brk^-/+;ab^-/+ F1 flies were examined for L5 phenotypes. None of the combinations of brk and ab alleles tested resulted in any dominant vein-loss phenotype in trans-heterozygotes. In addition, no enhancement of the homozygous ab¹/ab¹ L5 truncation phenotype was observed in brk^-/+; ab¹/ab¹ flies. By contrast, when trans-heterozygous interactions between ab and omb alleles were tested, consistent genetic interactions were observed. For example, omb¹/+; ab¹/+ flies exhibit truncations in the distal portion of L5 (with 3% penetrance, whereas neither ab¹/+ nor omb¹/+ heterozygotes ever show any L5 phenotype. Moreover, the omb¹ allele, which causes notching of the wing margin when homozygous but has no associated L5 phenotype, strongly enhances the ab¹/ab¹ L5 truncation phenotype. This interaction is evident in omb¹/+; ab¹/ab¹ females, and is very pronounced in omb¹/omb¹;ab¹/ab¹ double homozygous females or hemizygous omb¹/Y; ab¹/ab¹ males. These results suggest that omb and ab function in concert to promote L5 formation (Cook, 2004).

Additional detailed experiments have shown that (1) misexpression of omb and brk shifts or eliminates the L5 and L2 veins; (2) omb is required cell autonomously for L5 development; (3) brk is required for the production of an L5 inductive signal, and (4) ab acts downstream of brk in L5 development (Cook, 2004).

Thus, this study examined the role of two Dpp target genes, brk, which is expressed in a domain abutting the L5 primordium, and omb, which is expressed in a domain just including the L5 primordium, in establishing the position of this vein. The results suggest a model for how the BMP activity gradient induces formation of the L5 primordium in the posterior compartment of the wing. According to this model, L5 development is initiated within the posterior region of the wing where brk and omb are expressed in adjacent domains with a sharp border between them. Since brk^- clones induce vein development within the clone along the border with brk⁺-neighboring cells, it is suggested that brk-expressing cells produce a short-range vein-inductive signal, Y, to which they cannot respond. This signal acts on neighboring omb-expressing cells to initiate vein development. The additional cell-autonomous requirement for Omb activity to respond to this Brk-derived signal suggests that the intracellular effector of the vein inductive signal Y must act in combination with Omb to induce vein formation. Because Brk is a repressor of omb expression, the combined requirement for the short-range Brk-derived vein-inductive signal and Omb activity within responding cells constrains L5 initiation to omb-expressing cells adjacent to brk-expressing cells. In this scheme, Brk plays at least two distinct roles in L5 induction. First, as a repressor of omb, Brk defines the border between the brk and omb expression domains, and, second, brk-expressing cells are the source of a vein-inductive signal required to initiate L5 development within adjacent omb-expressing cells (Cook, 2004).

A key mediator of L5 induction is the Ab transcription factor, which is expressed in a narrow stripe along the brk/omb border, just within the omb expression domain. ab is required for expression of all known vein genes and for downregulation of intervein genes in the L5 primordium (Biehs, 1998). Similarly, the ability of brk^- clones to induce an ectopic posterior vein depends on ab function. In addition, localized misexpression of ab in small flip-out clones leads to induction of vein markers in wing imaginal discs and to the formation of ectopic patches of vein material. The vein-organizing activity of ab depends on its being expressed in a localized pattern, since ubiquitous expression of ab suppresses vein development throughout the wing disc. This effect of ubiquitous ab misexpression is similar to that observed previously for ubiquitous expression of kni or knrl, in which all distinctions between vein and intervein regions are lost although expression of other genes in the wing disc are not perturbed. One explanation for this vein-erasing phenotype is that kni/knrl and ab control the expression of a lateral inhibitory signal. Consistent with this possibility, small ab flip-out clones autonomously express the lateral inhibitory signal Dl. According to the model, establishment of the L5 primordium requires input from both omb (cell autonomous) and brk (cell non-autonomous), which collaborate to initiate ab expression in a narrow stripe along their borders (Cook, 2004).

