BIOLOGICAL OVERVIEW

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).

Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis

The deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. Previous studies on Drosophila melanogaster have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib) and overexpression of the BTB-ZF protein Abrupt (Ab). This study shows that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, the study shows that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis reveals that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems (Turkel, 2015).

This study has shown that over-expression of the Ab BTB-ZF protein cooperates with upregulation of RhoGEF2 or Src64B in tumorigenesis, whereas Ab and DRac1 do not cooperate. Furthermore, expression of Ab with each of these cytoskeletal regulators results in disruption to differentiation, in that the photoreceptor cell marker, Elav, and the early cell fate gene, Dac, are not expressed, although the antennal cell fate gene, Dll, is retained in all except ab Src64B co-expressing clones. Finally, a significant correlations was found in human epithelial cancer datasets between the high expression of BTB-ZF oncogenes, Bcl6 and ZBTB7A, and low expression of Dlg2 or lgl1 cell polarity genes or high expression of ArhGef11, ArhGef12, MAP2K4, MAP2K7, MAPK8, MAPK9, MAPK10, Src or Yes1 cytoskeletal genes. This data suggests that cooperation between these genes may occur in some human epithelial cancers (Turkel, 2015).

RhoGEF2 ab or Src64B ab tumours showed overgrowth during an extended larval period resulting in giant larvae and loss of differentiation. However, unlike scribab tumours there was also non-cell autonomous proliferation and the tumours did not appear to be as invasive as scrib ab tumours, although a more detailed analysis of this is required. By contrast, co-expression of DRac1 and ab did not result in cooperative tumorigenesis, but rather non-cell autonomous proliferation. Relative to the cooperation of these cytoskeletal genes with RasV12, RhoGEF2 or Src64B cooperation with ab showed similar properties. By contrast, DRac1 RasV12 tumours showed strong cell-autonomous overgrowth and invasive properties, whereas DRac1 ab expressing cells did not overgrow relative to wild-type tissue, but instead the surrounding wild-type tissue was induced to overgrow (Turkel, 2015).

The phenomenon of non-cell autonomous overgrowth observed in DRac1 ab mosaic eye-antennal discs (and to some extent in ab RhoGEF2 and ab Src64B mosaic discs) is similar to the effect that 'undead' cells (cells where apoptosis is initiated by activation of initiator caspases, but effector caspase activation is blocked - and thus cell death - by expression of the inhibitor, p35) have upon their surrounding wild-type neighbours. This occurs by the release of Wingless (Wg) and Decapentaplegic (Dpp) and perhaps other morphogens from the undead cells, which promote compensatory proliferation in the surrounding wild-type cells. The similarity of these phenotypes suggests that DRac1 ab expressing cells might be in an 'undead' state, and release Dpp and Wg, thereby inducing proliferative overgrowth of the surrounding wild-type cells. Alternatively, these cells might be deficient in mitochondrial function, which together with expression of a cell-survival factor, such as RasV12, results in non-cell autonomous overgrowth without evidence of caspase activation. In this scenario, the mitochondrial dysfunction results in increased reactive oxygen species (ROS) that activate JNK signalling, which subsequently inactivates Hippo pathway signalling, leading to increased expression of the target genes Wingless and Unpaired (Upd) that activate Wg signalling and Jak/Stat signalling, respectively, in the neighbouring wild-type cells. However, since TUNEL-positive cells in were observed DRac1 ab, RhoGEF2 ab and Src64B ab expressing clones, it is more likely that the first of these mechanisms is responsible for the non-cell autonomous overgrowth, however this requires further investigation (Turkel, 2015).

Interestingly, in undead cells JNK activation is required for Dpp and Wg production and non-cell autonomous overgrowth. Furthermore, strong activation of JNK signalling together with RasV12results in non-cell autonomous overgrowth, although at presumably lower levels of JNK activation, cell autonomous overgrowth occurs. Therefore it is possible that the different effects on non-cell autonomous versus autonomous cell overgrowth in DRac1 ab versus RhoGEF2 abor Src64B ab-expressing cells might depend on the level of JNK activation. Nonetheless, at early stages, ab-driven RhoGEF2, Src64B or DRac1 tumours were similar in inducing non-cell autonomous effects, but at later times the RhoGEF2 ab and Src64B ab-expressing cells showed more predominant autonomous cell overgrowth, whilst the DRac1 ab expressing cells did not, suggesting that there are likely to be molecular differences between DRac1 and RhoGEF2 or Src64B in their cooperative interactions with ab that impact on cell proliferation or survival of the tumour cells (Turkel, 2015).

Profiling of Ab targets and deregulated genes revealed that dac, dan, eya and ct eye-antennal differentiation genes were repressed, along with changes in expression of cell growth/proliferation and survival genes that would be expected to promote tumorigenic growth in cooperation with scrib loss-of-function. scrib ab tumours showed downregulation of Dac, but the antennal cell fate expression domain of Dll was not affected. Similarly, ab expression with either of the cytoskeletal genes resulted in repression of Dac, however Src64B ab tumours additionally repressed Dll, in contrast to DRac1 ab, RhoGEF2 ab and scrib ab tumours where Dll was unaffected. This data suggests that Src64B expression exerts an additional effect on ab-expressing cells to inhibit Dll gene expression and differentiation. Srcupregulation activates the JNK and Stat signalling pathways, affects adherens junction function and represses Hippo signalling. Furthermore, recent studies have shown that overexpression of Src64B in the Drosophila intestinal stem cells can alter differentiation and result in amplification of progenitor cell pools. scrib mutant cells also upregulate JNK, downregulate the E-cadherin/β-catenin adhesion complex and repress Hippo signalling. Furthermore, the Jak/Stat ligand, Upd3, is also upregulated in the scrib cells, where it drives tumour overgrowth, and is also required to activate Jak/Stat signalling in the wild-type neighbouring cells in cell competition. RhoGEF2 and DRac1 also upregulate JNK signalling, and might also repress Hippo signalling to promote tissue growth, since regulators of actin cytoskeletal tension, such as activated Rok and Myosin II regulatory light chain, induce Yki target gene expression. However, in Drosophila it is unknown if RhoGEF2 or DRac1 affect Jak/Stat signalling. Since scrib loss-of-function and Src activation deregulate similar pathways, the precise mechanism by which Src64B cooperates with abto block expression of Dll in the developing eye-antennal disc remains to be determined (Turkel, 2015).

The finding that there was a significant correlation between increased expression of human BTB-ZF oncogenic genes, Bcl6 or ZBTB7A, and downregulation of the cell polarity genes, Dlg2 and Llgl1, or homologs of JNKK(MAPK2K4, MAPK2K7), JNK (MAPK8, MAPK9, MAPK10), RhoGEF2 (ArhGEF11,ArhGEF12) or Src (Yes1, Src) cytoskeletal genes in various epithelial cancers, suggests that the concordant expression of these genes might be contributing to human epithelial cancer initiation and progression. Whilst this study only focused on two of the 47 BTB-ZF genes in the human genome, it raises the question of whether other BTB-ZF genes might also show correlations with the expression of cytoskeletal or cell polarity genes in human epithelial cancers. However, tissue and cancer-grade specific effects might be observed, as a recently published study revealed that ZBTB7A was commonly deleted in late stage oesophageal, bladder, colorectal, lung, ovarian and uterine cancers. Moreover, it was found that low ZBTB7A expression correlates with poor prognosis in colon cancer patients, suggesting that ZBTB7A plays a tumour suppressor function in these cancers. Interestingly, this study also found that in colon cancer xenografts, ZBTB7A represses the expression of genes in the glycolytic pathway, a metabolic pathway that is required for aggressive tumour growth, and that inhibition of this pathway reduces tumour growth. Pertinent to this finding, it was found that blocking glycolytic pathways in Drosophila polarity-impaired tumours, impedes tumour growth without substantially affecting normal tissues, suggesting that downregulation of the Scribble polarity module might upregulate glycolytic metabolic pathways and be dependent on them for tumour growth and survival. It is therefore possible that the cooperation between ab and scrib or cytoskeletal genes in Drosophila may also reflect a need for upregulation of the glycolytic pathway. In human epithelial cancers, the correlations observed between elevated ZBTB7A expression and reduced expression of the Scribble polarity module gene (or high expression of cytoskeletal genes) might also indicate a requirement for glycolytic pathway activation for tumorigenesis. Further studies are clearly required to examine the cooperative effects of Bcl6 or ZBTB7A with deregulated cytoskeletal or cell polarity genes in human epithelial cell lines and mouse models in order to discern whether the findings in Drosophila are indeed conserved in mammalian systems (Turkel, 2015).

