semaphorin-1a: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - semaphorin1a

Synonyms - Semaphorin I, D-Sema-I, Fasciclin-IV

Cytological map position - 29E1--29E2

Function - transmembrane protein

Keywords - axonogenesis

Symbol - sema1a

FlyBase ID: FBgn0011259

Genetic map position - 2-[30]

Classification - semaphorin family

Cellular location - cell surface

NCBI links: Entrez Gene

sema1a orthologs: Biolitmine
Recent literature
Hong, Y. G., Kang, B., Lee, S., Lee, Y., Ju, B. G. and Jeong, S. (2020). Identification of cis-regulatory region controlling Semaphorin-1a expression in the Drosophila embryonic nervous system. Mol Cells. PubMed ID: 32024353
The Drosophila transmembrane semaphorin Sema-1a mediates forward and reverse signaling that plays an essential role in motor and central nervous system (CNS) axon pathfinding during embryonic neural development. Previous immunohistochemical analysis revealed that Sema-1a is expressed on most commissural and longitudinal axons in the CNS and five motor nerve branches in the peripheral nervous system (PNS). This study uncovered three cis -regulatory elements (CREs), R34A03, R32H10, and R33F06, that robustly drove reporter expression in a large subset of neurons in the CNS. In the transgenic lines R34A03 and R32H10 reporter expression was consistently observed on both ISNb and SNa nerve branches, whereas in the line R33F06 reporter expression was irregularly detected on ISNb or SNa nerve branches in small subsets of abdominal hemisegments. Through complementation test with a Sema1a loss-of-function allele, it was found that neuronal expression of Sema-1a driven by each of R34A03 and R32H10 restores robustly the CNS and PNS motor axon guidance defects observed in Sema-1a homozygous mutants. The results suggest that in a redundant manner, the CREs, R34A03, R32H10, and R33F06 govern the Sema-1a expression required for the axon guidance function of Sema-1a during embryonic neural development.
Rozbesky, D., Monistrol, J., Jain, V., Hillier, J., Padilla-Parra, S. and Jones, E. Y. (2020). Drosophila OTK Is a Glycosaminoglycan-Binding Protein with High Conformational Flexibility. Structure. PubMed ID: 32187531
The transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. This study has determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. Detection of direct binding between Drosophila Plexin A (PlexA) and OTK failed, that was suggested previously. Instead of PlexA, OTK directly binds semaphorin 1a. This binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.
Rozbesky, D., Verhagen, M. G., Karia, D., Nagy, G. N., Alvarez, L., Robinson, R. A., Harlos, K., Padilla-Parra, S., Pasterkamp, R. J. and Jones, E. Y. (2020). Structural basis of semaphorin-plexin cis interaction. Embo j: e102926. PubMed ID: 32500924
Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. This study discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. High-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.
Clements, J., Buhler, K., Winant, M., Vulsteke, V. and Callaerts, P. (2021). Glial and Neuronal Neuroglian, Semaphorin-1a and Plexin A Regulate Morphological and Functional Differentiation of Drosophila Insulin-Producing Cells. Front Endocrinol (Lausanne) 12: 600251. PubMed ID: 34276554
The insulin-producing cells (IPCs), a group of 14 neurons in the Drosophila brain, regulate numerous processes, including energy homeostasis, lifespan, stress response, fecundity, and various behaviors, such as foraging and sleep. Despite their importance, little is known about the development and the factors that regulate morphological and functional differentiation of IPCs. This study describes the use of a new transgenic reporter to characterize the role of the Drosophila L1-CAM homolog Neuroglian (Nrg), and the transmembrane Semaphorin-1a (Sema-1a) and its receptor Plexin A (PlexA) in the differentiation of the insulin-producing neurons. Loss of Nrg results in defasciculation and abnormal neurite branching, including ectopic neurites in the IPC neurons. Cell-type specific RNAi knockdown experiments reveal that Nrg, Sema-1a and PlexA are required in IPCs and glia to control normal morphological differentiation of IPCs albeit with a stronger contribution of Nrg and Sema-1a in glia and of PlexA in the IPCs. These observations provide new insights into the development of the IPC neurons and identify a novel role for Sema-1a in glia. In addition, this study shows that Nrg, Sema-1a and PlexA in glia and IPCs not only regulate morphological but also functional differentiation of the IPCs and that the functional deficits are likely independent of the morphological phenotypes. The requirements of nrg, Sema-1a, and PlexA in IPC development and the expression of their vertebrate counterparts in the hypothalamic-pituitary axis, suggest that these functions may be evolutionarily conserved in the establishment of vertebrate endocrine systems.
Juarez-Carreno, S., Vallejo, D. M., Carranza-Valencia, J., Palomino-Schatzlein, M., Ramon-Canellas, P., Santoro, R., de Hartog, E., Ferres-Marco, D., Romero, A., Peterson, H. P., Ballesta-Illan, E., Pineda-Lucena, A., Dominguez, M. and Morante, J. (2021). Body-fat sensor triggers ribosome maturation in the steroidogenic gland to initiate sexual maturation in Drosophila. Cell Rep 37(2): 109830. PubMed ID: 34644570
Fat stores are critical for reproductive success and may govern maturation initiation. This study reports signaling and sensing fat sufficiency for sexual maturation commitment requires the lipid carrier apolipophorin in fat cells and Sema1a in the neuroendocrine prothoracic gland (PG). Larvae lacking apolpp or Sema1a fail to initiate maturation despite accruing sufficient fat stores, and they continue gaining weight until death. Mechanistically, sensing peripheral body-fat levels via the apolipophorin/Sema1a axis regulates endocytosis, endoplasmic reticulum remodeling, and ribosomal maturation for the acquisition of the PG cells' high biosynthetic and secretory capacity. Downstream of apolipophorin/Sema1a, leptin-like upd2 triggers the cessation of feeding and initiates sexual maturation. Human Leptin in the insect PG substitutes for upd2, preventing obesity and triggering maturation downstream of Sema1a. Data shows how peripheral fat levels regulate the control of the maturation decision-making process via remodeling of endomembranes and ribosomal biogenesis in gland cells (Juarez, 2021).
Nguyen, C. T., Nguyen, V. M. and Jeong, S. (2022). Regulation of Off-track bidirectional signaling by Semaphorin-1a and Wnt signaling in the Drosophila motor axon guidance. Insect Biochem Mol Biol 150: 103857. PubMed ID: 36244650
Off-track receptor tyrosine kinase (OTK) has been shown to play an important role in the Drosophila motor axon pathfinding. The results of biochemical and genetic interactions previously suggested that OTK acts as a component of Semaphorin-1a/Plexin A (Sema-1a/PlexA) signaling during embryonic motor axon guidance and further showed that OTK binds to Wnt family members Wnt2 and Wnt4 and their common receptor Frizzled (Fz). However, the molecular mechanisms underlying the motor axon guidance function of OTK remain elusive. This study concludes that OTK mediates the forward and reverse signaling required for intersegmental nerve b (ISNb) motor axon pathfinding and it was also demonstrated that the loss of two copies of Sema-1a synergistically enhances the bypass phenotype observed in otk mutants. Furthermore, the amorphic wnt2 mutation resulted in increased premature branching phenotypes, and the loss of fz function caused a frequent inability of ISNb motor axons to defasciculate at specific choice points. Consistent with a previous study, wnt4 mutant axons were often defective in recognizing target muscles. Interestingly, the bypass phenotype of otk mutants was robustly suppressed by loss of function mutations in wnt2, wnt4, or fz. In contrast, total ISNb defects of otk were increased by the loss-of-function alleles in wnt2 and wnt4, but not fz. These findings indicate that OTK may participate in the crosstalk between the Sema-1a/PlexA and Wnt signaling pathways, thereby contributing to ISNb motor axon pathfinding and target recognition.
Bustillo, M. E., Douthit, J., Astigarraga, S. and Treisman, J. E. (2023). Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis. bioRxiv. PubMed ID: 37609142
Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. This study found that in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of Semaphorin function indicates that Semaphorin 1a, provided by cells that include Tm5 neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A does not disrupt the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles. The axon guidance molecule Plexin A has two functions in Drosophila medulla development; morphogenesis of the neuropil requires its cytoplasmic domain, but establishing synaptic layers through Semaphorin 1a does not.