A curious phenotype associated with some brk^- clones generated in an ab¹/ab¹ background is the formation of diffuse wandering veins within the interior of the clone. A similar disorganized ectopic vein phenotype is also observed in a fraction of omb^- brk^- double mutant clones. This phenotype may reflect the lack of a lateral inhibitory factor (e.g. Dl) produced by ab-expressing cells to suppress vein formation in neighboring cells. The observation that ubiquitous expression of ab suppresses vein formation throughout the wing disc is consistent with this possibility. It is also possible that omb plays a role in promoting intervein development as well as in activating ab expression. Additional analysis will be needed to address this question (Cook, 2004).

Previous analysis of L2 initiation lead to a model in which sal-expressing cells produce a short-range vein-inductive signal (X) to which they cannot respond (Sturtevant, 1997). In response to signal X, neighboring cells outside of the sal domain express the L2 vein-organizing genes kni and knrl. In addition, analysis of an L2-specific cis-regulatory element of the kni/knrl locus provided indirect evidence for negative regulation by a repressor, possibly Brk, expressed in peripheral/lateral regions of the wing disc (Cook, 2004).

An interesting question regarding veins forming within more anteriorly located brk^- clones is whether they have an L2- or an L5-like identity. In one case, these veins express kni, but not Dl, suggesting that they have an L2-like identity. In the other, the ectopic veins induced anteriorly by brk^- clones require omb function, as do L5-like veins generated in the posterior compartment of the wing. This latter observation suggests that the brk^- border in anterior regions acts as it does in posterior regions of the wing disc, but that its effect may be mediated by the L2 organizing kni/knrl locus rather than the L5 organizing gene ab. This hypothesis might provide an explanation for why ectopic veins that form in various mutant backgrounds tend to form along a line running between the L2 vein and the margin (which is referred to as the P2 paravein) (Sturtevant, 1997). This sub-threshold vein promoting position may be defined by the anterior border of brk and omb expression. Further analysis of the identity of these ectopic veins will be required to resolve this question (Cook, 2004).

Since the L2 and L5 veins form at similar lateral positions within the anterior and posterior compartments of the wing, respectively, it is informative to compare the mechanisms by which positional information is converted into vein initiation programs in these two cases. The positions of these two veins are determined by precise dosage-sensitive responses to BMP signaling emanating from the center of the wing; these responses are mediated by the borders of the broadly expressed, Dpp signaling target genes sal and omb. Brk also plays a role in initiating both L2 and L5 development. In the posterior compartment, Brk leads to the production of a hypothetical vein-promoting signal Y, which has a function and range similar to the putative L2 vein-inducing signal X, produced by sal-expressing cells. It is not clear whether the signals X and Y are the same or different; however, an important difference between L2 and L5 initiation is that only L5 has an additional requirement for omb function. This dual requirement for omb function within the L5 vein primordium and a short-range inductive signal in neighboring brk-expressing cells provides a stringent constraint on where the L5 primordium forms. Brk may also directly repress expression of the vein-organizer gene ab in cells posterior to the L5 primordium, analogous to the proposed role as a repressor of kni/knrl anterior to L2. One possible rationale for induction of the L5 vein depending on inputs from both omb and brk is that these genes are expressed in partially overlapping patterns and neither pattern may carry sufficiently detailed information to specify the position of the L5 primordium alone. Although the omb and brk expression levels fall off relatively steeply (i.e. over a distance of six to eight cells), these borders are not as sharp as the anterior sal border (two to three cells wide), which alone is sufficient to induce the L2 primordium (Cook, 2004).

A final similarity between the initiation of L2 and L5 formation is that induction of both veins is mediated by a vein-organizing gene that regulates vein and intervein gene expression in the vein primordium. Although kni and ab are members of different subfamilies of Zn-finger transcription factors, they are both expressed in a narrow stripe of cells along their respective inductive borders, and ubiquitous misexpression of either gene results in elimination of vein pattern in the wing disc. Thus, the L2 and L5 veins are induced by remarkably similar mechanisms and principles of organization. Further comparison of the mechanisms of these developmental programs should provide insights into the degree to which general and specific vein processes define the L2 versus the L5 vein identity (Cook, 2004).