Identifying cooperative interactions in cancer is likely to provide novel therapeutic approaches in combating the tumour. Indeed, recently a small molecule inhibitor targeting Bcl6 has been developed, and combining this with a Stat3 inhibitor resulted in enhanced cell killing in triple negative breast cancer cell lines. Since in Drosophila and human cells, Src upregulates Stat activity, tumours showing high Bcl6 and Src or Yes1 expression would be predicted to be sensitive to this combined therapeutic regime. Interestingly, a predominance of the significant correlations that were observed in the human epithelial cancer datasets with either Bcl6 or ZBTB7A involved upregulation of JNKK and JNK family genes. Since JNK signalling is central to many cooperative interactions, inhibiting the JNK pathway in addition to Bcl6 in Bcl6-driven cancers might also be a promising therapeutic approach to combat these cancers. In summary, these functional studies in Drosophila and bioinformatics analysis of human cancers has shown that cooperative tumorigenic interactions occur between BTB-ZF genes and cell polarity or cytoskeletal genes, and warrants further investigation to determine whether restoring normal expression of these genes or downstream pathways in human cancer cells can reduce tumorigenesis (Turkel, 2015).

BTB-zinc finger oncogenes are required for Ras and Notch-driven tumorigenesis in Drosophila

During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers, Drosophila epithelial tumor models were used, that are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors a comparative microarray analysis was used to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, this work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states (Doggett, 2015).

This report has defined the transcriptional changes induced by JNK signaling within both scrib>RasACT and scrib>NACT tumors by carrying out comparative microarray expression arrays. This analysis that JNK exerts a profound effect upon the transcriptional profile of both Ras and Notch-driven tumor types. The expression of nearly 1000 genes was altered by the expression of bskDN in either Ras or Notch-driven tumors, and less than half of these changes were shared between the two tumor types, indicating that JNK signaling elicits unique tumorigenic expression profiles depending upon the cooperating oncogenic signal. Nevertheless, of the 399 JNK-regulated probe sets shared between Ras and Notch-driven tumors, it is hypothesized that these had the potential to provide key insights into JNK's oncogenic activity, and to prioritize these targets, it was considered that the expression of the critical oncogenic regulators would not just be altered by bskDN, but would be normalized to close to wild type levels. This subset of the 399 probe set was identified by comparing the expression profile of each genotype back to control tissue, thereby producing a more focussed JNK signature of 103 genes. Notably, this included previously characterized targets of JNK in the tumors, such as Mmp1,cherand Pax, thereby providing validation of the approach. Also amongst these candidates were 4 BTB-ZF genes; two of which were upregulated by JNK in the tumors (chinmo and fru), and two downregulated (br and ttk) (Doggett, 2015).

Focussing upon chinmo, chinmo overexpression was shown to be sufficient to prime epithelial cells for cooperation with RasACT in both the eye antennal disc and in the adult midgut epithelium, and that chinmo is required for cooperative RasACTor NACT-driven tumor overgrowth, although its function was only exposed when its knockdown was combined with knockdown of a functionally similar BTB-ZF transcription factor, abrupt. This family of proteins is highly oncogenic in Drosophila, since previous work has shown that ab overexpression can cooperate with loss of scrib to promote neoplastic overgrowth, and in these studies, it was also shown that overexpression of a fru isoform normally expressed in the eye disc is capable of promoting cooperation with RasACT and NACT in the eye-antennal disc, in a similar manner to chinmo overexpression. Thus, whether fru also plays a role in driving Ras or Notch-driven tumorigenesis warrants further investigation. Indeed, a deeper understanding of the oncogenic activity of these genes is likely to be highly relevant to human tumors, since of the over 40 human BTB-ZF family members, many are implicated in both haematopoietic and epithelial cancers, functioning as either oncogenes (eg., Bcl6, BTB7) or tumor suppressors (eg., PLZF, HIC1). Furthermore, over-expression of BTB7, can also cooperate with activated Ras in transforming primary cells, and its loss makes MEFs refractory to transformation by various key oncogenes such as Myc, H-rasV12 and T-Ag, suggesting that cooperating mechanisms between BTB-ZF proteins and additional oncogenic stimuli might be conserved (Doggett, 2015).

JNK signaling in Drosophila tumors is known to promote tumor overgrowth through both the STAT and Hippo pathways. Deregulation of the STAT pathway was evident in the arrays through the upregulation of Upd ligands by JNK in both Ras and Notch-driven tumors. In contrast, although cher was identified in the arrays as being upregulated in both tumor types and previous studies have shown that cher is partly required for the deregulation of the Hippo pathway in scrib>RasACT tumors, more direct evidence for Hippo pathway deregulation amongst the JNK signature genes was lacking. In part, this could be due to JNK regulating the pathway through post-transcriptional mechanisms involving direct phosphorylation of pathway components. However, the failure to identify known Hippo pathway target genes, and proliferation response genes in general, may simply highlight limitations in the sensitivity of the array assay and the cut-offs used for determining significance, despite its obvious success in correctly identifying many known JNK targets (Doggett, 2015).

Whether tumor overgrowth through STAT and Yki activity is somehow associated with a stem cell or progenitor-like state remains uncertain. Although imaginal discs exhibit developmental plasticity and regeneration potential, and JNK signaling is required for both of these stem-like properties, there is no positive evidence for the existence of a population of asymmetrically dividing stem cells within imaginal discs. Instead, symmetrical divisions of progenitor cells may be the means by which imaginal discs can rapidly generate enough cells to form the differentiated structures of the adult fly. To date, progenitor cells have only been characterized in the eye disc neuroepithelium. These cells have a pseudostratified columnar epithelial morphology and express the MEIS family transcription factor, Hth, which is downregulated as cells initiate differentiation and begin expressing Dac and Eya. Interestingly, they also require Yki for their proliferation, and can be induced to overproliferate in response to increased STAT activity. However, analysis of cell fate markers indicated that tumor overgrowth was not likley to be solely due to the overproliferation of these undifferentiated progenitor cells. Although scrib>RasACT/NACT tumors, were characterized by the failure to transition to Dac/Eya expression in the eye disc, blocking JNK in scrib > RasACT/NACT tumors did not restore tumor cell differentiation, despite overgrowth being curtailed, and Hth expression was not maintained in the tumors in a JNK-dependent manner. Nevertheless, a JNK-induced gene such as chinmo is likely to be associated with promoting a progenitor-like state, since it is a potential STAT target gene required for adult eye development that is expressed in eye disc progenitor cells in response to increased Upd activity and its overexpression alone is sufficient to block Dac/Eya expression. Furthermore, chinmo is also required for cyst stem cell maintenance in the Drosophila testis, and the current work has shown that chinmo overexpression promotes increased numbers of esgGFP expressing stem cells or enteroblasts in the adult midgut. As the BTB-ZF protein Ab is also highly oncogenic and expressed in the eye disc progenitor cells, it is hypothesize that the JNK-induced expression ofchinmo in scrib>RasACT/NACT tumors could cooperate with Ab to maintain a progenitor-like cell state in the eye disc, and that this is required for scrib->RasACT/NACT tumor overgrowth. However, although Ab was expressed in chinmo-expressing, JNK positive tumor cells, Ab does not appear to be a JNK-induced gene. What JNK-independent mechanisms control ab expression will therefore require further analysis (Doggett, 2015).

Interestingly, previous studies have observed that ab overexpression in eye disc clones upregulates chinmo expression and although the effect of chinmo expression upon ab is yet to be described, the data at least suggest that the control of their expression is interlinked in a yet to be defined manner (Doggett, 2015).

