baboon

DEVELOPMENTAL BIOLOGY

Embryonic

Baboon is maternally deposited into oocytes and widely distributed during development in the embryo and in the imaginal discs of the larva. The 4.0 and 3.6 kb mRNAs appear to be predominantly maternal while the 4.9 kb mRNA appears to be predominantly zygotic and is present throughout development (Wrana, 1994).

Larval

Metamorphosis of the Drosophila brain involves pruning of many larval-specific dendrites and axons followed by outgrowth of adult-specific processes. From a genetic mosaic screen, two independent mutations were recovered that block neuronal remodeling in the mushroom bodies (MBs). These phenotypically indistinguishable mutations affect Baboon function, a Drosophila TGF-ß/activin type I receptor, and Smad on X (Smox, or dSmad2), its downstream transcriptional effector. Punt and Wit, two type II receptors, act redundantly in this process. In addition, knocking out Activin-beta (dActivin) around the mid-third instar stage interferes with remodeling. Binding of the insect steroid hormone ecdysone to distinct Ecdysone receptor isoforms induces different metamorphic responses in various larval tissues. Interestingly, expression of the Ecdysone receptor B1 isoform (EcR-B1) is reduced in activin pathway mutants, and restoring EcR-B1 expression significantly rescues remodeling defects. It is concluded that the Drosophila Activin signaling pathway mediates neuronal remodeling in part by regulating EcR-B1 expression (Zheng, 2003).

It was of interest to identify possible ligands that participate in the remodeling process. Seven TGF-β type ligands are present in the Drosophila genome. Three of these, dpp, scw, and gbb, are clearly of the BMP family. The remaining, maverick (mav), myoglianin (myo), dActivin (dAct), and activin-like-protein (alp), have not been assigned either genetically or biochemically to a particular family or signaling pathway. Phylogenetic considerations place dAct clearly within the Activin subfamily, while Myo is most similar to BMP-11 and GDF-8, and Mav and Alp are equidistant from both the BMP and TGF-β/Activin subgroups. Therefore, possible involvement of dAct in the Babo signaling was examined (Zheng, 2003).

First, in situ hybridization revealed that dAct is widely expressed in larval brains. Next, when conditioned media from cells expressing dAct was added to S2 cells transfected with Smox, it was found that this ligand is able to stimulate phosphorylation of Smox, while the prototypical BMP ligand Dpp is not. Finally, attempts were made to knock out dAct activity using two independent approaches and dAct, like Babo, was found to be essential for both optic lobe development and EcR-B1 expression in larval brains. Since dAct mutations are currently unavailable, attempts were made to produce a partial loss-of-function condition by overexpression of a dominant-negative form of the protein or RNAi. All TGF-β type ligands that have been examined dimerize and are processed prior to secretion. Previous studies have shown that overexpression of a cleavage-defective form of a particular ligand can interfere with processing and secretion of endogenous ligand. Therefore, a cleavage defective form of dAct (CMdAct) was expressed using either a general GAL4 driver (tubP-GAL4) or an MB-specific driver (GAL4-OK107). CNS development was observed to be retarded only when the CMdAct is ubiquitously expressed and not when it is expressed in MBs. This suggests that dAct does not function within MBs in an autocrine-like fashion. Poor development of the optic lobes is apparent in the tubP-GAL4>CMdAct larval brains, similar to that observed in babo and punt mutant larvae. More importantly, EcR-B1 expression is largely absent in γ neurons of animals that ubiquitously express CMdAct, similar to what is observed in babo mutants. Hs-GAL4-mediated transient expression of CMdAct around the mid-third instar stage also blocks both optic lobe development and EcR-B1 expression (53%). Consistent results are obtained after induction of RNAi using a hairpin-loop dAct construct (UAS-HLdAct). For instance, no EcR-B1 expression was detected in 65% of the late third instar larval brains that were heat shocked to express UAS-HLdAct transiently around the mid-third instar stage. Again, absence of EcR-B1 expression is tightly associated with poor optic lobe development. Similar treatments yield no detectable phenotypes when UAS-CMdAct/UAS-HLdAct is absent or replaced with other UAS-transgenes, such as UAS-mCD8-GFP and UAS-antisense dActivin. In addition, punt mutants, despite having small brains, continue to show EcR-B1 expression. Taken together, these results suggest that dAct, like Babo and dSmox, is indispensable for EcR-B1 expression in the CNS of wandering larvae (Zheng, 2003).