Induction of Drosophila wing veins at borders between adjacent gene expression domains provides a simple model system for studying how information provided by morphogen gradients is converted into the stereotyped pattern of wing vein morphogenesis. Each of the four major longitudinal veins (L2-L5) is induced by a for-export-only mechanism in which cells in one region of the wing produce a diffusible signal to which they cannot respond. In the case of L3 and L4, an EGF-related signal (Vein) is produced between these veins in the central organizer where expression of the EGF receptor is locally downregulated. With respect to L2, response to the vein-inductive signal X is repressed in Sal-expressing cells that produce the hypothetical signal X. Finally, the L5 vein-inductive signal produced by brk-expressing cells depends on omb, the expression of which is repressed by Brk (Cook, 2004).

For-export-only mechanisms also underlie the induction of boundary cell fates in many other developmental settings. In the well-studied Drosophila wing, the earliest and most rigorously defined boundaries are the AP and DV borders, which are determined by Hh and Notch signaling, respectively. These compartmental borders define domains of non-intermixing groups of cells, and function as organizing centers by activating expression of the long-range morphogens Dpp and Wingless (Wg), respectively. In both cases, cells in one compartment produce a signal to which they cannot respond. This signal is constrained to act only on neighboring cells in the adjacent compartment. Other well-studied examples of for-export-only signaling include: induction of the mesectoderm in blastoderm stage Drosophila embryos by a likely cell-tethered Notch ligand expressed in the mesoderm; induction of parasegmental expression of stripe via Wg, Hh and Spi signaling in gastrulating Drosophila embryos; induction of mesoderm in Xenopus embryos by factors produced in the endoderm under the control of VegT, and formation of the DV border of leaves in plants controlled by the PHANTASTICA gene . The similar but distinct mechanisms for inducing the L2 and L5 vein primordia offers a well-defined system for examining these relatively simple cases in depth. These inductive events take place at the same developmental stage but within separate compartments of a single imaginal disc, and should provide general insights into the great variety of mechanisms that can be co-opted to accomplish for-export-only signaling (Cook, 2004).

cDNA clone length - 5.1 kb

Exons -There are three exons, extending over a 33 kb DNA interval.

Bases in 3' UTR - 1655

PROTEIN STRUCTURE

Amino Acids - 904

Structural Domains

Abrupt has two Cys2-His2 zinc fingers and three Asn-Pro repeats and an N-terminal BTB domain (Hu, 1995).

A novel zinc finger protein, ZID (standing for zinc finger protein with interaction domain) was isolated from humans. ZID has four zinc finger domains and a BTB domain, also know ans a POZ (standing for poxvirus and zinc finger) domain. At its amino terminus, ZID contains the conserved POZ or BTB motif present in a large family of proteins that include otherwise unrelated zinc fingers, such as Drosophila Abrupt, Bric-a-brac, Broad complex, Fruitless, Longitudinals lacking, Pipsqueak, Tramtrack, and Trithorax-like (GAGA). The POZ domains of ZID, TTK and TRL act to inhibit the interaction of their associated finger regions with DNA. This inhibitory effect is not dependent on interactions with other proteins and does not appear dependent on specific interactions between the POZ domain and the zinc finger region. The POZ domain acts as a specific protein-protein interaction domain: The POZ domains of ZID and TTK can interact with themselves but not with each other, or POZ domains from ZF5, or the viral protein SalF17R. However, the POZ domain of TRL can interact efficiently with the POZ domain of TTK. In transfection experiments, the ZID POZ domain inhibits DNA binding in NIH-3T3 cells and appears to localize the protein to discrete regions of the nucleus (Bardwell, 1994).

DEVELOPMENTAL BIOLOGY

Embryonic

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

Initial ab expression is in the CNS midline cells, beginning at stage 9 and lasting through stage 13 [Images]. Segmentally repeated stripes of ectodermal expression appear at stage 11 and becomes uniform throughout the entire epidermis by stage 12. During myoblast fusion and syncytial muscle formation at stage 14, Abrupt mRNA can be detected in the somatic muscle cells. By stage 16, all abdominal muscles express ab (Hu, 1995).

Larval

ab is expressed in imaginal discs, consistent with the wide ranging adult defects seen in mutants.

Effects of mutation or deletion

In abrupt mutants, a specific set of motorneurons (SNb) fail to make proper synaptic connections with their ventral longitudinal muscle targets. The axons form abnormal branches instead of forming their wild-type axonal extensions onto the muscle fibers. These aberrant branches wander over the prospective target muscles and occasionally form connections at ectopic sites. The targeting defects in ab mutants are limited to the ventral region of the embryo (Hu, 1995).