Consistent with Chinmo being important for scrib->RasACT/NACTv tumor overgrowth, chinmo overexpression itself is also highly oncogenic. Over-expression of chinmo with RasACT or NACT drives tumorigenesis in the eye-antennal disc, and also resulted in enlarged brain lobes, presumably due to the generation of overexpressing clones within the neuroepithelium of the optic lobes. In the adult midgut, the overexpression of chinmo with RasACT in the stem cell and its immediate progeny, the enteroblast, promoted massive tumor overgrowth, resulting in esgGFP expressing cells completely filling the lumen of the gut, and eventual host lethality. The luminal filling of esgGFP cells is reminiscent of the effects of RasACT expression in larval adult midgut progenitor cells. Together with the data linking Chinmo function to stem or progenitor cells, these data reinforce the idea that epithelial tumorigenesis can be primed by signals, such as chinmo over-expression, that promote a stem or progenitor cell state (Doggett, 2015).

The function of some Drosophila BTB-ZF proteins including Chinmo and Ab, has also been linked to heterochronic roles involving the conserved let-7 miRNA pathway and hormone signals, to regulate the timing of differentiation. Indeed, Ab can directly bind to the steroid hormone receptor co-activator Taiman (Tai or AIB1/SRC3 in humans), to represses the transcriptional response to ecdysone signaling. Thus, the capacity of BTB-ZF proteins to influence the timing of developmental transitions, particularly if they impede developmental transitions within stem or progenitor cells, could help account for their potent oncogenic activity. Indeed, ecdysone-response genes were repressed by JNK in the tumorigenic state, consistent with the failure of the larvae to pupate and a delay in developmental timing. Whether repressing the ecdysone response cell autonomously might contribute to tumor overgrowth and/or invasion will be an interesting area of future investigation, given the complex role of hormone signaling in mammalian stem cell biology and cancers (Doggett, 2015).

Previous studies have suggested that JNK-dependent tumor cell invasion is developmentally similar to the JNK-induced EMT-like events occurring during imaginal disc eversion. Thus the capacity of JNK to also promote tumor overgrowth is reminiscent of how EMT inducers such as Twist (Twi) and Snail (Sna) are associated with the acquisition of cancer stem cell properties. In Drosophila, however, twi and snawere not induced by JNK in the tumors, although transcription factors involved in mesoderm specification, including the NF-kappaB homologue, dl (a member of the 103 JNK signature), and Mef2 (a member of the 399 JNK signature), were amongst the up-regulated JNK targets. Mesoderm specification is not necessarily associated with a mesenchymal-like cell morphology, however, dl is involved in the induction of EMT during embryonic development, and both dl and Mef2 act with Twi and Sna to coordinate mesoderm formation. Interestingly, recent studies have identified dl in an overexpression screen for genes capable of cooperating with scrib > in Drosophila tumorigenesis, and Mef2 has been identified as a cooperating oncogene in Drosophila, and possibly also in humans, where a correlation exists between the expression of Notch and Mef2 paralogues in human breast tumor samples. It is therefore possible that dl and Mef2 either act in combination with Twi or Sna, or independently of them but in a similar oncogenic capacity, to promote a mesodermal cell fate in scrib > RasACT/NACT tumors. The potential relevance of this to the mesenchymal cell morphology associated with tumor cell invasion, as well as the acquisition of progenitor states is worthy of further investigation (Doggett, 2015).

In mef2-driven tumors both overgrowth and invasion depend upon activation of JNK signaling, suggesting that Mef2 is not capable of promoting invasive capabilities independent of JNK. In contrast, chinmo+RasACT/NACT tumors appeared non-invasive and retained epithelial morphology despite the massive overgrowth, although closer examination of cell polarity markers will be required to confirm this. Furthermore, the overgrowth of chinmo+RasACT/NACT tumors was not dependent upon JNK signaling, suggesting that the maintenance of a progenitor-like state could be uncoupled from JNK-induced EMT-effectors associated with invasion. Whether clear divisions between mesenchymal behaviour and progenitor states in tumors can be clearly separated in this manner is not yet clear, however, overall, it is likely that multiple JNK-regulated genes will participate in both promoting tumor overgrowth as well as migration/invasion. Although this study used the 103 JNK signature as a means to focus upon potential key candidates, an analysis of the 399 JNK-regulated probe sets common to both Ras and Notch-driven tumours has the potential to provide deeper insights into the multiple effectors of JNK signaling during tumorigenesis. Whilst the individual role of these genes can be probed with knockdowns, the complexity of the response, potentially with multiple redundancies and cross-talk, will ultimately need a network level of understanding to more fully expose key nodes participating in overgrowth and invasion. This approach has considerable potential to further expose core principles and mechanisms that drive human tumorigenesis, since it is clear that many fundamental commonalities underlie the development of tumors in Drosophila and mammals (Doggett, 2015).

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).

EVOLUTIONARY HOMOLOGS

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. The metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum was studied RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, the prediction was tested that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, the hypotheses was tested for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. The data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles (Angelini, 2012).

In D. melanogaster, Dll mutants lack maxillary structures and portions of the proboscis (i.e., labium), although Dll expression in the maxillary anlagen is weaker than in the leg or antennal discs. Paralleling the results for T. castaneum, in the horned beetle Onthophagus taurus distal regions of the adult mouthparts were deleted with larval Dll RNAi (Simonnet 2011). The embryonic and metamorphic functions of Dll in T. castaneum are also similar: the gene is required for the development of distal structures at both stages, and during embryogenesis Dll is expressed throughout the developing palps. Interestingly, removal of T. castaneum Dll expression earlier during larval life led to delayed metamorphosis, as well as changes in appendage morphology (Suzuki, 2009). Many insects delay molting after appendage loss to allow time for regeneration, and this dual role of Dll suggests a mechanism linking these processes (Angelini, 2012).

The data from T. castaneum provide evidence for a conserved gap gene role of dac during patterning of mouthparts and legs of this species. dachshund is not expressed in or required for development of the labial and maxillary anlagen of D. melanogaster. In T. castaneum embryos dac is expressed strongly in the proximal maxilla and part of the developing endite. Embryonic dac expression is weaker in the distal maxillary palp and the labium. The current data show a clear metamorphic requirement for dac in the intermediate regions of the maxillary and labial palps, as does a recent study of O. taurus (Simonnet, 2011). A function for dac in the development of an intermediate portion of the maxillary and labial appendages has so far only been observed in these two beetles, while data from two species with specialized mouthparts (the milkweed bug O. fasciatus and D. melanogaster) found that dac is not required for PD patterning of the mouthparts. Thus, comparative data from other species do not support the hypothesis that this mouthpart patterning role is ancestral. However, if mandibulate mouthparts evolved from leg-like structures similarities in the expression and function of genes patterning both legs and mouthparts are expected to be plesiomorphic. This hypothesis can be further tested by examining the role of dac in mouthpart development in additional insect orders, particularly those that retain mandibulate mouthparts, and in other arthropods (Angelini, 2012).

The effects of hth depletion are distinct in different species, but typically involve some degree of homeotic transformation. In D. melanogaster, hth is expressed in the labial discs, but without nuclear expression of its cofactor Extradenticle. Maxillary palps are retained in hth loss-of-function flies, but they may possess bristles typical of legs, indicating a partial proboscis-to-leg transformation. In the cricket Gryllus bimaculatus, which has mandibulate mouthparts, hth depletion causes transformation of proximal mouthpart structures towards antennal identity, with a loss of endites, while distal structures are transformed towards leg identity (Ronco, 2008). hth RNAi in T. castaneum transformed intermediate regions of the maxilla and labium towards distal mouthpart identity. Proximal regions also appeared transformed, but their identity could not be established, while distal regions appeared wild type. In the beetle O. taurus, proximal regions of the labium are transformed towards maxillary endite identity, but distal regions of the labium and the entire maxilla remain relatively unaffected (Angelini, 2012).

