Targets of Activity

A GATA/homeodomain transcriptional code regulates axon guidance through the Unc-5 receptor

Transcription factor codes play an essential role in neuronal specification and axonal guidance in both vertebrate and invertebrate organisms. However, how transcription codes regulate axon pathfinding remains poorly understood. One such code defined by the homeodomain transcription factor Even-skipped (Eve) and by the GATA 2/3 homologue Grain (Grn) is specifically required for motor axon projection towards dorsal muscles in Drosophila. Using different mutant combinations, genetic evidence is presented that both Grn and Eve are in the same pathway as Unc-5 in dorsal motoneurons (dMNs). In grn mutants, in which dMNs fail to reach their muscle targets, dMNs show significantly reduced levels of unc-5 mRNA expression and this phenotype can be partially rescued by the reintroduction of unc-5. It was also shown that both eve and grn are required independently to induce expression of unc-5 in dMNs. Reconstitution of the eve-grn transcriptional code of a dMN in dMP2 neurons, which do not project to lateral muscles in Drosophila, is able to reprogramme those cells accordingly; they robustly express unc-5 and project towards the muscle field as dMNs. Each transcription factor can independently induce unc-5 expression but unc-5 expression is more robust when both factors are expressed together. Furthermore, dMP2 exit is dependent on the level of unc-5 induced by eve and grn. Taken together, these data strongly suggests that the eve-grn transcriptional code controls axon guidance, in part, by regulating the level of unc-5 expression (Zarin, 2012).

Different transcriptional codes regulate axon guidance but the guidance systems they regulate are still unknown. The GATA factor, Grn, is a major determinant of guidance within dorsal MNs. A strong guidance phenotype occurs in grn mutants, in which ISN axons fail to reach their targets in >85% of the segments. However, none of the downstream molecules required for guidance downstream of grn has been identified to date. Several lines of evidence indicate that the Unc-5 receptor mediates guidance downstream of the GATA transcription factor Grn. First, both genes interact genetically in trans and this type of genetic interaction is often seen between two genes the gene products of which directly interact, such as Slit and Robo. Second, there is also a partial requirement of grn for unc-5 expression in dMNs as unc-5 mRNA levels are reduced in aCC and RP2 in grn mutants. Further support for the role of unc-5 downstream of grn comes from the partial rescue of the grn phenotype obtained by exogenously providing unc-5 specifically in aCC and RP2 (Zarin, 2012).

Among transcriptional codes that regulate motor-axon pathfinding, specific Lim-HD codes are required for the proper guidance of vertebrate motoneurons, in part, through the regulation of the EphA. In Drosophila, Nkx6 (HGTX -- FlyBase) is important for vMN specification and has been proposed to promote guidance through the expression of fasciclinIII (Fasciclin 3 -- FlyBase). Similarly, eve regulates guidance of dMNs through unc-5. In at least some of these situations the regulation is unlikely to be direct because the regulators are thought to mediate their function through a repressive activity. Indeed, eve repressive activity has been shown to be responsible for its guidance function in aCC and RP2, suggesting that its function might be to repress the expression of other transcription factors, such as HB9 (Exex -- FlyBase), which would confer those motoneurons with a ventral fate. Analysis of the unc-5 neuronal enhancer regulated by eve failed to identify any conserved Eve consensus homeodomain binding site, suggesting that eve regulation of unc-5 is not mediated through a direct binding to this element (Zarin, 2012).