Analysis of the mature embryonic muscle pattern in ab mutants reveals that most muscles appear normal, however a few show variably penetrant defects in the locations of their muscle attachments (Hu, 1995).

Abrupt function is required for the development of numerous adult structures. Viable mutant alleles show wing venation and macrochaete (bristle) defects. Legs may be severely gnarled. Mutants have a furrow at the midline of the dorsal thorax, and antennal aristae are also deformed (Hu, 1995).

Development of morphological diversity of dendrites is directed by Abrupt

Morphological diversity of dendrites contributes to specialized functions of individual neurons. In the present study, the molecular basis that generates distinct morphological classes of Drosophila dendritic arborization (da) neurons was examined. da neurons are classified into classes I to IV in order of increasing territory size and/or branching complexity. Abrupt (Ab), a BTB-zinc finger protein, is expressed selectively in class I cells. Misexpression of ab in neurons of other classes directs them to take the appearance of cells with smaller and/or less elaborated arbors. Loss of ab functions in class I neurons results in malformation of their typical comb-like arbor patterns and generation of supernumerary branch terminals. Together with the results of monitoring dendritic dynamics of ab-misexpressing cells or ab mutant ones, all of the data suggests that Ab endows characteristics of dendritic morphogenesis of the class I neurons (Sugimura, 2004).

Both misexpression and loss-of-function analyses support the hypothesis that selective expression of ab in class I da neurons plays a pivotal role in forming dendritic arbors, which are characteristic of the class I cells, and that development of more complex arbors of class II-IV neurons depends on the absence of Ab. This conclusion was drawn not only from quantification of the number of terminals and the area size of individual dendritic trees at single given developmental stages, but also from time-lapse recordings to monitor dynamic behaviors at embryonic and/or larval stages (Sugimura, 2004).

As far as analysis with molecular markers is concerned, neither ab misexpression nor its loss of function results in alteration of cell identity of da classes examined. The molecular tools employed were enhancer-trap markers for class I (ddaE) or class IV and the level of Cut that distinguishes between class I and II-IV. Thus, it appears unlikely that the dendritic phenotypes reported could be indirect consequences of cell identity alteration; an alternative hypothesis is preferred; that Ab is more immediately involved in regulating dendritic morphology through transcriptional regulation of its target genes (Sugimura, 2004).

ab misexpression decreases the number of branch terminals of class III and IV neurons, and conversely, ab mutant class I cells produce supernumerary branches. One possible interpretation of these results would be that Ab may negatively regulate dendritic branching in a broad range of neuronal types. However, this hypothesis would be difficult to reconcile with the following findings: in normal development, the territory size of class I dendrites is smaller than that of class II dendrites, while the dendritic trees of both classes are similarly complicated. ab misexpression in class II neurons reduces the territory size, but the terminal number does not change significantly. Furthermore, ab misexpression in any da neuron of class II-IV in the dorsal cluster reduces the size and/or the terminal number to values that are comparable to those of class I neurons of the same cluster. The easiest interpretation of these results would be that the misexpression morphologically alters class II-IV dendrites toward that of class I. It should be also noted that dendritic patterns are defined not only by the numerical parameters such as the terminal number or the territory size, but also by other properties such as the comb-like design of class I, spike protrusion of class III, and mutual avoidance of class IV. Effects of ab loss of function or misexpression are consistent with the notion that Ab endows every feature of class I dendritic patterning (Sugimura, 2004).

Although manipulations of ab misexpression cause severe and reproducible phenotypes, they do not necessarily provide evidence for almost complete morphological transformation from classes II-IV into class I neurons. Class II, III, or IV neurons that had misexpressed ab were morphologically recognized as such, in terms of the number and the direction of dendritic shafts that grew out of each cell body and the branching pattern within the region proximal to the soma. Time-lapse recordings showed that da neurons of class I and class IV use distinct strategies from the very beginning of dendritic birth from the soma that contribute to differences in their basic arbor patterns. The partial alteration of the arbor patterns by ab misexpression might be due to a late onset and/or a low level of ab transgene expression obtained by using the available postmitotic drivers (Sugimura, 2004).