Therefore, from forward genetic mosaic screens, it was found that the Babo TGF-β/Activin type I receptor and a well-known TGF-β/Activin receptor downstream effector, Smox, are both cell autonomously required for remodeling of MB neurons during metamorphosis, providing definitive evidence for involvement of TGF-β/Activin signaling in neuronal plasticity. No evidence exists for any cell fate change in babo mutant MB neurons. For instance, expression of multiple cell type-specific markers remains normal, and mutant γ neurons, unlike wild-type α'/β' neurons, consistently acquire mature dendritic morphological features before metamorphosis. In addition, MB γ neurons that are born at various stages all commit to expressing EcR-B1 in response to TGF-β signaling at the same time and after they all develop into morphologically mature neurons. Therefore, TGF-β signaling probably plays a direct role in programming neuronal plasticity and is not required for cell specification (Zheng, 2003).

TGF-β signaling is implicated in regulating neuronal plasticity in diverse organisms. For instance, environmental cues regulate synthesis of a TGF-β-related ligand (DAF-7) in a pair of chemosensory neurons in C. elegans to direct entry into and exit from an alternative third larval stage called the dauer larva. Dauer formation involves arrest of all postembryonic cell divisions and remodeling of various tissues throughout the body. Given that the DAF-7 TGF-β ligand is primarily sensed by neurons expressing appropriate TGF-β receptors and Smads, it is likely that changes in TGF-β activities directly mediate neuronal remodeling and in turn orchestrate diverse dauer entry/exit responses outside the nervous system. However, it remains to be elucidated whether and how TGF-β signaling regulates neuronal projections and connections in individual remodeling neurons during the entry into and exit from the dauer stage (Zheng, 2003).

In Drosophila, recent data suggest that a BMP signaling pathway controls synaptic growth and function at the neuromuscular junction (NMJ). Whether this pathway also contributes to activity-dependent remodeling at the NMJ remains to be determined. It is interesting to note, however, that in this pathway Wit acts as a BMP receptor, and it can not be substituted for by Punt. In contrast, the activin pathway described here appears to be able to utilize either Punt or Wit for signaling. This may reflect selectivity in the binding of some ligands to one receptor, but not the other. Additional studies will be required to resolve this issue. Since many components of several different TGF-β signaling pathways show pronounced expression in different parts of the developing and postnatal rodent brain, the demonstration that TGF-β/Activin signaling cell-autonomously controls plasticity of MB neurons may provide novel insights into how neuroplasticity is dynamically regulated in higher organisms (Zheng, 2003).

Changes in gene expression are believed to mediate most TGF-β-dependent biological processes. The observation that restoration of EcR-B1 expression significantly rescues remodeling defects in babo mutant neurons supports the model that the Babo/dSmox-mediated TGF-β signaling mediates neuronal remodeling via upregulation of the EcR-B1 expression. Interestingly, ecdysone has also been implicated in regulating synaptic efficacy at the Drosophila NMJ, as has BMP signaling. However, as yet no connection between TGF-β signaling and the ecdysone pathway has been established in this system. In C. elegans, the DAF-7 TGF-β ligand as well as the DAF-12 nuclear hormone receptor are involved in dauer formation. In response to hormonal signals, DAF-12 and EcR coordinate changes in diverse tissues during dauer formation and metamorphosis, respectively. Therefore it might be a common theme that TGF-β signaling patterns tissue-specific responses to steroid hormones in diverse organisms by regulating expression and/or activities of specific steroid hormone receptors (Zheng, 2003).