Combinatorial codes of transcription factors play an instructive role in the generation of subclass diversity within the vertebrate spinal cord. In invertebrates, in which it is possible to analyse individual motoneurons within a subclass, a further level of complexity is revealed. Within the subclass of motoneurons that project to dorsal muscles, aCC and RP2, subclass determinants (eve, grn and zfh1) can work in a sequential order in aCC specification or independently, in parallel pathways, within RP2. Whereas Zfh1 is a general factor required in all motoneurons, eve and grn are specific to dMNs. It is plausible that each one of them might be important for specific aspects of specification within the same subclass of neurons but together might be responsible for the regulation of common targets such as unc-5. Although grn is required in both aCC and RP2 for unc-5 expression, it is not the only factor required because unc-5 mRNA is not completely absent from those cells and it also requires the presence of eve. In fact, grn or eve can independently induce unc-5 expression in dMP2 neurons but only both factors expressed in combination are able to induce axon exit towards the muscle field. This combinatorial expression of eve and grn might bring unc-5 above the threshold required for exit. unc-5 levels are definitely important for dMP2 exit because removing 50% of the gene dosage significantly suppresses the exit phenotype and this suppression is almost complete in an unc-5 null background. Transheterozygous interactions identified between unc-5 and eve, or unc-5 and grn also suggest that their levels are tightly controlled. As both eve and grn can regulate unc-5 it is likely that their combined activity in aCC and RP2 is essential to express the required levels of unc-5 in both neurons. A model is presented of unc-5 regulation by grn and eve in each individual neuron (Zarin, 2012).

The combinatorial nature of substrate recognition during motoneuron guidance and targeting is well established. Mutants for guidance receptors or ligands often show partially penetrant phenotypes and this includes the unc-5 phenotype in dMNs. By contrast, phenotypes observed for transcription factor mutants that affect dMN guidance are far more severe. For example, Lim1 regulates EphA4 in vertebrate motoneurons but the EphA4 mutant phenotype is less severe than Lim1 mutant phenotype. Similarly, 100% of ISNs in eve mutants or 85% in grn mutants are affected, suggesting that they regulate several guidance systems. It will be interesting to investigate the full array of guidance systems regulated by the eve-grn code. One potential candidate is FasII (Fas2 -- FlyBase) as it is an essential molecule for the pioneer function of aCC and RP2 (Zarin, 2012).

In summary, this study has identified the guidance receptor Unc-5 as a novel target of the GATA transcription factor Grn. unc-5 is key common target that mediates guidance downstream of both eve and grn. Furthermore, both transcription factors can promote transcription of unc-5 independently, suggesting that their combined action is essential to attain the proper expression levels of the Unc-5 receptor in dMNs. Future investigations in cell type-specific expression profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis will hopefully unravel other guidance systems regulated by the eve-grn code in dMNs. This knowledge will bring us closer to understanding the cell-specific transcriptional regulation of guidance (Zarin, 2012).



The expression of Grain transcripts during early Drosophila embryos is not detectable until the cellular blastoderm stage. Initially, the RNA transcripts are evenly distributed and concentrated at the basal end of the cells. Within a short period of time, the transcripts become localized to three regions along the dorsal portion of the embryo. In the procephalic region, the grain gene is abundantly expressed and the transcripts are widely distributed, properly reflecting its later role in the development of the head region. The expressed transcripts are also detectable in the posterior third (15%-25% egg length) and middle third (40%-60% egg length) of the dorsal embryo. These regions give rise to the precursors of the posterior spiracles and the dorsal epidermis, respectively. In addition, a very faint signal can be seen in a small region of the ventral embryo (Lin, 1995).

As embryonic development reaches stage 11 and beyond, three organ systems clearly stain positive with the Grain probe. The developing posterior spiracles are most prominent, and the antisense probe could serve as a useful marker to trace the development of this structure. It is noticeable that as germ band shortening occurs, the posterior spiracles moved backward and outward toward their final position. Similarly, the strong but relatively diffuse signals of the anterior and posterior midgut primordia become discrete and approach the middle portion of the embryos. The expression of grain gene in the developing central nervous system is also seen after stage 11. Distinct signals corresponding to each segment of the embryo become evident at stages 12-13. From the ventral view, the probe-positive cells for each segment are distributed along both sides of the midline. In the head region, the brain and the developing optic lobes, as well as the anterior tip of the clypeolabrum, are also clearly stained (Lin, 1995).