In contrast to the formation of supernumerary branch terminals of class I neurons in the ab mutants, the same cells did not show obvious expansion of the arbor size compared with the control cells; this could be due to the possibility that the expansion, if any, was too small to be detected at the early larval stage when differences in the field size of class I and that of other classes were subtle compared with those at late larval stages. MARCM analysis was performed to explore phenotypes at late larval stages, and it was shown that ddaD increases its arbor size, but another class I neuron examined, ddaE, does not. This variation could be explained by perdurance of the wild-type protein in each mutant cell. Alternatively, dysfunction of Ab-dependent mechanisms might not be sufficient for expansion of the territorial field, and an additional mechanism, which works in classes II-IV neurons in normal development, may be required (Sugimura, 2004).

The Ab protein has two zinc fingers of the C₂H₂ class, which is one of the most common types of DNA binding domains; in addition, a BTB/POZ domain is found at the N terminus of the fingers. The BTB/POZ domain is an evolutionarily conserved protein-protein interaction domain, and BTB/POZ domains of several zinc finger proteins, such as PLZF and Tramtrack, have been shown to be responsible for transcriptional regulation. Recent studies have discovered several putative transcriptional factors of other families that regulate morphological heterogeneity of dendrites including branching complexity, field size, and targeting specificity in different model systems, suggesting that transcriptional regulation is a common mechanism to generate morphological diversity of dendrites. Therefore, it is likely that class-specific profiles of gene expression controlled by these factors are responsible for distinctive dendritic morphogenesis. Target genes of these transcriptional regulators in the context of dendritic pattern formation have not yet been found, and their future identification should give detailed pictures of the molecular machineries at work (Sugimura, 2004).

Ab and Cut provide striking contrasts to each other in terms of class-dependent levels of immunoreactivity and, furthermore, gain-of-function and loss-of-function phenotypes of dendritic morphology. Neither ab loss of function nor its misexpression is associated with alteration of cut expression, which does not provide evidence for a simple epistatic or mutually dependent relationship between the two genes at the level of gene expression. It could be that selective expression of Ab and Cut is operated by a mechanism that is separate, at least partially. When the two putative transcription factors were examined at the level of dendritic morphology as a final read-out, it was found that they can interfere with each other's function upon overexpression, which argues against a simple epistatic relationship between them. A couple of possibilities could explain this mutual interaction. For example, target genes of Ab and those of Cut may be partially overlapped, and the interference may be due to competitions between the two for cis regulatory elements of the same target gene. Alternatively, Ab's targets and those of Cut may operate on cytoskeletal reorganization in different ways. The molecular basis of the mutual interaction between Ab and Cut should be clarified by identifying their target genes (Sugimura, 2004).

Recent functional studies support the involvement of da neurons in thermosensation and/or pain sensation and in coordination of rhythmic locomotion. Interesting questions include whether distinct classes of da neurons or, more specifically, distinct class-specific morphological features of dendritic arbors, are responsible for distinctive physiological roles or not. This question might be addressed by monitoring the behavior of animals, in which all da neurons have class I-like dendritic patterns. Combinations of genetic and physiological approaches in this model system may shed light on a long-standing question of how each dendritic form relates to its function at the various levels, molecular, cellular, and whole body (Sugimura, 2004).

Abrupt suppresses dendritic branching in a neuronal subtype-specific and dosage-dependent manner

How dendrites of different neuronal subtypes exhibit distinct branching patterns during development remains largely unknown. Loss-of-function mutations in the abrupt (ab) gene have been identified and mapped that increase the number of dendritic branches of multiple dendritic (MD) sensory neurons in Drosophila embryos. Ab encodes an evolutionarily conserved transcription factor that contains a BTB/POZ domain and C2H2 zinc finger motifs. ab has a cell-autonomous function in postmitotic neurons to limit dendritic branching. Ab and the homeodomain protein Cut are expressed in distinct but complementary subsets of MD neurons, and Ab functions in a transcriptional program that does not require Cut. Deleting one copy of ab or overexpressing ab has opposite effects on the formation of higher-order dendritic branches, suggesting that the Ab level in a specific neuron directly regulates dendritic complexity. These results demonstrate that dendritic branching can be suppressed by neuronal subtype-specific transcription factors in a cell-autonomous and dosage-dependent manner (Li, 2004).