Several lines of evidence support the model that patterned EcR-B1 expression in the late third instar larval CNS is likely established as a consequence of stage-regulated, cell type-specific responses to TGF-β signaling: (1) dActivin is broadly expressed in the CNS, while expression of EcR-B1 is selectively restricted; (2) despite the persistent presence of dActivin expression during all developmental stages and the fact that γ neurons are born at different times, programming of EcR-B1 expression in γ neurons does not occur until the mid-third instar stage; (3) ubiquitous expression of activated Babo fails to activate EcR-B1 expression ectopically. Determining how TGF-β signaling induces such stage-specific, cell type-dependent responses will provide mechanistic cues for how EcR-B1 is differentially expressed to pattern metamorphosis of the CNS. Possible models might include differential expression of a Smox cofactor or the requirement for a second signal that cooperates with the dActivin signal. It will be important to determine how dActivin reaches its target MB neurons. As has been recently suggested for BMP signaling at the NMJ, this might involve retrograde signaling from the MB synapse or it may occur via a juxtacrine mechanism from nearby cells (Zheng, 2003).

No direct connection has been shown between TGF-β signaling and regulation of cytoskeleton dynamics. Consistent with vital roles of TGF-β pathways in regulating gene expression, the results suggest that Babo/Smox signaling might simply lead to activation of EcR-B1 expression to capacitate MB neuronal remodeling during metamorphosis. Although this study provides novel insights into how differential expression of EcR isoforms is achieved, the challenge now is how ecdysone-induced transcriptional hierarchies mediate complex cytoskeletal changes in remodeling neurons. Identifying mutations that block various aspects of the MB neuronal remodeling in mosaic organisms will continue to shed new light on the molecular mechanisms underlying neuronal plasticity (Zheng, 2003).

Tiling of R7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion

The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. The restriction of terminals to single columns is refered as tiling. In a genetic screen based on an R7-dependent behavior, the Activin receptor Baboon and the nuclear import adaptor Importin-α3 were identified as being required to prevent R7 axon terminals from overlapping with the terminals of other R7 projections in neighboring columns. This tiling function requires the Baboon ligand, dActivin (Activin-β), the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus (Ting, 2007).

Activin-β, the first invertebrate activin gene to be described, is located 102 F region of the Drosophila chromosome 4, and has a multibasic protease site that would generate a mature C-terminal peptide containing 113 amino acids showing >60% similarity to the vertebrate activin β_B(inhibin β_B) sequences. A TGF-β family signature as well as all 9 cysteine residues conserved in the vertebrate activins are also present in this mature peptide sequence. Northern blot and RT-PCR analyses indicated that the activin β gene is expressed in embryo, larva and adult stages of Drosophila. It is proposed that dActivin is an autocrine signal that restricts R7 growth cone motility, and it was demonstrated that dActivin acts in parallel with a paracrine signal that mediates repulsion between R7 terminals (Ting, 2007 and references therein).

Previous anatomical studies have highlighted two prominent features of neuropil organization in the fly visual system: (1) the axons of most neuron classes arborize in characteristic layers of the brain and (2) they then remain restricted either to one column or a small number of adjacent columns. To gain insight into the developmental mechanisms that regulate these aspects of axon targeting, focus was placed on the R7 photoreceptor neuron. Previous studies have characterized mechanisms controlling the precise layer termination of R7 growth cones. This paper analyzed the mechanisms that restrict R7 terminals to the correct columns. The latter process is regulated by two partially redundant pathways: a paracrine signal that mediates repulsion between adjacent R7 axons and an autocrine Activin signal that is transduced by retrograde transport and import of the transcription factor Smad2 into the nucleus by a component of the classical nuclear import pathway, Importin-α3 (Ting, 2007).

A prominent organizing feature of the medulla is the restriction of axons and their terminals, including those of R7, R8, and the lamina monopolar neurons (except L4), to single columns. This phenomenon is similar to the tiling of processes observed in both the peripheral and central nervous systems. Ablation experiments in both fly and zebrafish support the view that repulsive interactions between processes of different cells of the same class prevent overlap of dendritic and axonal receptive fields. Consistent with this model, wild-type R7 terminals only invade adjacent columns from which R7s have been removed. However, an R7 axon does not invade even empty columns unless it is under 'competitive pressure' from additional R7 axons within its own column, suggesting that a second, intrinsic mechanism also restricts R7 terminals (Ting, 2007).