At late blastoderm stage, grn transcripts are prominently expressed in the procephalic region that forms the acron. This region requires grn function during development as indicated by the head phenotypes observed in grn mutants. At the extended germ band, grn is expressed in the CNS, in the primordia of the anterior and posterior midgut, and in a dorsolateral stripe running anteroposteriorly at the level where the tracheal spiracular branches join to the ectoderm. In the posterior spiracles, expression is detected in a subset of the cells that give rise to the spiracular chamber, accounting for the abnormal filzkörper in grn mutants; and in an area coinciding with the sal-expressing cells, accounting for the abnormal stigmatophore (Brown, 2000).


At the wandering larva stage (just before pupariation) grn transcripts are detected in the imaginal discs from which the adult ectodermal structures form. In the leg discs, high levels are seen in the region that gives rise to the proximal parts of the leg (femur and tibia), with the exception of a small patch of cells. Lower levels of grn are expressed in the central parts of the disc that form the tarsal segments. In the wing and haltere discs, grn is expressed in the areas that form the hinge, with little or no expression in the wing pouch. grn is not expressed in the proximal regions that form the main body (notum and pleura). When the discs evert, grn is expressed in a similar area -- the wing expression now clearly restricted to the hinge and the triple row. In the larval nervous system grn is expressed in the optic lobe of the brain. Although grn is not expressed in the primordia of the discs during the embryonic stages, transcripts can be detected at early and mid-third instar larva stage (Brown, 2000).

Effects of Mutation or Deletion

Embryos mutant for grain have defects in the head skeleton and in the posterior spiracles. The head skeleton is mostly affected in the dorsal bridge and in the lateralgräten, structures that derive, respectively, from the acron and the mandibular segment of the embryo. The posterior spiracles have a shorter filzkörper and do not form the protruding stigmatophore. grn null mutant embryos have been analyzed using the neural marker 22C10 antibody and Hindsight, a protein expressed in the endoderm. Although grn is expressed during midgut formation and CNS formation, the gut epithelium forms normally in grn and the CNS shows no obvious abnormalities (Brown, 2000).

Since grn mutants are embryonic lethal, mosaic analysis was used to study the function of grn in imaginal tissues. Clones were induced using the minute technique, which generates clones occupying large areas of the adult tissue, owing to growth advantage from the surrounding cells. Clones of grn7J86, grn7L12 and grnSPJ9 alleles give identical results and will be considered together. No abnormalities were detected when the mutant clones occupied the wing or the distal leg segments. In contrast, grn clones on the proximal leg segments severely affect the shape of the femur and tibia (Brown, 2000).

Out of a total of 25 M + forked clones generated in the leg, 13 affected the shape of the femur and tibia, which became shorter and broader than in the wild type. In these cases the segments were approximately one third shorter and one third broader, but leg segmentation was normal, indicating that grn does not affect leg patterning. To find out if the reason for the different shape is due to an abnormal packaging of the cells, the leg trichomes were counted. In the leg, each trichome is formed by a single cell. Therefore, by comparing the distribution of the trichomes in a region occupied by a grn clone with the distribution of trichomes in the same region of a wild type leg, it could be indirectly determined if the abnormal leg shape is due to shape changes of the individual cells. The number of cells and their spatial distribution in grn clones do not differ from the wild type, indicating that the shape change is not caused by a change in cell density (Brown, 2000).

Since grn is proximal to the forked duplication used to label the clones, some clones marked with forked will not be grn. This may account for some of the forked clones that did not affect the leg shape, but it is unlikely that it will account for all. The high number of forked clones that did not affect leg shape could be explained if some grn clones were induced in areas of the leg where grn is not expressed. Alternatively, grn requirement could be non-cell-autonomous and some mutant clones could be rescued by the wild-type neighboring cells (Brown, 2000).