Ab was first identified as an important regulator that controls the specificity of neuromuscular connections between a subset of motoneurons and a subset of muscles. Interestingly, Ab is expressed in the nucleus of muscle cells but not motoneurons, indicating that it affects the targeting of motoneuron axon terminals in a non-cell-autonomous fashion. It remains unknown what downstream targets are misregulated in muscle cells in ab mutants that are responsible for mediating the interactions between motoneuron axon terminals and the muscle surface. Evidence is provided that Ab has a cell-autonomous function in neural development (Li, 2004).

Ab is expressed in the nucleus of a subset of postmitotic MD sensory neurons. Ab mutant embryos have a normal number of MD neurons that can still be labeled by a pan-MD marker Gal4109(2)80, suggesting that the ab gene does not control the MD fate of these neurons. This notion is in contrast to other transcription factors in Drosophila that have a dual function in both cell fate determination and dendritic morphogenesis. It is possible that Ab is a transcription factor dedicated to maintaining the less-branched dendritic trees of ddaE, ddaF, and dbd neurons in the dorsal cluster. Ab normal function limits rather than promotes dendritic branching in postmitotic neurons (Li, 2004).

MARCM analysis has demonstrated that Ab has a cell-autonomous function in postmitotic neurons to directly control dendritic branching during development. The unique features of MD neuron lineages ensure that the presence of a single mCD8-GFP-labeled MD neuron itself indicates that the somatic recombination occurs during the last cell division that gives rise to the MD neuron. Therefore, the dendritic phenotypes observed in single MD neuron clones reflect the gene function in postmitotic neurons. Although Ab is also expressed in muscle cells and in epidermis, it is unlikely that Ab has a non-cell-autonomous function in controlling dendritic branching of sensory neurons, since expression of UAS-ab only in ddaE, ddaF, and vpda neurons in ab mutants could rescue the dendritic phenotype (Li, 2004).

In the dorsal cluster, Ab and Cut are expressed in distinct but complementary subsets of DA neurons. Ab is expressed only in ddaE and ddaF neurons in addition to dbd neurons, while Cut is expressed in the four other DA neurons. Ab limits the dendritic branching of neurons with less-branched dendritic trees, while Cut promotes dendritic branching in other neurons with highly branched dendritic trees (Li, 2004).

Several lines of evidence indicate that Ab functions in a transcription program that does not require Cut. (1) Ab and Cut are expressed in distinct but complementary subsets of neurons in each dorsal cluster. (2) Cut is undetectable in normally Ab-positive, Cut-negative neurons in ab mutant embryos, suggesting that Ab does not function by suppressing Cut expression. (3) Cut expression is not affected in Ab-negative, Cut-positive neurons when Ab is ectopically expressed. Taken together, these data show that Ab controls dendritic branching through a transcriptional program that does not require Cut. In contrast, Ab expression is not detectable even in ddaB neurons where Cut expression is very low and is not upregulated in cut mutant neurons that are normally Ab negative and Cut positive. These findings further support the notion that, under normal circumstances, Ab and Cut control two transcription programs independent of each other. Ectopic expression of Cut in Ab-positive, Cut-negative neurons can suppress Ab expression; this suppression is probably due to the fact that Cut and several other transcription factors share common DNA binding sites. Actually, Ab itself and Cut can bind to some consensus DNA sequences at least in vitro, raising the possibility that the transcription of at least some common target genes is regulated by Ab in one subset of sensory neurons but by Cut in another subset. This notion is further supported by the finding that coexpression of Ab partially rescues the dendritic overgrowth phenotype caused by ectopic expression of Cut in Ab-positive, Cut-negative neurons (Li, 2004).

These studies provide strong evidence that different transcription factors specifically either promote or inhibit dendritic branching in a neuronal subtype-specific manner. A similar mechanism has been demonstrated in other model systems to control axonal branching. For instance, the zinc finger protein Brakeless controls axon terminal arborization of a subset of photoreceptors in Drosophila. In the spinal cord, the ETS class transcription factor PEA3 regulates axonal branching of specific motoneuron pools. In the Drosophila olfactory system, the POU domain transcription factors Acj6 and Drifter regulate both dendritic targeting specificity and axon terminal arborization. The BTB/POZ domain is known to mediate protein-protein interactions between heterodimers. It is likely that other transcription factors may collaborate with Ab or Cut to provide additional layers of specificity in controlling dendritic branching in a subtype-specific manner (Li, 2004).