Activin signaling is required for tiling of R7 terminals: loss of babo, dSmad2, or dActivin causes R7 axons to invade adjacent occupied targets. Because these mutant axons invade even when they are not under competitive pressure, it is hypothesized that Activin affects an intrinsic property of R7 terminals such as their motility or ability to initiate synaptogenesis. In support of this model, it was found that virtually all R7 axons lacking babo, dSmad2, or dActivin initially extend filopodia beyond the R7-temporary layer at 17 hr APF, although these retract by 40 hr APF, and expression of a constitutively active Baboon in R7s affects growth cone morphology. Although models in which Activin signaling also mediates repulsion among R7 terminals cannot be ruled out, R7s unable to respond to Activin are still partly repelled by their neighbors, indicating the existence of repulsive mechanisms that are redundant with Activin. Recent studies have demonstrated that Dscam2 mediates repulsion between L1 growth cones in a layer immediately distal to the R7 terminals (Millard, 2007). Because Dscam2 is not expressed in R7, other cell-surface proteins must mediate the repulsive interactions between adjacent R7 terminals. The identification of Activin's involvement in R7 tiling paves the way to identifying such molecules by allowing the removal of a pathway that is functionally redundant with them (Ting, 2007).

Members of the TGF-β superfamily have been widely implicated in regulating axon guidance and synaptogenesis by both transcription-dependent and -independent mechanisms. This study found that loss of dSmad2 from R7s resembles loss of Activin, suggesting that the tiling of R7 terminals requires changes in transcription. In support of this model, it was found that restriction of R7 terminals also requires imp-α3, which is required for the accumulation of dSmad2 in R7 nuclei. Whereas some previous vertebrate studies have suggested that individual Smads are imported by Importin-α, others argue instead that active Smad complexes can enter the nucleus by an importin-independent mechanism. The results provide the first genetic evidence that Smad function can require Importin-α-mediated nuclear import and may help reconcile previous results by demonstrating that different cell types import dSmad2 by different mechanisms (although R7s require imp-α3, pigment cells do not). It is noted that imp-α3 mutant R7s have more frequent defects in tiling than babo or dSmad2 mutant R7s, suggesting that imp-α3 may transport additional nuclear proteins that, redundantly with the Activin pathway, restrict R7 terminals (Ting, 2007).

In addition to their classical nuclear import function, Importins have been implicated in mediating retrograde transport of signals from growth cones to the nucleus. Both Imp-α3 and dSmad2 are found throughout the length of R7 axons, and like loss of Activin signaling, disrupting retrograde axonal transport affects the intrinsic mechanism that restricts R7 terminals. These results are consistent with a model in which the Activin signal is received by R7 growth cones, and dSmad2 bound to Imp-α3 is transported through the axon and ultimately into the nucleus (Ting, 2007).

Surprisingly, Activin appears to be required in the R neurons and likely in R7s themselves: disrupting Activin in all R neurons causes R7 terminals to invade adjacent targets, and among the photoreceptors, only R7 and R8 express Activin. An attempt to test whether Activin is specifically required in R7s met with only partial success: sevenless-Gal4 (sev-Gal4) was used to express UAS-Activin^DN in R7s but not R8s and it was found that the resulting R7s temporarily overshoot their initial target layer, a phenotype also caused by loss of Babo or dSmad2. Thus, Activin can function as an autocrine effector. Unfortunately, the sev-Gal4 driver is no longer expressed by 40-50 hr APF, the time at which Activin prevents R7s from invading adjacent columns, and sev-Gal4/Activin^DN R7s appear normal at this time point (Ting, 2007).