These results highlight the similarity between patterning of the maxilla, labium and legs in T. castaneum. Functional data from two species with highly derived mouthpart morphologies, D. melanogaster and the milkweed bug Oncopeltus fasciatus, suggest only limited similarity between mouthpart and leg patterning. One explanation for this low degree of conservation is that evolution of the ancestral patterning mechanism has occurred in concert with the functional and morphological diversification of these mouthparts. A correlation between generative mechanisms and structural morphology has been used as a common null hypothesis, although exceptions in which similar morphologies result from different developmental pathways are documented. Nevertheless, this hypothesis predicts that developmental patterning should be more highly conserved across appendage types in species that retain the ancestral mandibulate mouthpart morphology (Angelini, 2012).

The maxillary and labial palps are an interesting case of serial homology. Despite a difference in overall size, their shape and arrangement of sensillae are similar. The intermediate segments of each palp type are also similar, but differ in number, which suggests that segment number is regulated independently from other morphological traits. The RNAi depletion of pdm in T. castaneum caused the reduction and deletion of the third maxillary palp segment, producing a phenotype closely resembling the wildtype morphology of the labial palps. While a role for pdm in the labium cannot be excluded, the absence of observed labial phenotypes was significant compared to maxillary results. Therefore, it is hypothesized that the difference in the number of palp segments results from specific activation of pdm in the maxillary palp. Loss of function in the Hox gene Deformed during T. castaneum embryogenesis causes a transformation of the larval maxillae towards labial identity. Since Hox genes are the primary determinants of body segment identity, it is proposed that pdm is activated by Deformed, and repressed by the labial Hox gene Sex combs reduced. RNAi targeting pdm in another mandibulate insect, the cricket Acheta domesticus, generated defects in the antenna and legs, but no defects in the mouthparts, despite similar pdm expression in these appendages (Turchyn; 2011; Angelini, 2012).

Endites are a primitive component of arthropod appendages, and they are retained in insect mouthparts, as well as in the mouthparts and thoracic appendages of many crustaceans (Boxshall 2004). At least three hypotheses have been put forward for how endites are patterned, and these hypotheses are not mutually exclusive. The first hypothesis states that multiple PD axes result from redeployment of a PD axis patterning mechanism shared by palps and endites. A second hypothesis posits that endites and appendage segments form by the same mechanism, Notch-mediated in-folding of the cuticle. A third hypothesis states that dac expression initiates endite branching from the main appendage axis. The axis redeployment hypothesis predicts that depletion of genes involved in PD axis patterning will have similar effects on the development of palps and endites. Some support for this hypothesis comes from studies of endite morphogenesis and the expression and function of leg gap genes in the embryos of T. castaneum and the orthopteran Schistocerca americana, but not all data are consistent with it. The segmentation hypothesis predicts that endites will fail to differentiate if genes required for joints are depleted. This hypothesis was posed based on a comparative developmental study of segmented and phyllopodous crustacean limbs. Finally, the dac-mediated hypothesis predicts that depletion of dac will lead to reduced endites. This hypothesis emerged from the observation that dac expression is reiterated along the medial edges of larval endites in the crustacean Triops longicaudatus. Comparative expression data from the isopod Porcellio scaber are also consistent with the dac-mediated hypothesis (Angelini, 2012).

The current data are consistent with predictions of the axis redeployment and segmentation hypotheses but do not support a role for dac in endite metamorphosis. Adult endites were disrupted by depletion of Dll, Krn, the odd-related genes, and Notch signaling, and to a lesser degree hth. In the maxilla depletion of most of these genes led to the failure of the single larval endite to divide into two distinct branches, while in the labium, their depletion caused reduction of the ligula. Their requirement in the endites is consistent with the hypothesis that these structures are generated by redeploying appendage PD axis determinants. Depletion of Notch signaling components and the odd paralogs produced reductions and fusions between palp segments, between the palps and endites, and between the lacinia and galea. Thus, these data are compatible with both the hypothesis that a reiterated PD axis is used to pattern the endites and the hypothesis that endite formation is linked to joint formation. Normal endite development in dac-depleted specimens is inconsistent with the dac-mediated hypothesis (Angelini, 2012).

It is noteworthy that endite specification and the division of the single larval endite into the adult galea and lacinia appear to be separable functions. For example, Ser RNAi resulted in a single endite lobe with lacinia identity medially and galea identity laterally. In contrast, severe Dll RNAi individuals had a single endite that lacked also obvious lacinia identity (Angelini, 2012).

The mandibulate structure of Tribolium mouthparts is the pleisomorphic state for insects and is shared by a majority of insect orders. These mouthparts are characterized by robust mandibles, lacking segmentation. A classic debate in arthropod morphology concerns whether the mandibles of insects and myriapods are derived from a whole appendage or only from proximal appendage regions; the latter are called gnathobasic mandibles. Palps are retained on the mandibles of many crustaceans, making it clear that the biting regions of their mandibles are gnathobasic. Phylogenetic support for the gnathobasic hypothesis comes from phlyogenetic studies that place insects nested within crustaceans (Regier, 2010). The first developmental genetic support for the gnathobasic hypothesis came from the discovery that insect mandibles lack Dll expression. Furthermore, neither mutations in Dll nor its depletion through RNAi have been observed to alter mandible development in insects, including T. castaneum. This evidence has led to widespread acceptance of the gnathobasic hypothesis. Of the 13 genes depleted in this study, two (Krn and hth) produced results that would not be predicted by the most straightforward form of the gnathobasic hypothesis for mandible origins (Angelini, 2012).

Loss of EGF function in insects leads to distal appendage defects, including pretarsal or tarsal deletions. The role of EGF signaling in distal appendage regions is conserved in T. castaneum metamorphosis, since depletion of the EGF ligand Krn leads to reduction of the antennal flagellum, and maxillary and labial palps, as well as to deletion of the pretarsus and malformation of the tarsus. In light of the restriction of Krn’s role to distal appendage regions and regulation of distal EGF ligand expression by Dll in D. melanogaster, the gnathobasic hypothesis predicts that Krn should not be required for normal development of the mandible in T. castaneum. In contrast to this prediction, depletion of Krn produced a significant reduction in mandible length (Angelini, 2012).

The hypothesis of a gnathobasic mandible also predicts that hth depletion should produce effects in the mandible similar to those in the proximal regions of other appendage types. In T. castaneum, hth RNAi during metamorphosis caused homeotic transformation of proximal regions of the maxilla, labium and legs. However, the mandibles were not affected by hth depletion. In the beetle O. taurus, hth depletion slightly altered mandible shape, but also without apparent homeosis. In contrast, hth RNAi in embryos of the cricket G. bimaculatus transformed the mandible towards a leg-like structure distally and an antenna-like structure proximally, paralleling the transformation observed in other appendages. Because these results come from only two lineages and from different life stages, additional data are needed to determine whether a homeotic role for hth was present ancestrally in insect mandibles (Angelini, 2012).

These data must be weighed alongside other evidence bearing on the gnathobasic hypothesis. In T. castaneum, the lack of phenotypic effects on mandible metamorphosis of other genes in this study is consistent with the gnathobasic hypothesis. In particular, it was observed that mandible metamorphosis was normal following depletion of genes involved in distal growth and patterning or joint formation. Moreover, homology at one biological level, such as anatomy, does not preclude divergence at other levels, such as development. Nevertheless, since developmental genetic studies of Dll and other appendage-patterning genes have been used as strong support for the gnathobasic homology of the insect mandible, the findings of Krn function highlight the difficulties in establishing serial homology based solely on developmental data (Angelini, 2012).

This study provides a genetic model of adult mouthpart development in Tribolium castaneum based on 13 genes. While previous studies have examined patterning in species with derived mouthpart morphologies, T. castaneum retains the pleisomorphic, mandibulate state of insect mouthparts. These results demonstrate the conservation of many gene functions in the maxilla and labium, relative to the legs, thus supporting the interpretation of novel gene functions in groups with derived mouthpart morphology as indicative of their specialized morphogenetic roles in those species. Mandibulate mouthparts such as those of T. castaneum include medial maxillary and labial endites, and the current data are consistent with hypotheses of reiteration in the PD axis and specification by Notch signaling, but rule out a direct role for dac in branch generation or patterning at metamorphosis. Additionally these results demonstrate that a regulator of distal leg development, Krn, which encodes an EGF ligand, is required for normal mandible elongation. This finding underscores the complex relationship between homology at the levels of anatomy and developmental patterning (Angelini, 2012).