An important finding of this study is the dosage-dependent effect of Ab on dendritic branching in a given neuron. Ab-positive, Cut-negative neurons, but not Ab-negative, Cut-positive neurons, exhibit increased dendritic branching in ab heterozygous larvae or Df/+ larvae, while overexpression of Ab results in decreased dendritic branching in Ab-positive, Cut-negative neurons. These findings suggest that dendritic branching complexity is tightly regulated at the transcriptional level and that Ab is a key component in this regulatory pathway. The evolutionarily conserved BTB/POZ domain can promote transcriptional repression by recruiting corepressor proteins. Different levels of Ab may form qualitatively or quantitatively different complexes that in turn regulate the expression level of its target genes. Fine regulation of Ab availability or activity might be an effective way to control the dendritic branching complexity of a specific neuron in response to neuronal activity or different environmental stimuli (Li, 2004).

Abrupt was identified in a genome-wide analyses for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4²²¹) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4²²¹-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

In addition to genes with functions in promoting dendrite arborization, 20 group A genes were identified that regulate dendrite arborization by limiting dendrite growth and/or branching. Consistent with recent reports that loss of function of the BTB/POZ domain TF abrupt (ab) causes an increase in dendritic branching and altered distribution of branches, it was found that ab(RNAi) altered the arborization of class I dendrites. ab(RNAi) caused an increase in the number and length of lateral branches, expanding the coverage field most noticeably along the anteroposterior (AP) axis. In addition to these defects, ab(RNAi) also caused frequent cell death, consistent with the phenotype observed for a hypomorphic allele of ab (Parrish, 2006).

Since group A and B TFs regulate aspects of dendritic growth and branching, potential epistatic relationships among TFs was explored in these phenotypic classes. To do this, RNAi was used to knockdown expression of select TFs in Drosophila embryos carrying a loss-of-function mutation in either the group B/C gene senseless (sens) or the group A gene abrupt (ab). sens mutant class I dendrites overextend dorsally and have reduced lateral branching in addition to routing defects. In sens mutants, RNAi of the group A genes Su(z)12 and ab, which cause increased lateral branching following RNAi in wild-type embryos, led to an increase in lateral branching compared with injected controls. Therefore, Su(z)12 and ab function are still required to limit arborization in sens mutants, and the increased dendritic branching as a result of Su(z)12(RNAi) or ab(RNAi) is epistatic to the increased dorsal extension and reduced lateral branching of sens mutants. In contrast, RNAi of the group A genes cg1244 and cg1841, which caused reduced arborization following RNAi in wild-type embryos, led to a reduction in primary dendrite outgrowth and lateral dendrite branching compared with injected controls. Therefore, at least in the instances described above, loss of group A genes is epistatic to loss of group B genes (Parrish, 2006).

RNAi of group A genes either promoted or antagonized dendrite arborization; therefore, the effect was examined of simultaneously disrupting one group A gene that promoted and one group A gene that antagonized dendrite outgrowth and lateral branching. RNAi or a loss-of-function mutant of the group A gene ab caused increased dendritic branching and extension of class I dendrites. In addition, mutation of ab caused a significant reduction in the number of class I neurons labeled by Gal4²²¹ that was most pronounced in the dorsal cluster of PNS neurons, consistent with the results from our RNAi experiments. To facilitate epistasis analysis in ab mutants, dendrite arborization effects in vpda, the ventrally located class I neuron, were assayed. RNAi of the group A gene hmgD, which caused reduced primary dendrite outgrowth and reduced lateral branching when injected into wild-type embryos, caused a striking reduction in the number of dendritic branches and size of the receptive field of vpda in ab mutants. RNAi of the group A gene bap55 had similar effects in ab mutants, demonstrating that, at least in some cases, loss of group A genes that results in reduced arborization is epistatic to loss of group A genes that results in increased arborization. Therefore it is possible that the different classes of group A genes antagonistically regulate a common set of target genes required for dendrite arborization (Parrish, 2006).

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date revised: 15 March 2007  

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