To test if the absence of grn phenotypes was due to non autonomy, smaller non-minute grn clones were made and their size was compared with control clones generated at the same stages. Wild-type control clones generated at second larval stage occupy long and thin stripes of tissue when labelled either with forked or yellow cuticle markers. Of 17 control forked or yellow clones induced on the femur or tibia, all occupied more than one leg segment, with most of them (13) occupying a long and narrow line running along femur and tibia. None of the control clones generated at this stage occupied a single leg segment. In contrast, in a grn background, out of 18 forked or yellow clones induced on the femur or tibia, more than half of them were short and broad. Of the latter, eight occupied a single segment and the two that occupied two segments were localized straddling the femur and tibia junction. These results suggest that wild-type neighboring cells cannot rescue grn mutant clones and that the reason why only half of the clones show grn phenotypes is due to the fact that not all leg cells express grn (Brown, 2000).

The grn clones marked with forked were generated in a genotype that simultaneously induces a wild type yellow twin clone. By studying the shape of the yellow twin, it can be found out if the presence of a grn clone affects the neighboring cells. Out of 10 yellow twin clones, seven had normal elongated shapes. At least in two of these cases it can confidently be said that the yellow clones had been generated beside a grn mutant clone, since the leg shape was abnormal. Thus, the function controlled by grain is mostly cell autonomous, because neighboring yellow clones can grow as would be expected for a clone generated at that age (Brown, 2000).

To find out if the different shape of the clone is due to how the cells oriente during division, the grn clones in the leg discs were labeled using a ubiquitin-GFP nuclear construct. This allows the visualization of the area occupied by the grn clones and allows this area to be compared with that of the wild-type twin clones before leg eversion. The shape of 25 grn clones generated in the proximal area was similar to that of the twin, showing that the clone shape difference seen in the adult leg must occur during leg eversion, a period when most of the cell divisions have ended. Taken together these results show that the grn phenotype is not due to effects on cell viability, shape, density or orientation of the divisions, but rather on how the cells of the imaginal disc rearrange to make the leg (Brown, 2000).

To test the effect of ectopic grn expression UAS-grn constructs were made. An analysis was carried out to see if the constructs were capable of rescuing the grn mutant phenotype. Flies simultaneously mutant for grn and carrying the 459.2-GAL4 and UAS-grn constructs were made. UAS-grn expression driven by 459.2-GAL4 rescues the mutant phenotype in the stigmatophore, proving that the construct produces functional protein (Brown, 2000). The effects of expressing Grm in areas outside of its normal pattern were investigated. Ectopic expression of grn using several GAL4 driver lines causes lethality. Analysis of the embryos using different driver lines show some common features: the ventral denticle belts tend to disappear, head involution is distorted, and in a small percentage of the embryos, the tail invaginates abnormally (Brown, 2000).

These defects were studied in detail using the hairy-GAL4 line using either one or two UAS-grn inserts. hairy is a pair-rule gene that drives expression in alternate segments. As expected, the penetrance of the transformations increases with two UAS inserts, although the phenotypes are not qualitatively different. In most h-GAL4 UAS-grn larvae, the denticle belts of the alternating segments are reduced or missing, leaving a naked cuticle area. No indication that the denticle cells have invaginated or that this is a segmentation defect was seen, since staining with anti-En antibody reveals that the segments form normally. The head skeleton is abnormal in about 9%-32% of the embryos and tail invagination is observed in 6%-20%, depending on the number of inserts and the temperature of the culture. Closer examination of the embryos with invaginated tails reveals that the spiracles are now at the end of a long tubular structure formed by segments posterior to A8, which normally would lay on the outside of the embryo. This structure includes cells from A8, A9, A10 and the telson. Although, due the absence of specific cell markers, it cannot be know if these defects are due to abnormal cell rearrangement, these results show that ectopic grain expression affects morphogenetic movements during gastrulation (Brown, 2000).

