BIOLOGICAL OVERVIEW

Although there is compelling proof for the existence of invertebrate BMP-like signaling pathways, the evidence for invertebrate TGF-beta or Activin-like signaling pathways has been scant. Baboon (Babo) is an invertebrate Activin type I receptor: the characterization of Baboon may well be the first evidence for the existence of an Activin-like signaling pathway in Drosophila. Null mutations and germ-line clonal analysis demonstrate that babo is not required during embryogenesis but is essential for proper pupation and adult viability. Loss of babo function results in late larval or early pupal lethality. The major defect in these mutants is a reduction of cell proliferation within the primordia for adult structures, specifically imaginal discs and brain tissue. Activated Babo can signal to vertebrate TGF-beta/Activin, but not to BMP-responsive promoters in cell culture. Activated Babo cannot bind to or interact with Drosophila Mad in tissue culture but can utilize a new Drosophila Smad homolog, dSmad2 (Smad on X), which relates most closely to the vertebrate Smads 2 and 3. Drosophila dSmad2 is highly expressed in tissues that require babo function and can be phosphorylated by either overexpression of activated Babo or by overexpression of wild-type Punt and Babo together. On the basis of these results, it is proposed that an Activin-like signaling pathway exists in Drosophila, which is required for proper cell proliferation in many primordial adult tissues (Brummel, 1999).

Upon cloning of Baboon, it was first necessary to test whether the newly identified receptor acted as a receptor for the known BMP-type ligands in Drosophila. The reduced size of imaginal discs and brain tissue is not unique to babo mutations: this is also a characteristic feature of mutations in Drosophila BMP signaling pathways. In the embryo, all three characterized BMP ligands, Decapentaplegic (Dpp), Screw (Scw), and Glass bottom boat (Gbb), participate in specific developmental processes. Because Scw and Dpp are both critically important for formation of the amnioserosa tissue at the blastoderm stage, a Kruppel-lacZ line was used to follow the fate of amnioserosa tissue in babo mutants: such tissue was found to be unaffected. The role of Babo in midgut development was analyzed. Between stages 14 and 16, three constrictions form in the midgut producing a four-chambered vessel. Dpp is required for formation of the second midgut constriction, whereas gbb mutants lack the first constriction. Mutant embryos derived from babo germ-line clones exhibit a normal four-chambered midgut at stage 16. Furthermore, visceral mesoderm expression of the Gbb target gene Antennapedia, and the Dpp target gene Ubx, are both normal in babo mutants. In addition to these activities, both Dpp and Gbb are required for growth and patterning of imaginal discs and larval brains. Therefore, the expression of several target genes was examined in babo mutant discs. In wild-type discs, the genes optomotor blind (omb) and spalt (sal) respond to Dpp in a dose-dependent fashion. In addition, proper omb expression also requires input from the Gbb ligand. In leg discs, Dpp represses wg in the ventral portion of the disc. No alterations were found in the expression patterns of omb, sal, and wg in any babo mutant disc. These results indicate that babo does not likely function as a receptor for Dpp, Scw, or Gbb during Drosophila development (Brummel, 1999).

Clonal analysis of babo mutants as well as overexpression of constitutively activated Babo receptors was carried out to identify the developmental function of Babo. The results suggest that Babo primarily regulates cell proliferation and has only minimal affects on patterning. The consequences of expressing a constitutively active form of Babo (Babo*, Q302D) in imaginal discs via the UAS-Gal4 system was examined. Ubiquitous expression of the constitutively active receptor leads to tissue overgrowth in the wing, but only limited pattern abnormalities. Surface area measurements indicate an ~30% increase in wing size for a particular UAS-Babo* line. To determine whether this increase in wing surface area results from an increase in cell size or cell number, wing hairs (single apical extensions found on the surface of each wing blade cell) were counted within a fixed area at two locations on the dorsal surface of the wing. An ~20% increase in the density of cells at both positions was found, as compared with control flies. Assuming that there is no decrease in cell death, and taking into account the ~30% increase in wing size, these results suggest that one of every two cells undergoes an extra round of division during wing formation as a result of ectopically activated Babo expression (Brummel, 1999).