REGULATION

Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs: Let-7 regulates Abrupt

The let-7 and lin-4 microRNAs belong to a class of temporally expressed, noncoding regulatory RNAs that function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the rapid evolution of new morphologies. let-7 is highly conserved and in Drosophila is temporally coregulated with the lin-4 homolog, miR-125. Little is known, however, about their requirement outside the nematode or whether they universally control the timing of developmental processes. A Drosophila mutant that lacks let-7 and miR-125 activities has been generated; these mutants have a pleiotropic phenotype arising during metamorphosis. Loss of let-7 and miR-125 results in temporal delays in two distinct metamorphic processes: the terminal cell-cycle exit in the wing and maturation of neuromuscular junctions (NMJs) at adult abdominal muscles. The abrupt (ab) gene, encoding a nuclear protein, was identifed as a bona fide let-7 target, and evidence is provided that let-7 governs the maturation rate of abdominal NMJs during metamorphosis by regulating ab expression. It is concluded that Drosophila Iet-7 and miR-125 mutants exhibit temporal misregulation of specific metamorphic processes. As in C. elegans, Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit, indicating that its function as a heterochronic microRNA is conserved. The ab gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. These results suggest that let-7 and miR-125 serve as conserved regulators of events necessary for the transition from juvenile to adult life stages (Caygill, 2008).

The results indicate that in Drosophila, loss of the let-7 and miR-125 microRNAs leads to numerous defects that alter the morphology and behavior of adult flies. The expression of these microRNAs is restricted to the metamorphic stage of development, and both loss and premature expression of let-7 and miR-125 are detrimental to animal survival, indicating that temporal restriction of their expression is essential. Many defects in the mutants affect sensory and motor behaviors; the results indicate that neuromuscular connectivity in abdominal muscles is severely hampered and that there are direct consequences on the rate of adult eclosion. Both the NMJ and eclosion defects result from deregulated expression of a single let-7 target in abdominal muscles. Control of NMJ growth represents a new regulatory role for microRNAs in general, and given the extensive neurological problems of let-7, miR-125 mutants, it is possible that synapse development is broadly regulated by these particular micoRNAs (Caygill, 2008).

The data clearly implicate regulation of ab by let-7 as a critical factor in the rate of NMJ development during Drosophila metamorphosis. The aberrant persistence of Ab in muscle nuclei in the mutant significantly slows NMJ development, perhaps by interfering with synapse-strengthening signals exchanged between muscle and motoneurons. Because Ab expression is widespread during Drosophila development, the need for its downregulation late in development is in striking contrast to the requirement for Ab at earlier stages: in muscle for neuromuscular targeting and for epidermal attachment of specific muscle groups and in a class of embryonic and larval sensory neurons to limit dendritic branching. Based on its dose sensitivity, Ab has been proposed to function in a concentration-dependent manner. By examining the expression of Ab protein directly, it was found that Ab is downregulated to nearly undetectable levels in most abdominal-muscle nuclei by the pharate adult stage. Its decline in the myoblasts may begin as early as 26 hr APF, a time when let-7 expression is rising. However, the experiments indicate that deregulation of Ab cannot account for all of the mutant defects, indicating that other targets of let-7 and/or miR-125 must function as effectors for these processes. To date, Ab is the only target of Drosophila let-7 to be verified in vivo, and no miR-125 targets have been authenticated (Caygill, 2008).

In addition to its role in NMJ maturation, the results implicate let-7 in a wing defect that causes cells to continue to divide after wild-type wing cells have exited the cell cycle. This phenotype is strikingly similar to the reiterated divisions of hypodermal blast cells in C. elegans let-7 mutants. In both cases, the extra divisions do not continue indefinitely, nor do they occur in all tissues. Little is known about the mechanism of cell-cycle regulation by let-7 in either organism. In C. elegans, persistent expression of lin-41, encoding a RBCC (ring B-box coiled-coil) protein, in let-7 mutants accounts for much of the mutant phenotype. Drosophila homologs of lin-41 include dappled (shown to be a let-7 target in cell-culture assays: O'Farrell, 2008) and brat (brain tumor), a translational inhibitor. brat encodes a potent tumor suppressor whose absence causes metastatic brain tumors and that is predicted by one computational program to contain let-7 binding sites. Interestingly, overexpression of brat suppresses wing growth. Thus, a plausible hypothesis for the wing defect in let-7, miR-125 mutants is that loss of let-7 simultaneously deregulates a cell-cycle regulator(s) and brat and that the former drives additional cell divisions in the wing while the latter suppresses growth (Caygill, 2008).

The expression of many genes required during larval stages is downregulated during metamorphosis, in part because their presence during pupal development hinders the ordered progression of events such as the primary and secondary responses to ecdysone, the hormone that controls insect metamorphosis. Thus, a plausible role for let-7 and miR-125 is to rid cells of unnecessary larval mRNAs quickly at the transition to pupal development; such a role might be similar to the clearing of maternal mRNAs at the maternal-zygotic transition as recently demonstrated in zebrafish and in Drosophila. Heterochrony facilitates rapid evolution of new morphologies through changes in the timing of developmental processes. The pleiotropic phenotype associated with let-7, mir-125 mutants suggests that each microRNA contributes to multiple processes during metamorphosis, and the identification of additional targets of both is therefore an important future quest that should clarify their contribution to developmental timing and morphological evolution (Caygill, 2008).

In conclusion this study has generated a mutant of Drosophila Iet-7 and miR-125 that exhibits temporal misregulation of specific metamorphic processes. Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit and thus functions as a conserved heterochronic microRNA. The abrupt gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. let-7 and miR-125 could serve as conserved regulators of processes necessary for the transition from juvenile to adult life stages (Caygill, 2008).

Border-cell migration requires integration of spatial and temporal signals by the BTB protein Abrupt

During development, elaborate patterns of cell differentiation and movement must occur in the correct locations and at the proper times. Developmental timing has been studied less than spatial pattern formation, and the mechanisms integrating the two are poorly understood. Border-cell migration in the Drosophila ovary occurs specifically at stage 9. Timing of the migration is regulated by the steroid hormone ecdysone, whereas spatial patterning of the migratory population requires localized activity of the JAK-STAT pathway. Ecdysone signalling is patterned spatially as well as temporally, although the mechanisms are not well understood. In stage 9 egg chambers, ecdysone signalling is highest in anterior follicle cells including the border cells. abrupt was identified as a repressor of ecdysone signalling and border-cell migration. Abrupt protein is normally lost from border-cell nuclei during stage 9, in response to JAK-STAT activity. This contributes to the spatial pattern of the ecdysone response. Abrupt attenuates ecdysone signalling by means of a direct interaction with the basic helix-loop-helix (bHLH) domain of the P160 ecdysone receptor coactivator Taiman (Tai). Taken together, these findings provide a molecular mechanism by which spatial and temporal cues are integrated (Jang, 2009).

Embryonic development unfolds as a series of changes in gene expression that are regulated in both space and time. The fundamental mechanisms of spatial patterning have been established. Temporal patterns of gene expression can be regulated globally by circulating hormones or locally by the sequential actions of transcription factors on one another. What remains to be elucidated are the mechanisms by which spatial and temporal patterns are integrated. This study identifies Abrupt as playing such a part in border cells (Jang, 2009).

The following model is proposed for the molecular integration of spatial and temporal control of border cell migration. Early in stage 9 the ecdysone titer begins to rise. Although the precise pattern in which it is produced is not known, it may be uniform. At this stage, EcRB1 expression is enriched in anterior follicle cells, leading to an enhanced ecdysone response in these cells. In response to ecdysone signaling, the levels of Abrupt protein begin to fall in anterior follicle cells, leading to a feedback amplification of the ecdysone response in those cells, further reduction in Abrupt protein levels and thus a gradually decreasing level of nuclear Abrupt throughout stage 9. Since the asymmetry in EcRB1 expression is transient, this feedback mechanism is necessary to maintain the spatially localized effect in the absence of the initiating event. Abrupt protein levels also decrease in response to JAK/STAT signaling, which is sustained and highest in border cells (Jang, 2009).