Specification of Drosophila aCC motoneuron identity by a genetic cascade involving even-skipped, grain and zfh1

During nervous system development, combinatorial codes of regulators act to specify different neuronal subclasses. However, within any given subclass, there exists a further refinement, apparent in Drosophila and C. elegans at single-cell resolution. The mechanisms that act to specify final and unique neuronal cell fates are still unclear. In the Drosophila embryo, one well-studied motoneuron subclass, the intersegmental motor nerve (ISN), consists of seven unique motoneurons. Specification of the ISN subclass is dependent upon both even-skipped (eve) and the zfh1 zinc-finger homeobox gene. ISN motoneurons also express the GATA transcription factor Grain, and grn mutants display motor axon pathfinding defects. Although these three regulators are expressed by all ISN motoneurons, these genes act in an eve->grn->zfh1 genetic cascade unique to one of the ISN motoneurons, the aCC. The results demonstrate that the specification of a unique neuron, within a given subclass, can be governed by a unique regulatory cascade of subclass determinants (Garces, 2006).

Why do these three genes act in a unique fashion in aCC, and why is grn and zfh1 sensitive to Notch specifically in this ISN motoneuron? One explanation may be that the differential input from upstream regulators, such as Ftz, Pdm1, Hkb and Pros, acts to modify the genetic interactions between eve, grn and zfh1. Another possibility is that the relative level of each factor plays an important role in dictating different cellular fates. Studies of the related Isl1 and Isl2 LIM-homeobox genes suggest that their involvement in motoneuron subclass specification is not primarily the result of the unique activity of each gene, but rather by the combined 'generic', tightly temporally controlled, Isl1 and Isl2 levels. Similarly, the different expression levels of the transcription factor Cut have been shown to play instructive roles during the specification of neuronal cell identities within the PNS. Different levels of expression of Grn and Zfh1 have been observed; while Grn is strongly expressed in aCC and weakly in RP2, Zfh1 expression shows an opposite distribution. It is tempting to speculate that these levels may be instructive for ISN motoneuron specification (Garces, 2006).

In the VNC, mutually exclusive expression is observed between Grn and Hb9 (and Islet) in different subsets of interneurons and motoneurons. Cross-inhibitory interactions between eve and Hb9 has been shown to contribute to their mutually exclusive expression patterns, and functional studies demonstrate that eve and Hb9 regulate axonal trajectories of dorsally and ventrally projecting axons, respectively. These observations are reminiscent of the cross-repressive interactions between classes of regulators that act to determine, refine and maintain distinct progenitor domains along the dorsoventral axis of the vertebrate neural tube. eve is important for proper grn and zfh1 expression in aCC, but not in RP2. These results are consistent with previously reported observations that the requirement for eve in axonal guidance is somewhat more stringent in aCC than in RP2, leading the the proposal that there may be different target genes for Eve in these two motoneurons (Garces, 2006).

Zfh1 expression was previously shown to depend upon Notch signaling activity in the aCC/pCC sibling pair as mutations in spdo or mam, members of the Notch signaling pathway, lead to de-repression of Zfh1 in pCC. Using the same allelic combinations, de-repression of grn was also observed in the pCC. Whether or not grn is directly suppressed by the Notch pathway remains to be seen, but it is interesting to note that in vertebrates, gata2/3 have been identified as targets of Notch during the differentiation of specific hematopoietic lineages (Garces, 2006).