The semaphorin gene family includes both secreted and transmembrane proteins that are selectively expressed in unique subsets of neurons in developing invertebrate and vertebrate nervous systems. Semaphorins are defined by the presence of a large phylogenetically conserved 500 amino acid semaphorin (sema) domain. Secreted semaphorins have an immunoglobulin (Ig) domain C-terminal to the sema domain, while transmembrane semaphorins can have either an Ig domain or a thrombospondin type I repeat, or no identified structural motif C-terminal to their sema domain. Transmembrane semaphorins have relatively short cytoplasmic domains that contain no obvious catalytic domains. The existence of both secreted and transmembrane semaphorins suggests that semaphorins function over long and short distances. g Semaphorin I (G-Sema-I, formerly Fasciclin IV), was the first semaphorin to be identified. G-Sema-I is a transmembrane semaphorin containing no obvious structural domains C-terminal to the semaphorin domain and is one of several invertebrate and vertebrate semaphorins with this overall structure. In vivo antibody perturbation experiments show that G-Sema-I plays an important role in establishing the axonal trajectory of the well-characterized Ti1 pioneer neurons (Kolodkin, 1993). In addition to peripheral epithelial localization, G-Sema-I is also found on grasshopper embryonic CNS axons in a highly selective pattern, suggesting that it is also likely to function in axon guidance events during CNS development (Yu, 1998 and references).

Sema-1a is, thus far, the semaphorin in Drosophila most closely related to G-Sema-I. The two proteins are similar in overall structure and Sema-Ia shares 60% amino acid identity with G-Sema-1a over its sema domain. Analysis of Sema-Ia mRNA distribution during Drosophila neurodevelopment has shown that like G-Sema-Ia, Sema-1a mRNA is expressed in the nervous system (Kolodkin, 1993). Sema-1a appears to be expressed in a much larger subset of neurons in Drosophila than is G-Sema-I in the related grasshopper nervous system. Sema-1a is required for the generation of the precise pattern of embryonic neuromuscular connectivity and appears to function at motor neuron pathway choice points in the periphery. Further, Sema I is also required for CNS pathfinding events. Rescue and ectopic expression experiments strongly suggest that Sema-1a can function as a repulsive ligand for motor axons that normally require it to navigate peripheral choice points, supporting the idea that both transmembrane and secreted semaphorins can function in vivo as repulsive guidance cues (Yu, 1998).

The development of the stereotypic pattern of neuromuscular connectivity in embryonic Drosophila abdominal segments has provided an excellent system in which to study axon guidance events that include motor axon fasciculation and defasciculation, target region identification, target recognition and initial synapse formation, and the later events of synaptogenesis. Each hemisegment contains 30 identified muscles that are innervated by 40 motor neurons, several of which have been identified. The pathfinding events that bring these motor axons to their targets involve several discrete steps. These motor neurons initially exit the CNS as part of the ISN (intersegmental nerve) or SN (segmental nerve). They then divide among five major motor neuron branches that target different muscle groups, eventually elaborating synapses upon individual target muscles. This stereotypic pattern of motor axon branches can be easily observed in late stage 16/early stage 17 embryos [Images] by using the monoclonal antibody (mAb) 1D4, which is directed against the axonal glycoprotein Fasciclin II (Fas II) and serves as a robust marker for all motor axons in the periphery (Yu, 1998).