The gradual decrease in the concentration of Abrupt in border cell nuclei due to the combined action of ecdysone signaling and JAK/STAT leads to a gradual increase in ecdysone signaling throughout stage 9, producing a temporal gradient. The gradual nature of the effect may serve as a buffer against any excessively rapid increase in the ecdysone concentration that might occur. As was shown in Tai(βB) overexpression, very high levels of ecdysone signaling are not compatible with border cell migration and may even serve as a stop signal since the highest level of ecdysone reporter expression occur at stage 10, which is the stage at which border cells stop migrating (Jang, 2009).

Two other BTB domain proteins that function in developmental timing are Chinmo (Chronologically inappropriate morphogenesis) and BrC (Broad Complex). These factors contribute to the temporal sequence of neuronal cell fates during postembryonic development. Early neuronal precursors express high levels of Chinmo, which subsequently decay by a post-transcriptional mechanism. Loss of Chinmo from early neuroblasts converts their progeny to later identities whereas over-expression of Chinmo has the opposite effect. In the developing larval CNS, early born neurons express higher levels of Chinmo and later born cells express BrC in a largely complementary pattern. Thus temporal regulation may be a general property of proteins containing both BTB and Zn2+ finger domains (Jang, 2009).

Could the three temporal control mechanisms that have been studied largely separately, actually represent a single unified mechanism? The work presented here demonstrates that Abrupt protein levels respond to ecdysone signaling and in turn affect the ecdysone response in the Drosophila ovary. Very recently, Abrupt has also been shown to be a direct target for the let-7 microRNA in larval muscle cells. Drosophila let-7 is homologous to one of the original miRNAs identified as a heterochronic gene in C. elegans. Drosophila let-7 is widely expressed in ecdysone-responsive tissues including ovaries and like ecdysone, is required for metamorphosis and for female (not male) fertility. Drosophila let-7 expression may require EcR or ecdysone and let-7 may function in parallel pathways to regulate developmental timing. The border cells now represent a well-developed model in which spatial and temporal control can be examined at the single cell level so that precise molecular mechanisms can be unraveled. Further investigation of this model system could help determine whether hormone, microRNA and BTB domain transcription factors are all part of one unified developmental timing pathway or function in parallel (Jang, 2009).

Sensory-neuron subtype-specific transcriptional programs controlling dendrite morphogenesis: genome-wide analysis of Abrupt and Knot/Collier

The transcription factors Abrupt (Ab) and Knot (Kn) act as selectors of distinct dendritic arbor morphologies in two classes of Drosophila sensory neurons, termed class I and class IV, respectively. Binding-site mapping and transcriptional profiling of these isolated neurons were performed in this study. Their profiles were similarly enriched in cell-type-specific enhancers of genes implicated in neural development. A total of 429 target genes were identified, of which 56 were common to Ab and Kn; these targets included genes necessary to shape dendritic arbors in either or both of the two sensory subtypes. Furthermore, a common target gene, encoding the cell adhesion molecule Ten-m, was expressed more strongly in class I than class IV, and this differential was critical to the class-selective directional control of dendritic branch sprouting or extension. These analyses illustrate how differentiating neurons employ distinct and shared repertoires of gene expression to produce class-selective morphological traits (Hattori, 2013).

The transcriptional programs studied in this paper were predicted to be more specialized for controlling neuronal terminal differentiation at postmitotic stages, compared to those in which proneural genes, such as Asense and its vertebrate homolog Ascl1 (Mash1), regulate cell proliferation or cell-cycle arrest and also promote differentiation. Indeed, predicted molecular functions of Ab and Kn target-coded proteins are diverse, ranging from transcriptional control to cell adhesion, membrane trafficking, Ca2+ entry, and cytoskeleton regulation. Then how do these targets contribute to shaping dendrite arbors in a class-selective fashion? This genome-wide study strongly supports the notion that the class selectors do indeed control transcription of target genes selectively. In contrast, both TFs have chromatin features of the binding sites in common and show the same directional (up or down) regulation of every common target. To explain these findings, the possibility was intriguing that some common targets might be regulated by the two TFs in quantitatively differential fashions. As a precedent, Cut (Ct) is differentially expressed among the three classes (class II-IV), which controls formation of the different branching patterns and the growth of dendritic arbors of individual classes. In this study, compelling data for the above hypothesis was obtained by analyses of a common target, Ten-m. Its high-level expression in class I ddaE endowed its branches with the capability to respond to the decreasing level of Ten-m in the epidermis, thus setting the directional preference of branch sprouting. In contrast, a much lower expression in class IV ddaC ensured the directing of terminal branches rather radially in the distal area of each arbor, where the overlying epidermal Ten-m expression is low. These level-dependent roles of Ten-m could be related or analogous to a role of mouse Ten-m3 in navigating Ten-m3-high retinal projections to the high target region and those of tenurins in instructing synaptic partner matching in the Drosophila olfactory map. It awaits further study to reveal how the differential levels of Ten-m produce the class-selective directional properties of branch patterning, possibly by way of organization of cytoskeletons and membranes (Hattori, 2013).

The other experimental results are also consistent with the critical role of the quantitative control of target gene expression. First, the amount of Lola, one common target-gene product, was higher in class I ddaE than class IV ddaC in a wild-type background. Second, results of knockdown and overexpression of IGF-II mRNA-binding protein (Imp) and lola indicate that their expression levels must be strictly controlled to determine the arbor complexity. Third, there were superficially puzzling findings about downregulated targets (those with decreased expression upon ab or kn misexpression). In the narrowed-down list of Ab target genes, ten targets were downregulated by ab misexpression, and their knockdown in class I (which expresses Ab endogenously) yielded obvious abnormal phenotypes. Ab may keep the transcription of the downregulated targets weakly active and does not totally shut down the expression; moreover, this low-level expression may be required for normal class I development. To test these hypotheses, what would be required is class-selective quantitative expression profiling, ideally at multiple developmental stages, including the onset of primary dendrite formation and a subsequent branch growing phase (Hattori, 2013).

A total of 85% or more of the bound genes were not identified as exclusively Ab and/or Kn-dependent genes; and it could be that Ab and Kn may be able to control transcription of some of those in conjunction with other TFs. Candidate-bound genes of this group include Kn-bound genes Ubx and abd-A that are silenced in class IV by PcG proteins, which showed a similar binding profile with Kn, while an unknown transcriptional coactivator may drive expression of turtle, which is an Ab/Kn-bound gene necessary in class I and class IV. Furthermore, with respect to physiological functions of proprioceptive class I and multimodal nociceptive class IV, it should be mentioned that Gr28b encoding a bright blue light sensor was a Kn-bound gene. Additional profiling data sets, such as that in the copresence of Kn and Ct, will deepen understanding of the intricate transcription codes, with the ultimate goal of identifying the molecular links between the codes and the diverse architectures of dendritic arbors and neuronal functions (Hattori, 2013).

Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster

Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. This study reports critical roles for lin-28 during oogenesis. let-7 maturation was shown to be increased in lin-28 null mutant fly ovaries. lin-28 null mutant female flies display reduced fecundity, due to defects in egg chamber formation. More specifically, in mutant ovaries, the egg chambers were shown to fuse during early oogenesis resulting in abnormal late egg chambers. This phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. A model is suggested in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. These results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation (Stratoulias, 2014).

The Cold-Shock Domain (CSD) protein Lin28 was initially identified in Caenorhabditis elegans (C. elegans) as a component of the heterochronic pathway that regulates the timing of cell fate specification (Ambros, 1984). Subsequent discovery of gene expression regulation through small non-coding RNAs clarified the role of Lin28 in this pathway. The lin-28 mRNA is a conserved target of the let-7 micro-RNA (miRNA) family both in C. elegans and vertebrates. On the other hand, Lin28 inhibits let-7 processing. At the molecular level, Lin28 protein interacts with the let-7 precursor (pre-let-7), resulting in inhibition of let-7 maturation. The let-7 inhibition occurs through the physical interaction of the pre-let-7 loop and Lin28 protein, preventing further processing of pre-let-7 towards the mature form of let-7. Together, these interactions create a feedback loop between Lin28 and let-7, leading to a strict regulation of let-7 maturation (Stratoulias, 2014 and references therein).