Within the ISN subclass, the aCC motoneuron pioneers the ISN to innervate the dorsal-most muscle, muscle 1. A number of genetic and cell-ablation studies have convincingly shown that aCC plays an instructive pioneer role and guides the follower U motoneurons along the ISN nerve. These results lend support for the proposed instructive role of aCC in ISN formation. However, these studies indicate that aCC may not be essential for ISN formation. First, using RN2-GAL4 to visualize aCC and RP2, aberrant innervation of muscle 8 were frequently found (35% of hemisegments) in grn mutants. However, an axonal projection was simultaneously observed at the vicinity of the dorsal muscles 2/10. In grn mutants, zfh1 expression is specifically lost in aCC but maintained in RP2. Given the role for zfh1 in motor axon pathfinding, it is proposed that aberrant innervation of muscle 8 in grn mutants, is caused by aCC and not by RP2, and that RP2 pathfinds normally to the muscles 2/10. If so, RP2 may function as a pioneer motoneuron for muscle 2 and project there without the aCC axon. Second, although the rescue of grn mutants using RN2-GAL4 is complete, it was found that using CQ2-GAL4 to specifically rescue U motoneurons does lead to a partial rescue (54% muscles 1/9 innervated compared with 15% in grn mutants). Thus, even in the absence of aCC pioneer function, the Us (presumably U1) can still project to the dorsal-most muscles. This is in line with previous studies showing that in eve aCC/RP2 mosaic mutants and in aCC/RP2 cell ablation experiments, there is still partial innervation of muscle 1/9 (Garces, 2006).

grn is part of an eve --> grn --> zfh1 transcriptional cascade crucial for specification of aCC motoneuron identity. However, the failure of grn to rescue eve, and of zfh1 to completely rescue grn, combined with the misexpression results, indicate additional roles for both eve and grn. These roles could be either in the regulation of other aCC determinants and/or in the regulation of genes directly involved in aCC axon pathfinding. Although there are no obvious candidates for additional aCC determinants, recent studies point to a candidate axon pathfinding gene. The Drosophila unc-5 gene encodes a netrin receptor and is expressed in subsets of neurons in the VNC. Misexpression of unc-5 is sufficient to trigger ectopic VNC exit in subsets of interneurons. Recent studies now show that unc-5 is specifically expressed in eve motoneurons, and that eve is necessary, but only partly sufficient for unc-5 expression. In line with these findings, it was found that whereas single misexpression of eve or grn in dMP2 neurons has very minor effects, co-misexpression of eve and grn can efficiently trigger dMP2 lateral axonal exit. This combinatorial effect of eve/grn occurs without apparent activation of zfh1. However, misexpression of zfh1 can also trigger dMP2 lateral exit. Thus, these genes appear to be able to act in an independent manner to trigger VNC exit, but in a highly context-dependent manner. A speculative explanation for not only the mutant and rescue results, but also these misexpression results, would be that all three regulators are needed for robust and context-independent activation of axon pathfinding genes such as, for example, unc-5 (Garces, 2006).

grn encodes a GATA Zn-finger transcription factor and is the ortholog of the closely related vertebrate gata2 and gata3 genes. In vertebrates, gata2/3 are expressed in overlapping domains in the nervous system, but relatively little is known about their function. Expression data and evidence from gene targeting suggest an involvement in neurogenesis, neuronal migration and axon projection. A role in specifying neuronal subtypes within the context of neural tube patterning is emerging and recently a role for gata2/3 during 5-HT neuron development has been reported. The role of gata3 in the development of the inner ear has been of particular interest, and in humans, mutations in this gene have been linked to HDR syndrome, which is characterized by hypoparathyroidism, deafness and renal defects. In the mouse, gata3 is expressed in auditory but not vestibular ganglion neurons during development. The mouse gata3 mutant shows auditory ganglion neuron loss and efferent nerve misrouting, revealing that gata3 regulates molecules associated with neural differentiation and guidance. These vertebrate studies, combined with the current results, suggest that gata2/3 genes, similar to other transcription factors specifying neuronal identities, such as islet1/2, evx1/2 or Hb9, and their respective orthologs in Drosophila, have maintained similar functions throughout evolution (Garces, 2006).

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, a very highly specific marker was used that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. The data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, it was shown that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, a targeted genetic screen was performed to identify players involved in ABCA specification. It was found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, it was shown that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third s segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification (Gabilondo, 2011).