The establishment of the stereotypic pattern of motor pathways in muscle innervation requires a series of axon pathfinding events that guide growth cones to sites of synapse formation. Central among these are defasciculation events that occur at discrete locations along these motor pathways. Sema-Ia mutants have defects at several of these locations, consistent with motor axons requiring Sema-1a for choice-point defasciculation. Since Sema-1a is found on most, if not all, motor axons, Sema I mutant embryos were examined for defects in the establishment of neuromuscular connectivity. Homozygous Sema-1a P1 mutant embryos show dramatic and highly penetrant pathfinding defects in both the ISNb and SNa pathways. Normally, the ISN, ISNb, and ISNd branches exit the CNS as a single pathway. Just lateral to the CNS, in the vicinity of the ventral oblique muscles, the motor neurons of the ISNb and ISNd branches defasciculate from the ISN. The ISNd subsequently innervates muscles 15, 16, and 17, while the ISNb continues to extend dorsally. After encountering muscle 28, the ISNb extends along the external surface of ventral lateral muscles (VLMs) 6 and 7 and the internal surface of VLMs 14 and 30. The ISNb then projects along the internal surface of VLMs 13 and 12. Synaptic arborizations are formed by the defasciculation of several motor axons from the ISNb (the RP3 motor neuron between muscles 6 and 7; the RP1 and RP4 motor neurons on muscle 13, and the RP5 motor neuron on muscle 12). The ISN continues to extend dorsally and contacts its dorsal target muscles, resulting in the formation of three characteristic arborizations in the dorsal muscle field. In the absence of Sema-Ia, ISNb motor neurons often fail to extend from the external to the internal ventral lateral muscle surface, stalling at positions where motor axons normally defasciculate from the ISNb and form synapses on target muscles. The observed defects in ISNb pathway formation are likely not to reflect loss of RP motor neurons, which contribute to ISNb, or any inability on the part of these neurons to extend axons contralaterally across the midline and out of the CNS. Immunohistochemistry using mAb 7G10, which is directed against the axonal glycoprotein Fasciclin III, reveals no abnormalities in the RP fascicles in Sema-1a P1 mutant embryos, either within or exiting the CNS, or in RP motor neurons themselves. Thus the defects are due to the stall or defasciculation phenotype and not to a guidance defect (Yu, 1998).

These results show that Sema-1a is required for the generation of the precise pattern of neuromuscular connectivity in the Drosophila embryo. This requirement appears to be for the navigation of specific choice points in motor axon pathways and not a general requirement for process outgrowth. ISN defects are not observed in Sema-IaP1 mutants, and the defects observed in the SNa and the ISNb occur at discrete locations along these pathways where motor axons normally defasciculate from their main branch and either extend toward, or elaborate synaptic arborizations on, their target muscles. Analysis of the peripheral sensory neurons with mAb 22C10 (see Futsch) reveals no defects in their mature axonal trajectories, consistent with the fact that no Sema-1a protein is detected on these PNS sensory afferents (Yu, 1998).

Sema-1a is a transmembrane semaphorin with a small cytoplasmic domain. To address how Sema-1a functions in neurons to mediate defasciculation events, rescue experiments were performed using a modified form of Sema-1a protein that lacks the transmembrane and cytoplasmic domains (called Sema-IaEC). The level of ectopic Sema-IaEC, driven by neuronal promoters, assessed immunohistologically, is equal to or higher than that of Sema-1a observed in wild-type embryos. Expression of Sema-IaEC in all neurons results in a partial, but significant, rescue of embryonic neuronal Sema-1a P1 phenotypes and adult lethality. For example, there is a 46% reduction (from 92% to 50%) in the fraction of abnormal SNa pathways observed in Sema-1a P1 mutant embryos when Sema-IaEC expression is driven ectopically. This reduction is primarily a rescue of the defasciculation defect at position S1. Similar results have been observed using a different ectopic expression protocol in Sema-1a P1 mutant embryos. The partial rescue of Sema-1a P1 motor axon pathway phenotypes may reflect a requirement for Sema-1a localization or presentation for ISNb and SNa development that is not completely satisfied by the Sema-IaEC protein in the context of the ectopic expression system used here. In addition to partial rescue of SNa and ISNb defasciculation defects, neuronal expression of Sema-IaEC partially but significantly rescues the Sema-1a P1 embryonic CNS phenotype, resulting in a 46% reduction in the fraction of hemisegments with a discontinuous Fas II-positive third longitudinal connective, correcting a defect caused by Sema-Ia mutation. Finally, ectopic Sema-IaEC significantly rescues Sema-IaP1 adult lethality. However, increasing the dosage of Sema-IaEC results in a reduction in the rescue of Sema-1a P1 adult lethality, presumably reflecting deleterious effects of panneuronal Sema-IaEC expression. Taken together, these rescue experiments show that Sema-1a is required in neurons to mediate motor neuron defasciculation events during neurodevelopment. Further, these results strongly suggest that Sema-1a is a ligand for an as yet unidentified receptor on motor axons (Yu, 1998).

Studies in which Sema-1a was overexpressed suggest that Sema-1a can act as a repulsive axon guidance cue. Sema-1a was overexpressed in all muscles in both wild-type and Sema-1a P1 mutant embryos. Dramatic motor axon defects are observed when Sema-1a is expressed in all muscles in wild-type embryos: these defects are sensitive to the endogenous dosage of Sema-Ia. The ISNb phenotypes observed in the presence of ectopic expression of Sema-1a protein on all muscles in wild-type embryos are qualitatively similar to those observed in Sema-1a P1 mutant embryos alone. (1) There is a significant increase (from 0% in wild type to 24% when Sema-1a is expressed in all muscles in wild-type embryos) in the fraction of hemisegments with a bypass of ISNb with the ISN. These bypass events include both parallel bypass (PB) events, where the ISNb fails to enter the ventral muscle field and extends dorsally in close proximity to the ISN as a separate pathway, and fusion bypass (FB) events, where the ISNb fails to enter the ventral muscle field and extends dorsally along the ISN. (2) There are a significant number of ISNb stall events that are similar to those observed in Sema-1aP1 mutant embryos: they occur at discrete locations between either muscles 7 and 6 or between muscles 6 and 13. (3) ISNb pathways with no evidence of synaptic arborizations between muscles 6 and 7, or aberrant synaptic arborizations between muscles 7 and 6 or on muscle 12, are observed in a large percentage of hemisegments. The occurrence of these motor axon pathfinding defects, which appear to reflect defects in motor axon defasciculation, is critically dependent on the dosage of ectopic Sema-1a expressed in muscles. A single copy of Sema-1a transgene does not result in the significant ISNb defects observed with two copies of each transgene. However, these events are sensitive to the endogenous dosage of Sema-1a expressed on axons, since a single copy of each transgene in a Sema-1a P1 mutant background results in a dramatic enhancement of the ISNb phenotypes observed in Sema-1a P1 mutants alone. For example, there is an increase from 7% to 58% in the fraction of hemisegments showing a bypass of ISNb with the ISN. Unlike the approximately equal numbers of PBs and FBs observed following ectopic Sema-1a expression on muscles in a wild-type background, these bypass events are almost always FBs (Yu, 1998).