Lin28 raised further interest when it was used, along with Nanog, to replace the factors c-Myc and Klf4 in somatic cell reprogramming. These experiments, together with data from human embryonic stem cells, underscored the important role of lin-28 in pluripotency regulation and maintenance. Besides acting as a negative regulator of let-7 maturation, Lin28 has also been shown to have a direct effect on translation through the recruitment of the RNA Helicase A. This mode of function, independent of let-7 maturation, has been demonstrated in the case of Insulin-like Growth Factor 2 during mouse myogenesis. Lin28 binding on IGF-2 mRNA increases its translation efficiency and therefore facilitates skeletal myogenesis in mice (Stratoulias, 2014 and references therein).

The Lin28 protein is composed of four domains: a positively charged linker that binds two Cys-Cys-His-Cys (CCHC)-type zinc-binding motifs to the CSD. In mammalian genomes, two paralogs of lin-28 are found, Lin28A and Lin28B. While Lin28B represses let-7 processing in the nucleus to prevent the formation of the precursor form from the primary let-7, Lin28A also blocks cytoplasmic processing of let-7 (Piskounova, 2011). It has recently been shown in mouse that deletion of the Lin28 linker domain alters the protein’s three-dimensional structure and is sufficient to disrupt sequestration of the precursor form of let-7 (pre-let-7) (Stratoulias, 2014).

The miRNA let-7 family is conserved across diverse animals, functioning to control late temporal transitions during development. During the last decade, the involvement of let-7 in regulating cell differentiation has been analyzed in various contexts, including neural cell specification, stem cell maintenance and hematopoietic progenitor differentiation. While eight different let-7 miRNA genes are annotated in the human genome, only one is found in Drosophila melanogaster. Like in C. elegans, in Drosophila the loss of let-7 expression leads to the modification of temporal regulation of the metamorphosis process. During fly metamorphosis, the expression of let-7 complex (let-7C), a polycistronic locus encoding the let-7, miR-100 and miR-125 miRNAs, is under direct control by the steroid hormone ecdysone. Ecdysone is the central regulator of insect developmental transitions. Therefore, let-7 has been proposed to be part of a conserved, ecdysone regulated pathway that controls the timing of the larva to adult transition (Stratoulias, 2014).

In addition to affecting the metamorphosis clock, Sokol and colleagues have shown that the let-7 deletion also affects the neuromuscular remodeling that takes place during the larva to adult transition. During neuromuscular remodeling, and under normal conditions, the dorsal internal oblique muscles (DIOMs) disappear 12 hours after emergence of the adult fly from the pupa. However, the adult let-7 mutants retain the DIOMs through adulthood. Deletion of the let-7 gene is sufficient to induce this phenotype, while deletion of either miR-100 or miR-125 genes is not enough to recapitulate the DIOM phenotype. Furthermore, let-7 has been shown to govern the maturation of neuromuscular junction of adult abdominal muscles, through regulation of Abrupt expression (Stratoulias, 2014 and references therein).

While previous studies have demonstrated that the let-7 target Abrupt and ecdysone signaling are required for oogenesis in fruit fly ovaries, and that the let-7 miRNA family is abundantly expressed both in newborn mouse ovaries and in fly ovaries, no study has been conducted on the role of Lin-28/let-7 network in Drosophila ovaries. Therefore, a study was undertaken of the effects of lin-28 during Drosophila melanogaster development from the egg to the adult, and more particularly during oogenesis (Stratoulias, 2014).

A lin-28 mutant was generated, and the consequent increase of let-7 maturation was validated. lin-28 knockout resulted in reduced muscular performance and defects in DIOM morphogenesis. These results were in line with the let-7 knock out muscular phenotype described earlier. Moreover, this study identified multiple defects during oogenesis due to abnormal follicle and germline stem cell (FSCs and GSCs respectively) differentiation. A link is proposed between ovarian defects and ectopic expression of Fasciclin2 (Fas2), a known downstream target of the Ecdysone pathway, and a predicted let-7 target (Stratoulias, 2014).

Because of their role during stem cell differentiation, members of the let-7 miRNA family have been extensively studied. However, the role of lin-28 is still poorly documented. Deletion of let-7 in Drosophila impairs the musculature remodeling during the larva to adult metamorphosis. For instance the DIOMs, muscles which are required for eclosion and which are lost within 12 hours after eclosion, they are maintained during adulthood upon let-7 deletion. By generating the first lin-28 deletion in flies, this study has successfully confirmed the involvement of Lin-28/let-7 regulatory network in DIOM remodeling. This study has shown that deletion of lin-28 leads to over maturation of let-7, which negatively affects, and sometimes prevents DIOM formation. This drastic phenotype leads to a suboptimal muscular phenotype. However, due to a variable penetrance of the lin-28 deletion phenotype, a proportion of the flies could eclose and live as fertile animals (Stratoulias, 2014).

In addition, a link was discovered between Lin-28 function and oogenesis. The data indicates a role of let-7 during GSC differentiation and egg chamber formation. Because of the importance of these processes, let-7 maturation has to be strictly regulated by Lin-28 activity. It is suggested that a potential network involving Lin-28/let-7/Ecdysone signaling/Abrupt/Fas2 is needed during GSC differentiation and BC migration. The role of Abrupt in downregulating the steroid hormone Ecdysone has previously been demonstrated. Indeed, the loss of Taiman, a target of the transcription factor Abrupt and co-activator of Ecdysone receptor, leads to an increase of undifferentiated GSCs in the germarium due to disruption of Ecdysone signaling. Therefore, by regulating the expression pattern of Abrupt, Lin28/let-7 may adjust the domain of Ecdysone activity, providing a control over the GSCs differentiation and egg chamber maturation during the oogenesis. Indeed, it has been shown that the Ecdysone titre rises during oogenesis at stage 9. While the precise Ecdysone expression pattern is not known, it is suggested that the uniform EcR expression pattern in follicle cells in lin-28 mutants may break the Ecdysone signaling asymmetry needed during proper oogenesis (Stratoulias, 2014).

Furthermore, a previous study demonstrated the activation of let-7 expression via Ecdysone activity. This study showed that lin-28 deletion, resulted in the alleviation of Lin28's inhibitory role on let-7 maturation. This led to loss of Abrupt, which in turn inhibited Ecdysone activity and maintained Fas2 expression, resulting in BC migration impairment. To test whether the increase of Ecdysone signaling amplifies let-7 expression through a positive feedback loop, a system was generated in which there is no control of either let-7 expression nor of Ecdysone activity. This situation leads to an early cyst fusion, a loss of proper GSC differentiation and a mitotic defect, as was observed in the homozygous lin-28^dF30 ovaries. The accumulation of these defects may be enough to trigger apoptosis at mid-oogenesis, a well-known checkpoint previously described (Stratoulias, 2014).

Interestingly, the variable penetrance of the phenotype allows proper oogenesis and appearance of subfertile adult flies. This suggests a robust molecular network where feedback loops can rescue the system if one component disturbs the balance (Stratoulias, 2014).

By combining these results with previously published studies, a conserved link is suggested between hormonal signaling and germline stem cell differentiation, involving the let-7 miRNA family. This suggestion is reinforced in the last couple of years by the discovery of dormant ovarian follicles and mitotically active germ cells in adult mammalian ovaries, which are responsive to gonadotropin hormone. Moreover, it has been demonstrated that Lin-28 is involved in germline stem cell regulation in human ovary and in the ovarian surface epithelium of severe ovarian infertility patients axonal projection is critical for assembly of a functional sensory circuit (Stratoulias, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

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 the 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).

Centrosomin represses dendrite branching by orienting microtubule nucleation

Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

Neurons primarily receive inputs through their dendrite arbors. The shape and complexity of the dendrite arbor, which is elaborated during differentiation, enables the neuron to properly cover its receptive field and establishes the positions of inputs into the arbor. Disruptions to dendritic branching can precipitate intellectual disability and psychiatric disorders (Yalgin, 2015).