Because genetic data indicate that in Drosophila Babo is not a component of Dpp signaling, the specificity of Babo activity was examined in mammalian cell culture assays using several different pathway specific promoters. Using a TGF-beta/Activin responsive promoter, p3TP, fused to luciferase, cultured cells were cotransfected with the reporter alone or with wild-type forms of Babo or the mammalian TGFbeta type-I receptor, TbetaRI. In the absence of TGF-beta, only basal levels of luciferase activity are observed. Addition of TGF-beta or transfection with an activated TbetaRI results in a strong induction of the tagged promoter. A similar induction is observed when the constitutively active form of Babo is cotransfected with the tagged promoter. In contrast, cotransfection with the constitutively active Dpp receptor Tkv does not stimulate the TGF-beta/Activin responsive reporter. Similar results were obtained with a second Activin/TGF-beta responsive element (known as ARE). Cotransfection of cultured cells with the second tagged promoter and activated Babo results in a fivefold induction in luciferase activity. This stimulation is similar to levels of induction produced by the activated forms of mammalian receptors TbetaRI or ActRIB. Tkv fails to modulate induction of the second TGF-beta/Activin responsive promoter. Together these data indicate that Babo specifically activates a TGF/Activin-like pathway in mammalian cells and suggest that this receptor may regulate a Smad2/Smad3-like pathway in Drosophila (Brummel, 1999).

Baboon was shown to function through the newly identified dSmad2, a Drosophila homolog of mammalian Smad2 and Smad3, which function in Activin signaling. Activated Babo induces phosphorylation on the last two serines of dSmad2. These data suggest that dSmad2 is a downstream target of Babo and is phosphorylated on the last two serine residues in the carboxyl terminus. Babo-dependent phosphorylation of dSmad2 also induces association with Medea, a homolog of mammalian Smad4. Phosphorylation of dSmad2 on the last two serines is necessary for receptor-dependent induction of heteromeric complexes of dSmad2 and Medea. Mammalian Activin receptors require dimerization with a type II receptor for their function. Punt, the Drosophila type II receptor was shown to function to activate Babo. dSmad2 interacts transiently and specifically with Punt-Babo receptor complexes. Taken together, these functional and biochemical analyses strongly suggest that dSmad2 is a Drosophila homolog of Smad2/Smad3 and functions as a downstream signaling component that directly interacts with Babo (Brummel, 1999).

The simplicity of the babo loss-of-function phenotype in Drosophila is striking in light of the number of speculated roles for Activin signaling in vertebrates. In particular, the fact that babo loss-of-function mutations primarily affects cell growth and proliferation in late development, but not cell fate specification or patterning processes, stands in marked contrast to the situation in vertebrates in which Activin or Activin-like signaling pathways have been implicated in a wide range of early developmental events, as well as adult functions such as reproductive potential. Similarly, in Drosophila the BMP-like factor Dpp has been implicated in the control of cell fate, cell proliferation, and patterning processes. Thus, the Babo signaling pathway appears to be unique in that it seems to act primarily in only one of these interconnected processes. It should be recognized, however, that Babo signaling may contribute to some patterning processes and/or cell fate decisions. For example, in the brain, it has yet to be determined whether all of the morphological defects can be accounted for by reduced proliferation rates of certain neuroblast populations or whether there are also changes in cell fate specification. Likewise, the cause of the enlarged anal pads, observed in babo mutants, during larval development has not been investigated. As the anal organ is involved in regulating salt homeostasis in insects, the enlarged anal pads might simply represent swelling due to a salt imbalance or the inability to regulate water content. Larvae missing babo function also fail to exhibit a typical behavioral trait, which is to move away from moist food before pupation. Thus, the inability to assess hydration as the result of abnormal brain development may underlie both the enlarged anal pads and the failure to seek a drier climate for pupation (Brummel, 1999).

The Babo pathway appears to act in a positive manner to stimulate cell proliferation. How might Babo signaling couple to cell proliferation? Recent analysis of cell growth and division in the Drosophila wing imaginal discs has led to the conclusion that the cell division rate is normally coupled to increases in cell mass such that a relatively constant ratio between the two processes is maintained (Neufeld, 1998). Because mutations that affect protein synthesis, such as Minutes, retard cell proliferation without changing cell size, Neufeld favored a model in which tissue growth is upstream and dominant to cell cycle control. Consistent with this view, it was found that if the cell cycle is artificially stimulated to give a higher division rate, then the size of the cells decreases. Conversely, if mitosis in the wing is blocked by inactivation of the Drosophila cdc2 kinase, then wings of normal size are produced but they contain smaller numbers of larger cells (Weigmann, 1997). Overexpression of activated Babo in the wing appears to alter the process that couples growth and proliferation rates with the determination of tissue size. The simplest explanation for these results is that Babo signaling stimulates some aspect of the cell cycle resulting in an increased proliferation rate and reduced cell size similar to that described by Neufeld. However, the fact that larger wings are seen despite the smaller cell size suggests that Babo may independently affect tissue growth parameters as well as the cell cycle. Therefore, additional studies on the mechanism of Babo signaling could help reveal how cell growth and proliferation are linked to the determination of final tissue size (Brummel, 1999).

TGF-β signals regulate axonal development through distinct Smad-independent mechanisms

Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Transforming growth factor β (TGFβ) signals regulate axon growth. In most contexts, TGFβ signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFβ responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFβ receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFβ receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo (Ng, 2008).