Nonetheless, the finding that R7s and/or R8s are the source of Activin raises two mechanistic questions. First, if the R7 or R8 neurons themselves are providing the signal and the signal is simply transduced into the R7 nucleus, why might Activin, as has been argued, be secreted in the target region and received by the R7 growth cone (i.e., rather than being secreted and received by the cell body)? One possibility is that R7s use Activin to coordinate their developmental program with that of other cells within the medulla. For example, one could imagine that both R7 growth cones and their postsynaptic targets would encounter the Activin signal in the medulla at the same time, allowing them to coordinate their preparations for mutual synaptogenesis. A second question is, therefore, how might Activin signaling be coordinated with the R7 growth cones' arrival in the medulla? It was found that the Activin-processing enzyme Tolloid-related (Tlr) is located both at the R7-temporary target layer (at 17 hr APF) and at the final R7 target layer (at 50 hr APF) and that Tlr mutants exhibit severe R7 retinotopic map defects. One possibility is therefore that the medulla localization of Tlr might confer spatial and temporal specificity on Activin expressed by R7 and/or R8 (Ting, 2007).

Although Activin is also expressed in R8s, no evidence was found that Activin affects R8 axons, as shown by the fact that neither babo mutant R8s nor R8s expressing GMR-Gal4/UAS-Activin^DN exhibited connectivity defects. However, the possibility cannot be ruled out that redundancy obscures such a role (for example, there is no straightforward method of removing adjacent R8s) (Ting, 2007).

In the mushroom body, dActivin signaling results in upregulation of the ecdysone receptor gene, EcR-B1. Although EcR-B1 is expressed in essentially all photoreceptor neurons, three lines of evidence indicate that EcR-B1 is not the target gene of Activin signaling in R7s: the expression level of EcR-B1, as judged by anti-EcR-B1 staining, was not altered in babo mutant R7 clones; forced expression of EcR-B1 did not rescue babo mutant R7 defects; and USP R7 mutants did not phenocopy babo. In the dorsal cluster of Atonal-positive neurons, Babo-mediated signaling, via an EcR-independent pathway, mediates morphogenesis and axonal extension. The versatility of Activin signaling likely reflects its ability to regulate the expression of different genes in a context-dependent manner. It is speculated that dActivin signaling activates a transcriptional program that not only restricts growth cone motility once R7s are within their target layer but also promotes synaptogenesis. Such a model could explain the observed strong defects in R7-mediated behavior despite the infrequent specific defects in R7 tiling. Identifying the transcriptional targets of Activin signaling in R7s will likely provide insight into these processes (Ting, 2007).

Drosophila Activin-β and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain

The Drosophila Activin-like ligands Activin-β and Dawdle control several aspects of neuronal morphogenesis, including mushroom body remodeling, dorsal neuron morphogenesis and motoneuron axon guidance. This study shows that the same two ligands act redundantly through the Activin receptor Babo and its transcriptional mediator Smad2 (Smox), to regulate neuroblast numbers and proliferation rates in the developing larval brain. Blocking this pathway results in the development of larvae with small brains and aberrant photoreceptor axon targeting, and restoring babo function in neuroblasts rescued these mutant phenotypes. These results suggest that the Activin signaling pathway is required for producing the proper number of neurons to enable normal connection of incoming photoreceptor axons to their targets. Furthermore, as the Activin pathway plays a key role in regulating propagation of mouse and human embryonic stem cells, the observation that it also regulates neuroblast numbers and proliferation in Drosophila suggests that involvement of Activins in controlling stem cell propagation may be a common regulatory feature of this family of TGF-β-type ligands (Zhu, 2008).

It is not entirely clear how Activin signaling in neuroblast lineages maintains the wild-type number of brain neuroblasts. Approximately 100 neuroblasts per brain lobe are formed during embryogenesis, and most go quiescent at the embryonic/larval transition. From L1-L3 they progressively re-enter the cell cycle to resume their cell lineages. Perhaps the slower cell cycle of Activin pathway mutants inhibits exit from quiescence and promotes premature differentiation. Mutations in trol, which encodes a heparin sulfate proteoglycan Perlecan, prevent neuroblast reactivation and lead to a severe reduction in neuroblast numbers and brain size. Many TGF-β ligands bind to heparin sulfate proteoglycans, and thus part of the effect of Activin signaling on brain size might be mediated by Perlecan or other proteoglycans such as the glypican Dally (Zhu, 2008). The small brain size is not just caused by a reduced number of neuroblasts, however; babo mutant clones that contained a single neuroblast produced fewer daughter cells in a given time window than did wild-type neuroblasts, presumably due to the increased expression of Cyclin A and the delay in metaphase exit. One additional possibility is that the Activin signal may affect neuroblast temporal identity progression in larval neuroblast lineages, similar to the effect of temporal identity mutations on embryonic neuroblast lineages, leading to the failure to produce early, mid or late subsets of larval lineages. Testing this hypothesis awaits the development of markers for different neurons within optic lobe neuroblast lineages (Zhu, 2008).