Effects qualitatively similar to those described above for ISNb were also observed for SNa following ectopic expression of Sema-1a on all muscles both in wild-type and Sema-1a P1 mutant embryos. A significant number of S1 stall events is observed when Sema-1a is ectopically expressed on muscles in wild-type embryos. SNa defects also include a dramatic enhancement of Sema-1a P1 SNa phenotypes when Sema-1a is expressed in all muscles in this genetic background using only a single copy of each transgene. These enhanced defects include SNa fusion bypass events in which SNa fails to enter the ventral muscle field and instead extends dorsally along the ISN. SNa FBs are never observed in wild-type or Sema-1a P1 mutant embryos in the absence of ectopic Sema-1a muscle expression. In this genetic background, there is a significant increase in the fraction of hemisegments that exhibit a complete loss of the entire major dorsal SNa branch, and in those pathways that do still have this branch a larger fraction stall at the initial bifurcation of this branch in the lateral muscle group (Yu, 1998).

In addition to the enhancement of ISNb and SNa phenotypes observed in Sema-1a P1 mutants, ectopic expression of Sema-1a on muscles in this genetic background results in ISN defects. In the vicinity of the dorsal muscles, the ISN normally forms a stereotypic pattern of three arborizations. The formation of these arborizations is not disrupted in Sema-1a P1 mutants or by ectopic expression of Sema-1a in wild-type embryos using a single copy of each transgene. In Sema-1a P1 mutant embryos that express Sema-1a on all muscles, however, the first and second arborizations of ISN were not observed in 29% and 32% of hemisegments, respectively. No significant stalling of the ISN is observed in this genetic background. These ISN phenotypes are consistent with the observed ISN expression of Sema-1a (Yu, 1998).

The motor axon guidance defects produced by ectopically expressing Sema-1a in all muscles are specific to motor axons; they do not affect peripheral sensory axon pathfinding, and do not appear to be the result of changes in neuronal or muscle cell fate or muscle morphology (assessed as described above for Sema-1a P1 mutants). The motor axon phenotypes observed by expressing Sema-1a on all muscles demonstrates an ability of Sema-1a to prevent motor axons from entering regions of Sema-1a expression. This results in a failure of motor axon defasciculation at specific choice points and strongly suggests that Sema-1a can act as a repulsive axon guidance cue (Yu, 1998).

How does Sema-1a function to direct the navigation of axons past choice points in motor axon pathfinding? The effect of Sema-1a on a wild-type background has a profound, dosage-sensitive effect on motor axon pathfinding. These results show that Sema-1a on muscles can prevent the entry of ISNb axons into the ventral muscle field. Since both ISNb fusion and parallel bypass events are observed, one might predict that endogenous axonal Sema-1a often still functions in this situation to allow ISNb defasciculation from the ISN. This prediction is supported by the observation that lack of endogenous Sema-1a renders the ISNb sensitive to a low dose of ectopic Sema-1a in muscles, which in a wild-type background has no effect. The bypass events observed in this sensitized background are mostly fusion bypasses, not parallel bypasses, and this provides further support for Sema-Ia's role in mediating defasiculation events during normal motor axon pathfinding. Taken together, all of these data support a model in which neuronal Sema-1a acts as a ligand for an as yet unidentified receptor on motor axons to mediate repulsive axon-axon interactions at motor pathway defasciculation choice points (Yu, 1998).

Though the overall penetrance of defective motor axon pathways in Sema-1a P1 mutants is very high, the penetrance of specific defects along these pathways is variable. The results presented here strongly suggest that the Sema-1a P1 mutant is a null or very severe loss-of-function Sema-Ia allele. Therefore, Sema-1a function is not absolutely required for motor axon defasciculation events to occur, and it is important to consider Sema-1a in the context of the complex panoply of motor axon guidance cues that have already been shown to function in these guidance events. Several genes have been identified that affect defasciculation events in motor pathways. These genes, which are expressed in many motor axon branches, include FasII, which encodes an Ig superfamily member that is a homophilic cell adhesion molecule (CAM), PTP69D and PTP99A, which encode receptor protein tyrosine phosphatases (RPTPs), and beaten path (beat), which encodes Beat, a novel secreted protein expressed by motor neurons. Mutations in the gene sidestep (side), which has yet to be cloned, also affect defasciculation of the ISNb from the ISN. The importance of modulating axon-axon adhesive interactions during motor axon pathfinding is underscored by the genetic interactions between FasII and beat. ISNb axons in beat mutants often fail to defasciculate from the ISN; however, this phenotype can be suppressed by hypomorphic FasII mutations, suggesting that Beat somehow modulates CAM function in axons to promote defasciculation at choice points. Sema-1a is not the only semaphorin in Drosophila capable of acting as a repulsive cue for motor axons. Sema-IaI, a secreted semaphorin, can act as a selective repellent for the RP3 motor axon when ectopically expressed in ventral abdominal muscles. In Sema-IaI mutants, embryonic motor axon guidance appears normal. Sema-IaI expression is very high in a single thoracic ventral muscle. Recent observations show that Sema-IaI is also found at low levels in all muscles, and this general expression is important for motor axon guidance (M. Winberg and C.S. Goodman, personal communication to Yu, 1998). Sema-1a can act as a repulsive guidance cue for motor axons that require it for pathfinding, which suggests that Sema-Ia-mediated repulsive growth cone guidance normally plays an important role in motor axon pathfinding. Future studies will address whether or not Sema-IaI exerts its repulsive effect on RP3 through a different, or the same, signaling pathway as does Sema-1a (Yu, 1998 and references).

Multiple interactions control synaptic layer specificity in the Drosophila visual system

How neurons form synapses within specific layers remains poorly understood. In the Drosophila medulla, neurons target to discrete layers in a precise fashion. This study demonstrates that the targeting of L3 neurons to a specific layer occurs in two steps. Initially, L3 growth cones project to a common domain in the outer medulla, overlapping with the growth cones of other neurons destined for a different layer through the redundant functions of N-Cadherin (CadN) and Semaphorin-1a (Sema-1a). CadN mediates adhesion within the domain and Sema-1a mediates repulsion through Plexin A (PlexA) expressed in an adjacent region. Subsequently, L3 growth cones segregate from the domain into their target layer in part through Sema-1a/PlexA-dependent remodeling. Together, these results and recent studies argue that the early medulla is organized into common domains, comprising processes bound for different layers, and that discrete layers later emerge through successive interactions between processes within domains and developing layers (Pecot, 2013).