Once growing axons reach the correct postsynaptic target, axon outgrowth terminates and synaptogenesis begins. These studies suggest that TGFβ signals play a role. When Babo is inactivated, MB axon growth does not terminate properly and overextends across the midline. Consistent with this, CA Babo expression results in precocious termination, forming axon truncations. How Babo is spatially and temporally regulated remains to be determined. Analogous to the Drosophila NMJ, MB axon growth might be terminated through retrograde signalling. Target-derived TGFβ ligands could signal to Babo (on MB axon growth cones) and stop axons growing further. In an alternative scenario, TGFβ ligands might act as a positional cue that prevents MB axons from crossing the midline. Recent data have shown that Babo acting through Smad2 restricts individual R7 photoreceptor axons to single termini. Loss of Babo, Smad2, or the nuclear import regulator Importin α3 (Karyopherin α3 - FlyBase), results in R7 mutant axons invading neighbouring R7 terminal zones. With the phenotype described in this study, Babo could similarly be restricting MB axons to appropriate termination zones, its loss resulting in inappropriate terminations on the contralateral side (Ng, 2008).

In contrast to MB neurons, Babo inactivation in AL and OL neurons resulted in axon extension and targeting defects. This might reflect cell-intrinsic differences in the response in different neurons to a common Babo signalling program. This may be the case for MB axon pruning and DC axon extension, which require Babo/Smad2 signals. Whether these differences derive from cell-intrinsic properties, or from Babo signal transduction, they underline the importance of Smad-independent signals in many aspects of axonal development (Ng, 2008).

The results suggest that Smad-independent signals involve Rho GTPases. One caveat in genetic interaction experiments is that the loss of any given gene might not be dosage-sensitive with a particular assay. Nevertheless, all the manipulations together suggest that Babo-regulated axon growth requires Rho1, Rac and LIMK1. How Babo signals involve Rho GTPases remains to be fully determined. In addition to LIMK1, which binds to Wit, one possibility, as demonstrated for many axon guidance receptors, is that the RhoGEFs, RhoGAPs and Rho proteins might be linked to the Babo receptor complex. Thus, ligand-mediated changes in receptor properties would lead to spatiotemporal changes in Rho GTPase and LIMK1 activities (Ng, 2008).

The data suggest that a RhoGEF2/Rho1/Rok/LIMK1 pathway mediates Babo responses. Whether Rac activators are required is unclear, as tested RacGEFs do not genetically interact with babo. In this respect, rather than through GEFs, Babo might regulate Rac through GAPs, by inhibiting Tumbleweed (Tum) activity (Ng, 2008).

Do mutations in Rho1 and Rac components phenocopy babo phenotypes? β lobe overextensions are observed in Rok, Rho1 and Rac mutant neurons. In MB neurons, Rac GTPases also control axon outgrowth, guidance and branching. Rho1 also has additional roles in MB neurons. Although Rho1 mutant neuroblasts have cell proliferation defects, single-cell αβ clones do show β lobe extensions. RhoGEF2 strong loss-of-function clones do not exhibit axon overextension. As there are 23 RhoGEFs in the Drosophila genome, there might well be redundancy in the way Rho1 is activated. LIMK1 inactivation in MB neurons was reported previously. Axon overextensions were not observed as LIMK1 loss results in axon outgrowth and misguidance phenotypes. This suggests that LIMK1 mediates multiple axon guidance signals, of which TGFβ is a subset in MB morphogenesis (Ng, 2008).

Although their phenotypes are similar, several lines of evidence indicate that CA Babo does not simply reflect LIMK1 misregulation in MB neurons. First, whereas LIMK1 genetically interacts with most Rho family members and many Rho regulators, CA babo is dosage-sensitive only to Rho1 and Rac and specific Rho regulators, suggesting that Babo regulates LIMK1 only through a subset of Rho signals (Ng, 2008).

Second, the LIMK1 misexpression phenotype is suppressed by expression of wild-type cofilin (Twinstar Tsr), S3A Tsr, or the cofilin phosphatase Slingshot (Ssh). By contrast, only wild-type Tsr, but not S3A Tsr or Ssh, suppresses CA Babo. The suppression by wild-type Tsr might reflect a restoration of the endogenous balance or spatial distribution of cofilin-on (unphosphorylated) and -off (phosphorylated) states within neurons. Indeed, optimal axon outgrowth requires cofilin to undergo cycles of phosphorylation and dephosphorylation. Since S3A forms of cofilin cannot be inactivated and recycled from actin-bound complexes, wild-type cofilin is more potent in actin cytoskeletal regulation (Ng, 2008).