The importance of Activin in regulating neuroblast proliferation is reminiscent of the positive role that Activin/Nodal signaling plays in regulating the cell cycle of mouse and human ES cells. In those cells, as in Drosophila neuroblasts, Activin/Nodal signaling enhances, but is not absolutely required for, cell proliferation. Another point of potential similarity is that the mES cells endogenously produce an Activin/Nodal signal leading to an autocrine/paracrine regulation of proliferation. While in situ data are not of sufficient resolution to unambiguously assign expression of actβ and daw to particular cell types, both are expressed in the optic proliferation zones where neuroblasts are highly concentrated. It is also possible that some ligand may be supplied by the innervating photoreceptors. Activin is strongly expressed in R7 and 8 and like hh and spitz may provide a tropic signal that simulates proliferation in the target tissue (Zhu, 2008).

Lastly, it is interesting to note that Activins are not the only TGF-β-like factors required for proliferation of Drosophila neuroblasts. The BMP family member Dpp is expressed in four regions in each brain lobe. Two lie in the dorsal and ventral margins of the posterior optic zone neuroepithelium near what has been termed the lamina glial precursor region, whereas the other two smaller zones are more interior at the base of the inner proliferation zone. In the brain, the dpp loss-of-function phenotype is remarkably similar to that seen in babo mutants. A potential trivial explanation for the similarity in phenotypes might be that Activin signaling is required for dpp expression, or vice versa. However, dpp is still expressed in babo mutants, and daw and actβ are both still expressed in dpp mutants, although it is difficult to know in each case whether the levels are equivalent. Thus, both Dpp and Activin signaling appear to be required to stimulate brain neuroblast proliferation (Zhu, 2008).

In addition to regulating proliferation in the brain, Dpp signaling plays a major role in regulating proliferation in other tissues, including the imaginal discs. Once again, Activins may collaborate with BMPs in regulating proliferation in this tissue. In particular, it is noted that babo mutants show ectopic P-H3 staining within the morphogenetic furrow of the eye disc, which is also observed in loss-of-function mutants in the Dpp receptor Tkv. Furthermore, babo mutant wing disc clones can grow large in contrast to clones mutant in dpp signaling components, although the overall sizes of babo mutant discs are not affected proportionally as much as is the brain. Therefore, the way in which BMP and Activin inputs regulate the cell cycle might be different in discs versus the brain, or the two tissues might exhibit different sensitivities to common inputs (Zhu, 2008).

How Activins and BMPs affect the cell cycle is not entirely clear. In the wing disc, Dpp signaling through Tkv/Mad has been shown to promote the G1-S transition. In the brain, babo mutants exhibit a decrease in the M/S ratio, which could be due to a decrease in cells at the G2/M phase of the cell cycle. Consistent with this view, it was found that Cyclin A levels are enhanced in babo mutants and that heterozygosity for a Cyclin A mutation suppresses the babo phenotype. This is very similar to that seen in dally mutants, which also affect brain development by causing a delay in the G2-M transition within the outer proliferation centers. Just as was found for babo mutants, heterozygosity for Cyclin A suppresses the dally cell cycle defect (Zhu, 2008).

One attractive model for how both BMPs and Activins contribute to cell cycle progression is that they regulate the cycle at different points: Activins at G2-M and BMPs at G1-S. Alternatively, since previous work has suggested that Cyclin A probably has roles in regulating both G2-M and G1-S transitions in Drosophila and since Smads can form heterotrimers, it may be that a composite signal composed of a Smad2/Mad/Medea heterotrimer acts at several points in the cell cycle. Interestingly, several potential target genes that are regulated by both Activin and BMP signals in the larval brain have been identified by microarray studies using activated receptors, but no obvious candidates for genes that might influence proliferation are evident within the list (Zhu, 2008).