Although the growth cones of L1, L3, and L5 neurons target to different layers, they initially overlap within a common domain in the outer medulla. Based on biochemical interactions and the mistargeting phenotypes and protein expression patterns described in this paper, it is envisioned that CadN-dependent adhesive interactions restrict processes to the outer medulla and that PlexA-expressing tangential neurons prevent Sema-1a expressing growth cones from projecting into the inner medulla. L2 and L4 growth cones also appear to initially target to a common domain within the distal outer medulla, but do not require Sema-1a and CadN for this targeting step and thus utilize an alternative mechanism. Interestingly, the morphology of L2 and L4 neurons does rely on Sema-1a and CadN function, indicating that within lamina neurons, these molecules regulate different aspects of targeting. This is supported by the expression of Sema-1a and CadN in all lamina neuron subclasses during development (Pecot, 2013).

In mice separate channels encoding light increments (ON) and decrements (OFF) are spawned in the outer retina and relayed to different sublaminas of the inner plexiform layer (IPL). The current findings are reminiscent of recent studies in the mouse IPL (Matsuoka, 2011) in which Kolodkin and colleagues demonstrated that the processes of different subclasses of PlexA4-expressing amacrine cells are segregated to different OFF layers and that this requires both PlexA4 and Sema6A. Although these proteins act in a more traditional fashion as a receptor and ligand, respectively, they are expressed in a complementary fashion early in development when the developing neuropil is very thin, with PlexA expressed in the nascent OFF layer and Sema6A in the developing ON layers. This raises the intriguing possibility that, as in the medulla, different cells initially target to common domains, from which they then segregate into discrete layers. As Cadherin proteins are differentially expressed in a layered fashion in the developing IPL and defects in targeting are incomplete in both Sema6A and PlexA4 mutants (Matsuoka, 2011), it is possible that, as in the medulla, Semaphorin/Plexin repulsion acts in parallel with cadherin-based adhesion to control layer-specific patterning within the developing IPL (Pecot, 2013).

Taken together, these studies suggest that the restriction of processes to a common domain prior to their segregation into distinct layers may be a developmental strategy used in both the medulla and the vertebrate IPL. This step-wise process may represent a more general strategy for reducing the molecular diversity required to establish synaptic connections by limiting the potential synaptic partners that growth cones and nascent dendritic arbors encounter within the developing neuropil (Pecot, 2013).

After targeting to a common domain within the outer medulla, L3 growth cones undergo stereotyped changes in shape and position that lead to segregation into the M3 layer. Initially, L3 growth cones are spear-like, spanning much of the depth of the incipient outer medulla. They then expand and elaborate a myriad of filopodia before resolving into flattened synaptic terminals within the M3 layer. This transformation is marked by two prominent steps: extension of processes from one side of the lateral region of the growth cone into the incipient M3 layer and retraction of the leading edge of the growth cone from the incipient M5 layer (part of the domain shared by L1 and L5 growth cones) (Pecot, 2013).

It has been suggested that CadN may regulate the extension within M3, as this step is partially perturbed in CadN mutant growth cones. However, as CadN mutations affect the initial position of L3 growth cones within the outer medulla, the extension defect within the M3 layer may be indirect. By contrast, in sema-1a mutant growth cones, initial targeting is indistinguishable from wild-type, so defects in retraction away from the incipient M5 layer are likely to reflect a direct role for Sema-1a in this later step in growth cone reorganization. PlexA RNAi phenocopies a sema-1a null mutation and, thus, PlexA is also required for retraction and is likely to function on medulla tangential fibers, where it is most strongly expressed. In support of this, the tip of the L3 growth cone that retracts is in close proximity to these PlexA-expressing fibers (Pecot, 2013).

The function of Sema-1a/PlexA signaling in sculpting L3 growth cones appears to be distinct mechanistically from the earlier role it plays in confining the growth cones to a common domain. During initial targeting, PlexA acts as a barrier to L3 growth cones and prevents them from projecting beyond the outer medulla. Thus, at this early step, Sema-1a/PlexA interaction provides a stop signal for the leading edge of L3 (uncovered in double mutants with CadN). In the second step, however, Sema-1a/PlexA signaling promotes retraction into the M3 layer. How these diverse outputs of Sema-1a/PlexA signaling arise is unclear. Sema-1a may be coupled to different downstream effectors at each step, modified by association with other receptor subunits, or may be modulated by other extracellular signaling pathways (Pecot, 2013).

CadN may also play a role in the retraction of L3 growth cones away from the domain shared with L1 and L5 growth cones. In early pupal stages, disrupting CadN function, while leaving growth cone morphology largely spear-like, causes L3 axons to project deeper within the medulla. Under these conditions, Sema-1a function is sufficient to prevent the growth cones from extending beyond the outer medulla. Subsequently, CadN mutant L3 growth cones fail to move away from the outer medulla's proximal edge into the developing M3 layer and thus remain within the most proximal layer, M6. This suggests that CadN, while acting in parallel with Sema-1a to restrict L3 growth cones to the outer medulla initially, may also be required at later stages for movement of the L3 leading edge into the M3 layer. As CadN has been shown previously to regulate neurite outgrowth over cultured astrocytes, it may be required for L3 growth cones to move along adjacent processes. However, the initial projection of L3 axons into the medulla is not affected by CadN mutations, indicating that other components control this process. It also remains possible that the defect in growth cone retraction results indirectly from CadN's earlier role in targeting; this earlier role may account for the defects in growth cone extension within M3 (Pecot, 2013).

Disrupting CadN function in different neurons affects targeting in unique ways. For example, L5 axons lacking CadN target to the proper layer, but extend inappropriately within the layer into neighboring columns (Nern, 2008). In addition, CadN mutant R7 growth cones display abnormal morphology and, in contrast to mutant L3 growth cones, initially target correctly, but retract to a more superficial medulla region. Collectively, these findings demonstrate that CadN regulates divergent features of growth cone targeting in different contexts. This likely reflects molecular diversity between different growth cones and illustrates the importance of understanding how molecules act in combination to generate target specificity (Pecot, 2013).