The findings strongly suggest that the Capaergic abdominal ABCA neuron arises from NB 5-3. This conclusion is based on the expression in ABCA cells of gsb, wg and unpg, and the absence of the markers lbe(K) and hkb. However, even though gsb expression is known to be maintained specifically in the lineage of rows 5 and 6 NBs, whether expression of the other genetic markers used to identify NBs at stage 11 changes late in embryogenesis remains unanswered. Nonetheless, the specific combination of NB markers found in ABCA cells and their position in the hemineuromere are consistent with their birth from NB 5-3. Previous accounts showed that this NB gives rise to a lineage of 9–15 cells. Additionally, observations derived from studies in which PCD was blocked showed that NB 5-3 can potentially produce a large lineage (ranging from 19 to 27 cells), suggesting that it could generate 13 or 14 GMCs. The lack of a NB 5-3 specific-lineage marker prevented resolution of its complete lineage, and thus determining the birth order of the ABCA cell (Gabilondo, 2011).

Recent findings on the NB 5-6 and NB 5-5 demonstrate that cas and grh act together as critical temporal genes to specify peptidergic cell fates at the end of these lineages. cas mutants lack ABCA cells and Cas is expressed in these cells, while the normal pattern of ABCA cells is found in grh mutants, and Grh is not present in ABCA neurons. These data strongly support the birth of ABCA cells in a cas-only temporal window. This is different from the s Capaergic SECA cells, which while also arising from NB 5-3, show a reduction in cell number in both pdm and cas mutants, demonstrating birth at a mixed pdm/cas temporal window. Previous studies in other lineages have shown that when a temporal gene is mis-expressed, all progeny cells posterior to that temporal window can be transformed to the specific fate born at that particular temporal window. However, cas mis-expression failed at inducing ectopic ABCA cells, suggesting that cas in necessary but not sufficient to specify the ABCA fate (Gabilondo, 2011).

Programmed cell death (PCD) is a basic process in normal development. The results suggest that the ABCA and its sibling are equivalent cells committed to achieve the ABCA fate. First, it was shown that the ABCA-sibling cell dies by apoptosis, but produces an ABCA-like Capaergic neuron if PCD is inhibited. Second, when PCD is blocked, NB 5-3 also produces a GMC generating two ABCA-like Capaergic cells in the A5–A7 segments. These data indicate that a segment-specific mechanism prevents death of the ABCA cells in A2–A4 neuromeres. Segment specific cell death has been previously reported for the NB 5-3 lineage, and detailed studies on segment-specific apoptosis of other lineages have shown that this process is under homeotic control. In addition, the results show a different timing in the PCD undergone by the ABCA sibling and the ABCA cells born in A5–A7. This interpretation is based on the differential effect of p35 expression when cas-Gal4 or elav-Gal4 drivers were used. Although elav-Gal4 is transiently express in NBs and GMCs, robust and maintained driver expression commences in differentiating neurons. In contrast, cas expression starts in the NB and is maintained in the GMC and neuronal progeny. Therefore, the finding that death of the ABCA-sibling cell can be prevented by directing p35 with cas-Gal4, but not with elav-Gal4, suggests that the death of the ABCA sibling occurs earlier in development than the death undergone by the ABCA cells in A5–A7 segments (Gabilondo, 2011).

In the ABLK/LK peptidergic fate (derived from the NB 5-5), activation of Notch (N) signaling in the peptidergic cell prevents its death, while its sibling, NOFF cell undergoes apoptosis. On the contrary, in the EW3/Crz peptidergic fate (derived from the NB 7-3), silencing of N signaling is essential for the neuron survival, and therefore for it proper specification. The current results are in accordance with the last scenario, in which the ABCA cell is NOFF, and it sibling, which undergoes apoptosis, is NON. Therefore, Notch signaling must be switch off for the proper specification of the ABCA neuron (Gabilondo, 2011).