CA Babo might not simply misregulate LIMK1 but also additional cofilin regulators. Recent data suggest that extracellular cues (including mammalian BMPs) can regulate cofilin through Ssh phosphatase and phospholipase Cγ activities. In different cell types, cofilin phosphorylation and phospholipid binding (which also inhibits cofilin activity) states vary and potently affect cell motility and cytoskeletal regulation. Whether a combination of LIMK1, Ssh and phospholipid regulation affects cofilin-dependent axon growth remains to be determined (Ng, 2008).

Third, by phalloidin staining, LIMK1, but not CA Babo, misexpression results in a dramatic increase in F-actin in MB neurons. Thus, CA Babo does not in itself lead to actin misregulation. Fourth, Babo also regulates axon growth independently of LIMK1 (Ng, 2008).

This study differs significantly from the canonical model of Smad signalling, in which type 1 receptors function downstream of the ligand-type 2 receptor complex. In this study, the gain- and loss-of-function results suggest that type 2 receptors act downstream of type 1 signals. Since ectopic only Wit and Put suppress the babo axon overextension phenotype, this implies that Smad-dependent and -independent signals have distinct type 1/type 2 receptor interactions. How these interactions propagate Smad-independent signals remains to be fully determined. Babo could act as a ligand-binding co-receptor with Wit and Put. In addition, Babo kinase activity could regulate type 2 receptor or Rho functions. The results suggest, however, that provided that Wit or Put signals are sufficiently high, Babo is not required. Whatever the mechanism(s), it is likely that Babo requires the Wit C-terminus-LIMK1 interaction to relay cofilin phosphoregulatory signals. How Put functions is unclear. Since the put¹³⁵ allele (used in this study) carries a missense mutation within the kinase domain, this suggests that kinase activity is essential. put does not genetically interact with LIMK1. Since Put lacks the C-terminal extension of Wit that is necessary for LIMK1 binding, this suggests that Put acts independently of LIMK1. One potential effector is Rac, which, in the context of Babo signalling, also appears to be Pak1- and thus LIMK1-independent (Ng, 2008).

In MB neurons, Wit and Put can function interchangeably. In other in vivo paradigms, type 2 receptors are not interchangeable. However, since the Wit C-terminal tail is required to substitute for Put, this suggests that Wit axon growth signals are independent of its kinase activity. Together, this suggests that Smad-independent signals involve LIMK1-dependent and -independent mechanisms (Ng, 2008).

This study shows that Babo mediates two distinct responses in related MB populations. How do MB neurons choose between axon pruning and axon growth? The babo rescue studies suggest that whereas Babo_a or Babo_b elicits Smad-independent responses, only Babo_a mediates Smad-dependent responses. Since Babo isoforms differ only in the extracellular domain, differences in ligand binding could determine Smad2 or Rho GTPase activation. However, it is worth noting that in DC neurons, either isoform mediates axon extension through Smad2 and Medea. In addition, although expressed in all MB neurons, CA babo misexpression (which confers ligand-independent signals) perturbs only αβ axons. Thus, cell-intrinsic properties might also be essential in determining Babo responses (Ng, 2008).

Many TGFβ ligands signal through Babo. For example, Dawdle, an Activin-related ligand, patterns Drosophila motor axons, whereas Activin (Activin-β, FlyBase) is required for MB axon pruning. Whether these ligands regulate Babo MB, AL and OL axonal morphogenesis is unclear. Taken together, the evidence suggests that Babo signalling is varied in vivo and is involved in many aspects of neuronal development (Ng, 2008).

TGFβ signals are responsible for many aspects of development and disease and, throughout different models, Smad pathways are closely involved. Although Smad-independent pathways are known, their mechanisms and roles in vivo are unclear. TGFβ signals often drive cell shape changes in vivo. During epithelial-to-mesenchymal transition (EMT), cells lose their epithelial structure and adopt a fibroblast-like structure that is essential for cell migration during development and tumour invasion. TGFβ-mediated changes in the actin cytoskeleton and adherens junctions are necessary for EMT. Although Smads are crucial, TGFβ signals also involve the Cdc42-Par6 complex, resulting in cell de-adhesion and F-actin breakdown through Rho1 degradation. In other studies, however, TGFβ-mediated EMT has been shown to require Rho1, which can be regulated by Smad activity (Ng, 2008).

Many TGFβ-driven events in Drosophila are Smad-dependent. Whether Smad-independent roles exist beyond those identified in this study remains to be tested. This study therefore provides a framework to understand how non-Smad signals regulate cell morphogenesis during development (Ng, 2008).