These studies add to previous findings suggesting that column assembly relies on a precisely orchestrated sequence of interactions between different neuronal cell types (Nern, 2008; Timofeev, 2012). This study shows that, as L1, L3, and L5 growth cones expressing Sema-1a enter the medulla, they meet the processes of newly arriving tangential fibers expressing PlexA, which acting in parallel with CadN, prevents extension of these growth cones into the inner medulla. This timing may permit other Sema-1a-expressing growth cones to extend into the inner medulla at earlier stages; these growth cones may then use Sema-1a/PlexA signaling for patterning connections in the inner medulla or deeper neuropils of the lobula complex. Subsequent sculpting of the L3 growth cone, mediated by Sema-1a/PlexA and perhaps CadN, leads to its reorganization into an expanded terminal within M3. As L3 growth cones become restricted to the M3 layer, Netrin, secreted from L3 growth cones, becomes concentrated within the M3 layer, and this, in turn, attracts R8 growth cones to the M3 layer, as recently described by Salecker and colleagues (Timofeev, 2012; Pecot, 2013 and references therein).

Given the extraordinary cellular complexity of the medulla neuropil, with over 100 different neurons forming connections in different medulla layers, and the few mechanistic clues to layer specific targeting that have emerged so far, a complex interplay between different sets of neurons is envisioned to be required to assemble the medulla circuit. The availability of specific markers for many of these neurons, techniques to follow the expression of even widely expressed proteins at the single cell level as is described in this study, and the ability to genetically manipulate single cells during development provide a robust system for uncovering the molecular logic regulating the layered assembly of axon terminals, dendritic arbors, and synaptic connectivity (Pecot, 2013).


Amino Acids - 771

Structural Domains

Two new complete insect sequences (Tribolium-Sema-I and Drosophila-Sema-IIa) encode proteins with signal sequences, as does Grasshopper-Sema-I. T-Sema-I and D-Sema-1a have transmembrane domains, as does G-Sema-I. However, D-Sema-IIa has no transmembrane domain, and its C-terminus shows no indication of a potential phospholipid linkage, and thus it is likely to be secreted. In addition, at its C-terminus, D-Sema-II has a single C2-type immunoglobulin (Ig) domain. All four insect Semaphorins share a highly conserved extracellular domain of 500 amino acids that is characterized by 16 conserved cysteines, one conserved potential N-linked glycosylation site, and numerous blocks of conserved amino acids throughout the 500 amino acid domain. In their Semaphorin domains (comprising two-thirds of each protein), the three putative homologs (G-Sema-I, T-Sema-I, and D-Sema-Ia) are most similar to one another and all are more divergent from D-Sema-IaI in terms of both percent identity and the absence of several blocks of amino acids that are found in D-Sema-IaI. The existence of two different sema genes in the same species (Drosophila) suggests that they define a novel gene family encoding both transmembrane and secreted proteins (Kolodkin, 1993).

Semaphorins share a common ~500-amino-acid semaphorin domain and are grouped into six classes. Class I consists of transmembrane semaphorins, including Drosophila Sema-Ia. Class II has a single member, Drosophila Sema-II. Class III consists of secreted vertebrate semaphorins, including Sema D (Coll-I/Sema-IaII), Sema A (Sema V), Sema E (Cooll-3), Sema-IV, Coll-2 and Sema H (Coll-5), that all possess an immunoglobulin doman (Ig) and a basic C-terminal domain, lacking in Drosophila Sema-IaI. Class IV semaphorins are also transmembrane proteins that possess an extracellular Ig domain. Class V semaphorins, including Sema F are transmembrane proteins with a set of tandem thrombosponding type I (TSP domains). Class VI is defined by the recently described Sema KI, a glycosylphosphatidylinositol (GPI)-anchored protein (Chen, 1998; Takahashi, 1998).


For information on other Semaphorin family members see Semaphorin II.

Drosophila Semaphorins

Transmembrane and secreted glycoproteins of the semaphorin family are typically classified as inhibitory neuronal guidance molecules. However, although chemorepulsive activity has been demonstrated for several semaphorin family members, little is known about the function of the numerous transmembrane semaphorins identified to date. The extracellular semaphorin domain of a transmembrane semaphorin, semaphorin-1a, can actively perturb axon pathfinding in vivo when presented homogenously as a recombinant freely soluble factor. When ectopic overexpression is limited to defined epithelial regions, semaphorin-1a can directly steer axons by acting as an attractive guidance molecule (Wong, 1999).

The sequence and expression analysis of two new Drosophila members of the Semaphorin family is reported. Both proteins show the presence of Semaphorin domains and transmembrane domains. Both genes are expressed maternally and in embryos, and reveal distinct expression patterns much earlier than the onset of neurogenesis. Elements of two novel Semaphorin-like sequences were observed in the STS and EST databases of Drosophila. Cloning and sequence analyses for these two cDNAs reveals the presence of the Sema domain with its conserved cysteine residues. This led to their classification as members of the Semaphorin family. A conceptual open reading frame (ORF) for the first gene contains 759 amino acid residues. The initiating methionine is followed by a stretch containing structural regions characteristic of a secretory signal sequence. A second long hydrophobic stretch at the C terminus suggests a transmembrane domainfollowed by a short cytoplasmic tail. The protein shows the closest resemblance to Drosophila Sema 1a with 46% identity within the Sema domain, and has therefore been named Sema 1b. The second Semaphorin is a protein of 1081 amino acids. As in Sema 1b, the N-terminus of this molecule shows the presence of a signal sequence and the C-terminus has transmembrane spanning segment and a short cytoplasmic tail (Khare, 2000).

Sequence comparison reveals no Drosophila counterpart, but it is most homologous to murine Sema 5B with 41% identity within the Sema domain. Therefore the Drosophila protein has been named Sema 5c. It contains seven thrombospondin type I (TspI) and thrombospondin-like repeats that are characteristic of class V Semaphorins. Phylogenetic analysis of all known Semaphorins using the Sema domain for comparison shows that the family can be grouped into 3 classes. Class I and II Semas exist in duplicates, while only one member of class V Sema has been detected in the Drosophila genome so far. A comparison between Drosophila and C. elegans Semas shows that most likely class I Semaphorins constitute the ancestral form because they can also be found in C. elegans. C. elegans does not contain any class II or class V Semaphorins. Class V Semaphorins appear more closely related to class I than class II. Class II Semas appear as a distinct group, with two closely related molecules, which may have arisen by a recent gene duplication. Both Semas reside within the same chromosomal sub-band, and are likely to be less than 250 kb apart. Furthermore, the duplication must have occurred after the separation of class II and class V Semas (Khare, 2000).