Arbor morphology is regulated for each neuron class to support its structural and functional requirements3; it is genetically encoded, being linked to class specification by transcriptional programs. For example, in Drosophila, the single unbranched dendrite of external sensory neurons is specified over an alternative multipolar dendritic arborization (da) neuron fate by the Prdm transcription factor Hamlet. Similarly, the proneural transcription factor Ngn2 regulates multiple aspects of pyramidal neuron development in the mammalian cortex, including the specification of a characteristic apical dendrite, whereas Cux1, Cux2 and SatB2 link dendrite development to cortical layer-specific developmental program (Yalgin, 2015).

Dendrite development is controlled in a neuron class-specific manner to create differences in arbor morphology and complexity. Class-specific dendrite targeting is regulated via the activity of transmembrane adhesion proteins. For example, in C. elegans, class-specific expression patterns of the transcription factors MEC-3, AHR-1 and ZAG-1 regulate the morphology of mechanosensory neurons, and MEC-3 promotes differential expression of the Claudin-like membrane protein HPO-30 to enable lateral branch stabilization. Drosophila da neurons exist in four classes, of which class I neurons express Abrupt (Ab), which defines their simple arbor shape, and class IV express Knot and Cut, which together promote the complex morphology of this class. The EGF-repeat factor Ten-m is co-regulated by both Knot and Ab to control the direction of branch outgrowth in both class I and IV neurons (Yalgin, 2015).

Contrasting activities of Knot, Cut and Ab in da neurons emphasize that altering dendrite branching is fundamental for regulating arbor complexity. Knot and Cut promote branch formation; conversely, Ab represses branch formation. Little is understood about how modulatory control over branching is achieved (Yalgin, 2015).

Microtubules polymerize via the addition of Tubulin dimers, primarily at the plus end. In axons, microtubules polymerize in an anterograde direction, providing a protrusive force for outgrowth. Microtubule polymerization also drives axon branch formation, as precursors only transform into branches after microtubule invasion. Mature dendrites have a predominantly minus-ends-out microtubule array, nevertheless recent studies have identified that anterograde microtubule polymerization events can initiate or extend branches, or modulate the size of dendritic spines. In addition, the re-initiation of dendrite growth and branch formation following injury uses upregulation of microtubule polymerization and its polarization in the anterograde direction (Yalgin, 2015).

This study examined whether class-specific transcription factors regulate branch promotion and repression by controlling microtubule organization during arbor development. In da sensory neurons, microtubule nucleation and polarity can be assayed in vivo using transgenic markers. Using genetic manipulation of class I and class IV da neurons, this study found that Ab controls class-specific differences in the localization of microtubule minus-end-directed markers in the da neuron arbor. By assaying Ab-mediated changes in the expression of a set of candidate microtubule regulators and using chromatin immunoprecipitation (ChIP), this study identified Cnn (Centrosomin) as an effector of Ab action. Cnn-centered control mechanisms, analogous to those that cluster microtubule nucleation events to create the mitotic spindle, are used in growing dendrites to regulate branching and to create class-specific arbor complexity (Yalgin, 2015).

Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

REFERENCES

Angelini, D. R., Smith, F. W., Aspiras, A. C., Kikuchi, M. and Jockusch, E. L. (2012). Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum. Genetics 190(2): 639-54. PubMed Citation: 22135350

Bardwell, V. J. and Treisman, R. (1994). The POZ domain: a conserved protein-protein interaction motif. Genes Dev. 8: 1664-1677. 7958847

Biehs, B., Sturtevant, M. A. and Bier, E. (1998). Boundaries in the Drosophila wing imaginal disc organize vein-specific genetic programs. Development 125: 4245-4257. 9753679

Boxshall, G. A. (2004). The evolution of arthropod limbs. Biological Reviews 79: 253-300. PubMed Citation: 15191225

Caygill, E. E. and Johnston, L. A. (2008). Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs. Curr. Biol. 18(13): 943-50. PubMed Citation: 18571409

Cook, O., Biehs, B. and Bier, E. (2004). brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila. Development 131: 2113-2124. 15073155

Doggett, K., Turkel, N., Willoughby, L. F., Ellul, J., Murray, M. J., Richardson, H. E. and Brumby, A. M. (2015). BTB-zinc finger oncogenes are required for Ras and Notch-driven tumorigenesis in Drosophila. PLoS One 10: e0132987. PubMed ID: 26207831

Hattori, Y., Usui, T., Satoh, D., Moriyama, S., Shimono, K., Itoh, T., Shirahige, K. and Uemura, T. (2013). Sensory-neuron subtype-specific transcriptional programs controlling dendrite morphogenesis: genome-wide analysis of Abrupt and Knot/Collier. Dev Cell 27(5): 530-44. PubMed ID: 24290980

Hu, S., Fambrough, D., et al. (1995). The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections. Genes Dev. 9(23): 2936-48. PubMed Citation: 7498790

Jang, A. C., Chang, Y. C., Bai, J. and Montell, D. (2009). Border-cell migration requires integration of spatial and temporal signals by the BTB protein Abrupt. Nat. Cell Biol. 11(5): 569-79. PubMed Citation: 19350016

Sokol, N. S., Xu, P., Jan, Y. N. and Ambros, V. (2008). Drosophila let-7 microRNA is required for remodeling of the neuromusculature during metamorphosis. Genes Dev. 22: 1591-1596. PubMed Citation: 18559475

Li, W., et al. (2004). BTB/POZ-Zinc finger protein Abrupt suppresses dendritic branching in a neuronal subtype-specific and dosage-dependent manner. Neuron 43: 823-834. 15363393

O'Farrell, F., Esfahani, S. S., Engstrom, Y. and Kylsten, P. (2008). Regulation of the Drosophila lin-41 homologue dappled by let-7 reveals conservation of a regulatory mechanism within the LIN-41 subclade. Dev. Dyn. 237: 196-208. PubMed Citation: 9299407

Parrish, J. Z., Kim, M. D., Jan, L. Y. and Jan, Y. N. (2006). Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites. Genes Dev. 20(7): 820-35. Medline abstract: 16547170

Regier, J. C., et al. (2010). Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature 463: 1079-1083. PubMed Citation: 20147900

Ronco, M., et al. (2008). Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus. Dev. Biol. 313: 80-92. PubMed Citation: 18061158

Simonnet, F., and Moczek, A. P. (2011). Conservation and diversification of gene function during mouthpart development in Onthophagus beetles. Evol. Dev. 13: 280-289. PubMed Citation: 21535466

Stratoulias, V., Heino, T. I. and Michon, F. (2014). Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster. PLoS One 9: e101141. PubMed ID: 24963666

Sturtevant, M. A. and Bier, E. (1995). Analysis of the genetic hierarchy guiding wing vein development in Drosophila. Development 121: 785-801. 7720583

Sturtevant, M. A., Biehs, B., Marin, E. and Bier, E. (1997). The spalt gene links the A/P compartment boundary to a linear adult structure in the Drosophila wing. Development 124: 21-32. 9006064

Sugimura, K., et al. (2004). Development of morphological diversity of dendrites in Drosophila by the BTB-zinc finger protein Abrupt. Neuron 43: 809-822. 15363392

Suzuki, Y., Squires, D. C. and Riddiford, L. M. (2009). Larval leg integrity is maintained by Distalless and is required for proper timing of metamorphosis in the flour beetle, Tribolium castaneum. Dev. Biol. 326: 60-67. PubMed Citation: 19022238

Turchyn, N., et al. (2011) Evolution of nubbin function in hemimetabolous and holometabolous insect appendages. Dev. Biol. 357: 83-95. PubMed Citation: 21708143

Turkel, N., Portela, M., Poon, C., Li, J., Brumby, A. M. and Richardson, H. E. (2015). Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis. Biol Open 4(8):1024-39. PubMed ID: 26187947

Yalgin, C., Ebrahimi, S., Delandre, C., Yoong, L. F., Akimoto, S., Tran, H., Amikura, R., Spokony, R., Torben-Nielsen, B., White, K. P. and Moore, A. W. (2015). Centrosomin represses dendrite branching by orienting microtubule nucleation. Nat Neurosci 18(10):1437-45. PubMed ID: 26322925

date revised: 30 October 2015

The Interactive Fly resides on the
Society for Developmental Biology's Web server.