Extrinsic activin signaling cooperates with an intrinsic temporal program to increase mushroom body neuronal diversity

Temporal patterning of neural progenitors leads to the sequential production of diverse neurons. To understand how extrinsic cues influence intrinsic temporal programs, Drosophila mushroom body progenitors (neuroblasts) were studied that sequentially produce only three neuronal types: γ, then α'β', followed by αβ. Opposing gradients of two RNA-binding proteins Imp and Syp comprise the intrinsic temporal program. Extrinsic activin signaling regulates the production of α'β' neurons but whether it affects the intrinsic temporal program was not known. This study shows that the activin ligand Myoglianin from glia regulates the temporal factor Imp in mushroom body neuroblasts. Neuroblasts missing the activin receptor Baboon have a delayed intrinsic program as Imp is higher than normal during the α'β' temporal window, causing the loss of α'β' neurons, a decrease in αβ neurons, and a likely increase in γ neurons, without affecting the overall number of neurons produced. These results illustrate that an extrinsic cue modifies an intrinsic temporal program to increase neuronal diversity (Rossi, 2020).

The building of intricate neural networks during development is controlled by highly coordinated patterning programs that regulate the generation of different neuronal types in the correct number, place and time. The sequential production of different neuronal types from individual progenitors, i.e. temporal patterning, is a conserved feature of neurogenesis. For instance, individual radial glia progenitors in the vertebrate cortex sequentially give rise to neurons that occupy the different cortical layers in an inside-out manner. In Drosophila, neural progenitors (called neuroblasts) also give rise to different neuronal types sequentially. For example, projection neurons in the antennal lobe are born in a stereotyped temporal order and innervate specific glomeruli. In both of these examples, individual progenitors age concomitantly with the developing animal (e.g., from embryonic stages 11-17 in mouse and from the first larval stage (L1) to the end of the final larva stage (L3) in Drosophila). Thus, these progenitors are exposed to changing environments that could alter their neuronal output. Indeed, classic heterochronic transplantation experiments demonstrated that young cortical progenitors placed in an old host environment alter their output to match the host environment and produce upper-layer neurons (Rossi, 2020).

The adult Drosophila central brain is built from ~100 neuroblasts that divide continuously from L1 to L3. Each asymmetric division regenerates the neuroblast and produces an intermediate progenitor called ganglion mother cell (GMC) that divides only once, typically producing two different cell types. Thus, during larval life central brain neuroblasts divide 50-60 times, sequentially producing many different neuronal types. All central brain neuroblasts progress through opposing temporal gradients of two RNA-binding proteins as they age: IGF-II mRNA binding protein (Imp) when they are young and Syncrip (Syp) when they are old. Loss of Imp or Syp in antennal lobe or Type II neuroblasts affects the ratio of young to old neuronal types. Imp and Syp also affect neuroblast lifespan. Thus, a single temporal program can affect both the diversity of neuronal types produced and their numbers (Rossi, 2020).

Since central brain neuroblasts produce different neuronal types through developmental time, roles for extrinsic cues have recently garnered attention. Ecdysone triggers all the major developmental transitions including progression into the different larval stages and entry in pupation. The majority of central brain neuroblasts are not responsive to ecdysone until mid-larval life when they begin to express the Ecdysone Receptor (EcR). Expressing a dominant-negative version of EcR (EcR-DN) in Type II neuroblasts delays the Imp to Syp transition that normally occurs ~60 hr after larval hatching (ALH). This leads to many more cells that express the early-born marker gene Repo and fewer cells that express the late-born marker gene Bsh (Rossi, 2020).

To further understand how extrinsic signals contribute to temporal patterning, Drosophila mushroom body neuroblasts were studied because of the deep understanding of their development. The mushroom body is comprised of ~2000 neurons (Kenyon cells) that belong to only three main neuronal types that have unique morphologies and play distinct roles in learning and memory. They receive input mainly from ~200 projection neurons that each relays odor information from olfactory receptor neurons. Each projection neuron connects to a random subset of Kenyon cells and each Kenyon cell receives input from ~7 different projection neurons. This connectivity pattern requires a large number of mushroom body neurons (~2,000) to represent complex odors. To produce this very large number of neurons, mushroom body development is unique in many respects. Mushroom body neurons are born from four identical neuroblasts that divide continuously (unlike any other neuroblast) from the late embryonic stages until the end of pupation (~9 days for ~250 divisions each). Furthermore, the two neurons born from each mushroom body GMC are identical. The neuronal simplicity of the adult mushroom body makes it ideal to study how extrinsic cues might affect diversity since the loss of any single neuronal type is obvious given that each is represented hundreds of times (Rossi, 2020).