In Northern analyses, Sema 1b transcripts are expressed more widely during embryogenesis than 5c. Both genes are expressed maternally, are virtually absent during third larval stage and reappear in late pupa during the last stage of metamorphosis. Sema 1b is first expressed in early oocytes, and staining is observed in ovarioles and nurse cells. In stage 2 embryos staining is uniform due to the presence of a smaller, putatively maternal RNA species. At stage 5, two lateral stripes, one to two cells wide, appear. These meet at stage 8 due to ventral furrow invagination as part of gastrulation. This movement of cells during this period is similar to the migration of the meso-ectodermal cells that line the ventral neurogenic region. A similar pattern has also been seen in the expression of the single-minded gene (sim). Interestingly, Sema 1b expression decays rapidly at the time point when sim expression becomes apparent, suggesting that Sema 1b may induce sim expression. No altered Sema 1b pattern was observed in sim mutants, suggesting that Sema 1b is acting upstream or in parallel to sim. At later embryonic stages, the expression is seen primarily in the ectoderm and becomes diffused as development proceeds. No staining is observed in either the PNS or the CNS in any of the later stages suggesting that this molecule is probably not involved directly in neural functioning (Khare, 2000).

The Sema 5c RNA is present right from the earliest developmental stages. The weak expression in the stage 10 oocyte and uniform expression in the stage 2 embryo suggests the presence of maternal contribution. At the blastoderm stage, a pattern of six stripes emerges: in this pattern the anterior three stripes are considerably stronger than the posterior three stripes. In addition, dorsal lateral extensions of the stripes are observed. During germband extension, the stripe pattern refines to 12 stripes that also become weaker. At this stage, expression is also observed in the region of amnioserosa. At stage 10, expression is most prominent in the mesoderm. Late stages of embryogenesis show strong expression at muscle attachment sites, the visceral mesoderm of the anteriormost part of the midgut and in the dorsal vessel. Several genes have been reported to be expressed in two bilateral stripes: ventral nervous system defective (vnd), intermediate neuroblast defective (ind), and the Enhancer of split E(spl) genes. Of these, only the E(spl) genes show overlapping expression with sema 1b. Sema1b has been shown to bind biochemically to plexins that are neuronal surface molecules. When expressed ectopically, it can also interact genetically with plexins. These data do not show any substantial expression of Sema 1b in the CNS. It is possible, however, that the levels of Sema 1b transcripts are below the level of detection, and that the genetics of interaction is much more sensitive. However, it is also plausible to assume that one Sema can replace the function of another; consequently, another Sema of the same class expressed at the right location could exert the expected function (Khare, 2000).

C. elegans semaphorins

The semaphorin family comprises secreted and transmembrane proteins involved in axon guidance and cell migration. Deletion mutants of C. elegans semaphorin 1a (Ce-sema-1a or smp-1) and semaphorin 1b (Ce-sema-1b or smp-2) genes have been isolated and characterized. Both mutants exhibit defects in epidermal functions. For example, the R1.a-derived ray precursor cells frequently fail to change anterior/posterior positions completely relative to their sister tail lateral epidermal precursor cell R1.p, causing ray 1 to be formed anterior to its normal position next to ray 2. The ray cells, which normally separate from the lateral tail seam cell (SET) at the end of L4 stage, remain connected to the SET cell even in adult mutant males. The ray 1 defects are partially penetrant in each single Ce-sema-1 mutant at 20°C, but are greatly enhanced in Ce-sema-1 double mutants, suggesting that Ce-Sema-1a and Ce-Sema-1b function in parallel to regulate ray 1 position. Both mutants also have defects in other aspects of epidermal functions, including head and tail epidermal morphogenesis and touch cell axon migration, whereas, smp-1 mutants alone have defects in defecation and brood size. A feature of smp-1 mutants that is shared with mutants of mab-20 (which encodes Sema-2a) is the abnormal perdurance of contacts between epidermal cells (Ginzburg, 2002).

Vulva development in C. elegans involves cell fate specification followed by a morphogenesis phase in which homologous mirror image pairs within a linear array of primordial vulva cells form a crescent shape as they move sequentially towards a midline position within the array. The homologous pairs from opposite half vulvae in fixed sequence fuse with one another at their leading tips to form ring-shaped (toroidal) cells stacked in precise alignment one atop the other. The semaphorin 1a SMP-1, and its plexin receptor PLX-1, are required for the movement of homologous pairs of vulva cells towards this midline position. SMP-1 is upregulated on the lumen membrane of each primordial vulva cell as it enters the forming vulva and apparently attracts the next flanking homologous PLX-1-expressing vulva cells towards the lumen surface of the ring. Consequently, a new ring-shaped cell forms immediately ventral to the previously formed ring. This smp-1- and plx-1-dependent process repeats until seven rings are stacked along the dorsoventral axis, creating a common vulva lumen. Ectopic expression of SMP-1 suggests it has an instructive role in vulva cell migration. At least two parallel acting pathways are required for vulva formation: one requires SMP-1, PLX-1 and CED-10; and another requires the MIG-2 Rac GTPase and its putative activator UNC-73 (Dalpe, 2005).