To search for genes involved in specification of the ABCA neural fate, a reduced set of mutants was screened of genes that are expressed in the embryonic CNS at stage 11, a time at which distinctly defined sublineages are being generated from all active NBs. Even though this method will certainly overlook important genes, the results reveal that it is in fact a very effective way to find genes involved in specification of a particular neural fate. Indeed, the ratio of success has been very satisfactory: 33.3% of the genes analyzed display a significant phenotype. Moreover, the set of identified genes could be further expanded by, for example, searching in interactome databases, and performing the subsequent screen on those putative interactors (Gabilondo, 2011).

It is assumed that the specification of a concrete cellular fate requires the combination of several transcription factors, namely a genetic combinatorial code. Recently, a detailed combinatorial code has been reported for three neuropeptidergic fates: ap4/FMRFa, ap1/Nplp1 and ABLK/Lk. However, very little is known about the specification of the rest of the 30 peptidergical fates. This study has identified several genes involved in the specification of the ABCA fate, which fit into three categories. First, genes were found whose loss-of-function produces a relevant increase of the number of ABCA cells. Most remarkable are the klu and rn phenotypes, which consist of duplications of the ABCA cells. These phenotypes suggest that these two transcription factors repress the ABCA fate in other neural cells (or/and NBs/GMCs). Interestingly, the normal phenotype of nab mutants indicates that, contrary to its mode of action in the wing, Rn does not work with the transcription cofactor Nab in this context (Gabilondo, 2011).

Second, genes were found whose loss-of-function produces a significant decrease of the number of ABCA neurons. In this category, the zhf2, ftz and grain phenotypes stand out. The effects of mutations on ftz are in agreement with its early role in segmentation: ftz is a pair-rule segmentation gene that defines even-numbered parasegments in the early embryo, and absence of ABCA cells was found in the A3 segment in ftz mutants. However, zfh2 and grain seem to be part of the specific combinatorial code of the ABCA cells. The Drosophila GATA transcription factor Grain has been reported to be involved in the specification of other cell fates, such as the aCC motoneuron fate. Based on its expression, the zinc finger homeodomain protein zfh2 has been proposed to mediate specification of the serotoninergic fate, but this has not been further demonstrated. Interestingly, during wing formation, zfh2 is required for establishing proximo-distal domains in the wing disc, and it does so partly by repressing gene activation by Rn. The opposite phenotypes that was observed in rn and zfh2 mutants suggest that similar interactions occur during ABCA specification. Analyses aimed to test this hypothesis are currently being performed (Gabilondo, 2011).

Third, two genes were found whose loss-of-function abolishes the ABCA fate: Grunge and hh. Grunge encodes a member of the Atrophin family of transcriptional co-repressors that plays multiple roles during Drosophila development. Taken together, studies from C. elegans to mammals suggest that Atrophin proteins function as transcriptional co-repressors that shuttle between nucleus and cytoplasm to transduce extracellular signals, and that they are part of a complex gene regulatory network that governs cell fate in various developmental contexts. Similarly, Hh is an extracellular signaling molecule essential for the proper patterning and development of tissues in metazoan organisms. It is noteworthy that two genes implicated in extracellular signaling pathways, Grunge and hh, are absolutely required for ABCA fate. Further studies will be needed to identify at which step/s they exert their actions, and to unravel possible interactions between them and with other players of the combinatorial code for ABCA specification (Gabilondo, 2011).


Brown, S. and Castelli-Gair Hombria, J. (2000). Drosophila grain encodes a GATA transcription factor required for cell rearrangement during morphogenesis. Development 127: 4867-4876. PubMed Citation: 11044401

Gabilondo, H., et al. (2011). A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons. Mech. Dev. 128(3-4): 208-21. PubMed Citation: 21236339

Garces, A. and Thor, S. (2006). Specification of Drosophila aCC motoneuron identity by a genetic cascade involving even-skipped, grain and zfh1. Development 133: 1445-1455. 16540509

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grain: Biological Overview | Regulation | Developmental Biology | Regulation | Effects of Mutation | References

date revised: 10 December 2012

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