The three main neuronal types that make up the adult mushroom body are produced sequentially during neurogenesis: first γ, followed by α'β', and then αβ neurons (see α'β' neurons are not generated from babo mutant neuroblasts), representing the simplest lineage in the central brain. The γ temporal window extends from L1 (the first larval stage) until mid-L3 (the final larval stage) when animals attain critical weight and are committed to metamorphosis; the α'β' window from mid-L3 to the beginning of pupation, and the αβ window from pupation until eclosion (the end of development). Like all other central brain neuroblasts Imp and Syp are expressed by mushroom body neuroblasts, but in much shallower gradients through time, which accounts for their extended lifespan. Imp and Syp are inherited by newborn neurons where they instruct temporal identity. Imp positively and Syp negatively regulate the translation of chronologically inappropriate morphogenesis (chinmo), a gene encoding a transcription factor that acts as a temporal morphogen in neurons. The first-born γ neurons are produced for the first ~85 cell divisions, when Imp levels in neuroblasts, and thus Chinmo in neurons, are high. α'β' neurons are produced for the next ~40 divisions, when Imp and Syp are at similar low levels that translate into lower Chinmo levels in neurons. Low Chinmo then regulates the expression in neurons of maternal gene required for meiosis (mamo), which encodes a transcription factor that specifies the α'β' fate and whose mRNA is stabilized by Syp. αβ neurons are generated for the final ~125 neuroblast divisions, when Syp levels are high, Imp is absent in neuroblasts, and thus Chinmo and Mamo are no longer expressed in neurons (Rossi, 2020).

Extrinsic cues are known to have important roles in regulating neuronal differentiation during mushroom body neurogenesis. The ecdysone peak that controls entry into pupation regulates γ neuron axonal remodeling. Ecdysone was also proposed to be required for the final differentiation of α'β' neurons. EcR expression in γ neurons is timed by activin signaling, a member of the TGFβ family, from local glia. Activin signaling from glia is also required for the α'β' fate (Marchetti, 2019): Knocking-down the activin pathway receptor Baboon (Babo) leads to the loss of α'β' neurons. It was proposed that activin signaling in mushroom body neuroblasts regulates the expression of EcR in prospective α'β' neurons and that when the activin pathway is inhibited, it leads to the transformation of α'β' neurons into later-born pioneer-αβ neurons (a subclass of the αβ class) (Marchetti and Tavosanis, 2019) (Rossi, 2020).

Although there is strong evidence that extrinsic cues have important functions in neuronal patterning in the Drosophila central brain, it remains unknown how extrinsic temporal cues interface with the Imp and Syp intrinsic temporal program to regulate neuronal specification. This question was addressed using the developing mushroom bodies. Activin signaling from glia was shown to be required for α'β' specification. However, this study also showed that activin signaling lowers the levels of the intrinsic factor Imp in mushroom body neuroblasts to define the mid-α'β' temporal identity window. Removing the activin receptor Babo in mutant clones leads to the loss of α'β' neurons, to fewer last-born αβ neurons, and to the likely generation of additional first-born γ neurons without affecting overall clone size. This appears to be caused by a delayed decrease in Imp levels, although the intrinsic temporal clock still progresses even in the absence of activin signaling. This study also demonstrated that ecdysone signaling is not necessary for the specification of α'β' neurons, although it might still be involved in later α'β' differentiation. These results provide a model for how intrinsic and extrinsic temporal programs operate within individual progenitors to regulate neuronal specification (Rossi, 2020).

Mushroom body neurogenesis is unique and programmed to generate many copies of a few neuronal types. During the early stages of mushroom body development, high Imp levels in mushroom body neuroblasts are inherited by newborn neurons and translated into high Chinmo levels to specify γ identity. As in other central brain neuroblasts, as development proceeds, inhibitory interactions between Imp and Syp help create a slow decrease of Imp and a corresponding increase of Syp. However, at the end of the γ temporal window (mid-L3), activin signaling from glia acts to rapidly reduce Imp levels in mushroom body neuroblasts without significantly affecting Syp, establishing a period of low Imp (and thus low Chinmo in neurons) and also low Syp. This is required for activating effector genes in prospective α'β' neurons, including Mamo, whose translation is promoted by Syp (Liu, 2019). The production of αβ identity begins when Imp is further decreased and Syp levels are high during pupation (see Model of how activin signaling defines the α'β' temporal identity window.). Low Chinmo in αβ neurons is also partly regulated by ecdysone signaling through the activation of Let-7-C, which targets chinmo for degradation. Based on this model, α'β' neurons could not be rescued by knocking-down Imp in babo clones, since low Imp is required for α'β' specification while the knockdown reduces its level below this requirement. It would be expected to rescue α'β' neurons if Imp levels were specifically reduced to the appropriate levels at L3. However, reducing Imp levels might not be the only function of activin signaling, which may explain why α'β' neurons are not simply made earlier (e.g., during L1-L2) when Imp is knocked-down (Rossi, 2020).

In babo mutant clones, it is speculated that additional γ neurons are produced at the expense of α'β' neurons since Imp levels in neuroblasts (as well as Chinmo in neurons) are higher for a longer time during development; There was also a significant decrease in the total number of αβ neurons in babo mutant clones that contrasts with a recent report by Marchetti (2019) that instead concluded that additional pioneer-αβ neurons are produced. It is believed that there is both an increase in the number of γ neurons and of the pioneer-αβ neuron subclass because pioneer-αβ neurons are the first of the αβ class to be specified (when Imp is still present at very low levels) during pupation. It is speculated that pioneer-αβ neurons are produced during the extended low Imp window that was detected during pupation in babo clones. However, this does not leave the time for the remaining population of αβ neurons to be formed, which explains why their number is reduced (Rossi, 2020).