Lastly, it is noted that daw plays a role in motoneuron axon guidance in the embryo, while actβ has been implicated in mushroom body remodeling and in regulating the terminal steps in photoreceptor R8 targeting during pupal stages. Since both ligands are expressed in each of these tissues, it is possible that there may be functional redundancy that limits the severity of the previously observed phenotypes. Consistent with this view, both actβ and daw modulate neurotransmission at the neuromuscular junction, and the double mutant phenotypes are more severe than those seen in the single mutants, similar to their redundant function in regulating neuroblast proliferation. In conclusion, all available data suggest that Activin signaling plays at least two important roles in Drosophila nervous system development. First, it ensures that the proper numbers of cells are produced in the CNS; and second, it helps establish correct functional connections between neurons and their synaptic partners (Zhu, 2008).

Effects of Mutation or Deletion

Homozygous babo mutant larvae develop more slowly than wild-type larvae, are smaller in length, and exhibit enlarged anal pads. The penetrance for all these phenotypes approaches 100%. A significant fraction of mutant homozygotes pupate and survive to pharate adults. On dissection, they are found to have normal thoracic cuticle and adult appendages, but portions of the abdominal cuticle are missing, suggesting a possible defect in the proliferation of the abdominal histoblasts. In contrast, very few babo mutants progress to the pharate stage. All babo alleles tested exhibited two additional defects: the failure to leave food and the failure to evert the anterior spiracles upon pupation. Because babo is maternally expressed, germ-line clones were generated with both the null and hypomorphic alleles. Maternally depleted animals derived from both alleles showed a lethal phase similar to the zygotic mutants (Brummel, 1999).

To investigate the cause of the lethality, the internal organs were examined from mutant third instar larvae. No significant abnormalites were noted in the morphology of the gut, fat body, or Malphigian tubules. However, a severe reduction in the size of the brain hemispheres was observed as well as a partial reduction in the size of the imaginal discs. The small discs gave rise to small adult structures in the dead pharates. Because the brain lobes appeared to be small, the development of the visual center was examined in babo mutants. As the eye disc matures, axons from photoreceptor cells posterior to the morphogenetic furrow fasciculate and send projections into the brain in a specific pattern. The axon bundles of photoreceptors one through six extend into the lamina, whereas those of photoreceptors seven and eight extend deeper into the medulla. In babo mutants, photoreceptor differentiation and axon bundling proceed normally, but the axons fail to extend into the brain. This phenotype is consistent with a number of explanations, the simplest of which is that the medulla and lamina targets in the optic lobe fail to form (Brummel, 1999).

The small size of the brains and discs might result either from a decrease in cell proliferation or an increase in cell death. To test for a reduction in cell division, larval tissue was labeled with BrdU, which is incorporated into S-phase cells. Wild-type brains display a characteristic mitotic replication pattern with high levels of cell division in the optic proliferation center, whereas the mutant brains show little or no DNA replication during the time interval of BrdU incorporation. No striking difference in BrdU incorporation into imaginal discs is observed consistent with the observation that the discs are only ~30% smaller. This slight reduction in size could be due to a more subtle change in cell proliferation or cell death. To test whether cell death might also be contributing to the reduced size of the mutant brains and discs, dying cells were stained with acridine orange. No increase in the number of dying cells is observed in either mutant brain or discs. An analysis was performed to see if the observed reduction in cell proliferation is the result of secondary changes in the expression levels or patterns of several important growth factors. When the expression patterns of dpp, wg, and hh in brains and discs are examined by in situ hybridization, no significant alterations in expression levels or patterns of these genes are observed. Taken together, these results suggest that loss of babo strongly reduces cell proliferation within the brain, whereas cell proliferation in imaginal discs is affected in a more subtle way. This is accomplished without an increased cell death or noticeable alterations in the expression of several patterning genes within these tissues (Brummel, 1999).

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date revised: 10 April 2010

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