This study has focused on the three main classes of mushroom body neurons although at least seven subtypes exist: 2 γ, 2 α'β' and 3 αβ. The subtypes are specified sequentially suggesting that each of the three broad mushroom body temporal windows can be subdivided further, either by fine-scale reading of the changing Imp and Syp gradients, by additional extrinsic cues, or perhaps by a tTF series as in other neuroblasts (Rossi, 2020).

Postembryonic central brain neuroblasts are long-lived and divide on average ~50 times. Unlike in other regions of the developing Drosophila brain, rapidly progressing series of tTFs have not yet been described in these neuroblasts. Instead, they express Imp and Syp in opposing temporal gradients. Conceptually, how Imp and Syp gradients translate into different neuronal identities through time has been compared to how morphogen gradients pattern tissues in space. During patterning of the anterior-posterior axis of the Drosophila embryo, the anterior gradient of the Bicoid morphogen and the posterior Nanos gradient are converted into discrete spatial domains that define cell fates. Since gradients contain unlimited information, differences in Imp and Syp levels through time could translate into different neuronal types. Another intriguing possibility is that tTF series could act downstream of Imp and Syp, similarly to how the gap genes in the Drosophila embryo act downstream of the anterior-posterior morphogens. This study has shown that another possibility is that temporal extrinsic cues can be incorporated by individual progenitors to increase neuronal diversity. In mushroom body neuroblasts activin signaling acts directly on the intrinsic program, effectively converting two broad temporal windows into three to help define an additional neuronal type. It is proposed that subdividing the broad Imp and Syp temporal windows by extrinsic cues may be a simple way to increase neuronal diversity in other central brain neuroblasts (Rossi, 2020).

This study has also shown that activin signaling times the Imp to Syp transition for mushroom body neuroblasts, similar to the function of ecdysone for other central brain neuroblasts. In both cases however, the switch still occurs, indicating that a separate independent clock continues to tick. This role for extrinsic cues during Drosophila neurogenesis is reminiscent of their roles on individual vertebrate progenitors. For example, hindbrain neural stem cells progressively produce motor neurons followed by serotonergic neurons before switching to producing glia. The motor neuron to serotonergic neuron switch is fine-tuned by TGFβ signaling. It would be interesting to determine if hindbrain neuronal subtypes are lost in TGFβ mutants, similar to how α'β' identity is lost in the mushroom bodies in babo mutants (Rossi, 2020).

The specification of α'β' neurons begins at mid-L3 with the onset of Mamo expression. In contrast, high levels of EcR are detected in mature mushroom body neurons starting at late L3. At this stage, both γ and α'β' neurons already exist and new α'β' neurons are still being generated. Thus, Mamo expression precedes EcR expression. These non-overlapping expression patterns suggest that ecdysone signaling does not regulate Mamo and therefore cannot control the specification of α'β' neurons. Furthermore, expression of UAS-EcR-RNAi or mutants for usp do not lead to the loss of α'β' neurons. It is noted that usp results contradict the loss of α'β' neuron reported by Marchetti (2017) in usp clones. However, α'β' neurons were seen in these clones based on the morphology of these neurons but the remodeling defect of γ neurons makes α'β' neurons difficult to identify. Nevertheless, ecdysone might still function later during α'β' differentiation, particularly during pupation when all mushroom body neurons express EcR (Rossi, 2020).

This study and that of Marchetti both show that expression of UAS-EcR-DN leads to the loss of α'β' neurons by acting in mushroom body neurons but not in neuroblasts. However, EcR must be first be expressed in the target cells of interest in order to make any conclusions about ecdysone function using UAS-EcR-DN. Since this study could not detect EcR protein in Mamo+ cells at L3, but expressing UAS-EcR-DN inhibits Mamo in those cells, it is concluded that EcR-DN artifactually represses Mamo and leads to the loss of α'β' neurons. This explains why expressing UAS-EcR-B1 does not rescue α'β' neurons in babo clones. However, Marchetti did rescue babo-RNAi by expressing EcR (Marchetti, 2019). This is likely because the current experiments were performed using babo MARCM clones in which the loss of α'β' neurons is much more severe than with babo-RNAi used in their experiments. Indeed, when attempts were made to eliminate α'β' neurons using a validated UAS-babo-RNAi construct, γ neurons did not remodel but there was only a minor (but significant) decrease in the number of α'β' neurons. This indicates that knocking-down babo with mb-Gal4 that is only weakly expressed in neuroblasts and newborn neurons is not strong enough to inhibit α'β' specification. Thus, it is speculated that the LexA line used by Marchetti (GMR26E01-LexA) may not be a reliable reporter for α'β' neurons upon babo knockdown, and that it might be ecdysone sensitive later in α'β' differentiation. Since EcR expression in all mushroom body neurons at L3 may be dependent on activin signaling directly in neurons, as it is in γ neurons for remodeling, expressing UAS-EcR-B1 together with UAS-babo-RNAi using OK107-Gal4 might both reduce the effectiveness of the RNAi while also allowing for the re-expression of GMR26E01-LexA (Rossi, 2020).

Glia are a source of the activin ligand myo, which is temporally expressed in brain glia starting at L3 to initiate the remodeling of mushroom body γ neurons (Awasaki, 2011) and α'β' specification (this study and Marchetti, 2019). However, knocking-down Myo from glia is not as severe as removing Babo from mushroom body neuroblasts. This might be due to incomplete knockdown of myo or to other sources of Myo, potentially from neurons. For example, in the vertebrate cortex, old neurons signal back to young neurons to control their numbers. It is also possible the Babo is activated by other activin ligands, including Activin and Dawdle. An intriguing hypothesis is that the temporal expression of myo in glia beginning at mid-L3 is induced by the attainment of critical weight and rising ecdysone levels. It would be interesting to determine whether blocking ecdysone signaling in glia leads to the loss of α'β' specification, similar to how blocking ecdysone reception in astrocytes prevents γ neuron remodeling (Rossi, 2020).

It is well established that extrinsic cues play important roles during vertebrate neurogenesis, either by regulating temporal competence of neural stem cells or by controlling the timing of temporal identity transitions. Competence changes mediated by extrinsic cues were demonstrated in classic heterochronic transplantation studies that showed that young donor progenitors produce old neuronal types when placed in older host brains. Recent studies show that the reverse is also true when old progenitors are placed in a young environment (Rossi, 2020).

Mechanisms of intrinsic temporal patterning are also conserved. For example, vertebrate retinal progenitor cells use an intrinsic tTF cascade to bias young, middle, and old retinal fates. Two of the factors (Ikaros and Casz1) used for intrinsic temporal patterning are orthologs to the Drosophila tTFs Hb and Cas. tTF series might also exist in cortical radial glia progenitors and even in the spinal cord. Recent results also show the importance of post-transcriptional regulation in defining either young or old cortical fates, which can be compared to the use of post-transcriptional regulators that are a hallmark of neuronal temporal patterning in Drosophila central brain neuroblasts. These studies highlight that the mechanisms driving the diversification of neuronal types are conserved (Rossi, 2020).

GENE STRUCTURE

Both BABO transcripts are encoded within a 9-kb genomic fragment and contain seven exons with exon 3a/b being differentially spliced to produce the two isoforms (Brummel, 1999)

Number of exons - 7

PROTEIN STRUCTURE

Amino Acids - 601

Structural Domains

A transmembrane protein serine/threonine kinase, Baboon, that is structurally related to receptors for members of the transforming growth factor-beta (TGF-beta) family has been cloned from Drosophila. The spacing of extracellular cysteines and the cytoplasmic kinase domain of Baboon resemble most closely those of the recently described mammalian type I receptors for TGF-beta and activin. The kinase domain of Baboon bears a 60% to 72% amino acid identity to mammalian type I receptors. The Baboon kinase domain is more distantly related (37% to 40% amino acid sequence identity) to those of the type II receptors, including Drosophila Punt. The extracellular domain of Baboon shows little sequence similarity to other receptors and is larger than those of serine/threonine kinase receptors from vertebrates. The spacing of the 10 extracellular cysteines in Baboon resembles the spacing in the other type I receptors, and includes the cysteine box motif near the transmembrane region that is characteristic of the serine/threonine kinase receptor family. Comparison of Baboon with other type I receptors reveals the presence of a characteristic 30-amino-acid domain immediately upstream of the kinase region in all these receptors. This domain, of unknown function, contains a repeated Gly-Ser sequence and is therefore referred to as the GS domain. Two alternative forms of Baboon have been identified that differ in an ectodomain region encompassing the cysteine box motif characteristic of receptors in this family. The second class of cDNA is represented by one clone of 4.9 kb encoding a product in which a 70-amino-acid sequence replaces a 49-amino-acid sequence in the extracellular region near the transmembrane domain. Aside from the cysteine box, which is partially included in this region, there is virtually no similarity between the two alternative sequences. The larger size of Baboon compared with mammalian type I receptor (Baboon contains a 94 amino acid N-terminal region that is not present in the mammalian proteins) correlates with the presence of a large extracellular region and more N-linked glycosylation sites in Baboon (Wrana, 1994).

date revised: 5 November 2023

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