Developmental expression of cell recognition molecules in the mushroom body and antennal lobe of the locust Locusta migratoria

This study examined the development of olfactory neuropils in the hemimetabolous insect Locusta migratoria with an emphasis on the mushroom bodies, protocerebral integration centers implicated in memory formation. Using a marker of the cyclic adenosine monophosphate (cAMP) signaling cascade and lipophilic dye labeling, new insights were obtained into mushroom body organization by resolving previously unrecognized accessory lobelets arising from Class III Kenyon cells. Antibodies against axonal guidance cues, such as the cell surface glycoproteins Semaphorin 1a (Sema 1a) and Fasciclin I (Fas I), were utilized as embryonic markers to compile a comprehensive atlas of mushroom body development. During embryogenesis, all neuropils of the olfactory pathway transiently expressed Sema 1a. The immunoreactivity was particularly strong in developing mushroom bodies. During late embryonic stages, Sema 1a expression in the mushroom bodies became restricted to a subset of Kenyon cells in the core region of the peduncle. Sema 1a was differentially sorted to the Kenyon cell axons and absent in the dendrites. In contrast to Drosophila, locust mushroom bodies and antennal lobes expressed Fas I, but not Fas II. While Fas I immunoreactivity was widely distributed in the midbrain during embryogenesis, labeling persisted into adulthood only in the mushroom bodies and antennal lobes. Kenyon cells proliferated throughout the larval stages. Their neurites retained the embryonic expression pattern of Sema 1a and Fas I, suggesting a role for these molecules in developmental mushroom body plasticity. This study serves as an initial step toward functional analyses of Sema 1a and Fas I expression during locust mushroom body formation (Eickhoff, 2012).

Semaphorin 1a-mediated dendritic wiring of the Drosophila mushroom body extrinsic neurons

The adult Drosophila mushroom body (MB) is one of the most extensively studied neural circuits. However, how its circuit organization is established during development is unclear. This study provides an initial characterization of the assembly process of the extrinsic neurons (dopaminergic neurons and MB output neurons) that target the vertical MB lobes. The cellular mechanisms guiding the neurite targeting of these extrinsic neurons were probed, and it was demonstrate that Semaphorin 1a is required in several MB output neurons for their dendritic innervations to three specific MB lobe zones. This study reveals several intriguing molecular and cellular principles governing assembly of the MB circuit (Lin, 2022).

The MB is one of the most intensively studied structures in the fly brain. Its complex and organized circuit architecture has provided important clues to its operational logic. However, in contrast to the extensive investigations of its functions, how the MB circuit architecture is established during development has been little explored. This study provides an initial characterization of MB circuit assembly and identifies Sema1a as an important guidance molecule that directs dendritic innervations of multiple MBONs in three MB lobe zones. Below, several implications of this study relating to the wiring principles of the MB circuit are discussed, and a hypothetical model for how DAN axons and MBON dendrites are modularly assembled into the MB lobes is presented (Lin, 2022).

The most intriguing feature of the organization of the MB circuit is the zonal innervation of the MB lobes by DAN axons and MBON dendrites. The borders of the zones are distinct, with minimal overlap between DAN axons or MBON dendrites in the neighboring zones. Given such a highly organized neural network, elaborate interactions among the extrinsic neurons might be expected. For example, dendritic tiling, as observed between dendritic arborization (da) neurons in fly larvae, might be required for the formation of zonal borders between MBON dendrites, and match-ups between DAN axons and MBON dendrites in the same zone might be important for these neurites to establish proper zonal innervation patterns. However, the results suggest that the targeting and elaboration networks of DAN axons and MBON dendrites are largely independent, at least for those projecting to the MB vertical lobes. In the α'2 zone, where innervation by DAN axons precedes that by MBON dendrites, ablation of DANs does not affect zonal elaboration of the MBON dendrites. Moreover, upon ablation of one type of DAN or MBON in a given zone, morphologies of the neighboring neurites appear to be normal. Therefore, the extent and location of zonal network elaboration by DAN axons and MBON dendrites in the vertical lobes do not depend on interactions between these extrinsic neurons (Lin, 2022).

In each MB lobe zone, the DAN axons and MBON dendrites form synapses with each other and the KC axons. Given that DANs and MBONs do not depend on each other to form zonal networks, could KCs be responsible? The results support the importance of the KCs in the zonal organization of the DAN axons and MBON dendrites. Aberrant branching of KCs in alpha lobes absent (ala) mutant brains resulted in some MBs lacking the vertical lobes. When this occurred, most DAN axons and MBON dendrites that normally innervate these lobes do not form zonal arborization. Without the MB vertical lobes, MBON-α'2 dendrites are rerouted to other zones in the horizontal lobes and form potential synaptic connections with the local DAN axons. Importantly, this reorganization of the MBON dendrites requires the presence of the KCs (Lin, 2022).

It is still possible that the KC axons and the lobes they form simply provide an anchoring point on which DAN axons and MBON dendrites grow, and that the extent of their arborization is determined cell-autonomously as an intrinsic property. However, the axonal innervation pattern of PPL1 DANs argues against this possibility. PPL1-α'3 and PPL1-α'2α2 axons enter the MB vertical lobes at almost the same location but specifically occupy distinct zones on opposite sides of the entry point, suggesting the existence of local positional cues in the lobes to guide the innervation of DAN axons. Furthermore, overexpression of sema1a in DANs directs their dendrites to specific MB lobe zones, and importantly, the arborization of these rerouted dendrites is confined to their respective zones. Since these DAN dendrites normally do not innervate the MB lobe, there likely exist local positional cues that interact with Sema1a-expressing dendrites to guide their zonal arborization (Lin, 2022).

What could be the sources of these positional cues? KCs are good candidates because they synapse with DAN axons and MBON dendrites and are essential for zonal arborization of these neurites. However, since KCs provide the main framework of the MB lobes, their manipulation may affect the organization of other cell types in the MB lobes that could also be potential sources of the positional cues. Electron microscopy-based reconstructions of the MB circuit have provided a comprehensive catalog for the neurons that innervate the MB lobe. In addition to KCs, DANs, and MBONs, the MB lobes are innervated by one dorsal paired medial (DPM) neuron, one anterior paired lateral (APL) neuron, two SIFamide-expressing neurons, and two octopaminergic neurons. These neurons do not exhibit zonal innervation patterns in the MB lobes; the SIFamide- and octopamine-expressing neurons only sparsely innervate the MB lobes, and the neurites of APL and DPM ramify the entire MB lobes. The MB lobes are also populated by glia, which is another potential source of the positional cues. Although their sources remain undetermined, the results suggest that the MB lobes are likely prepatterned with positional cues to guide the zonal elaboration of the MBON and DAN neurites (Lin, 2022).

Sema1a is an evolutionarily conserved guidance molecule that functions as a ligand or receptor depending on the cellular context. The data suggest that Sema1a functions as a receptor in MBONs to regulate dendritic targeting in a zone-specific manner. Loss of sema1a activity preferentially affects MBON dendrites in the α'3, α'1, and β'2 zones. Even for MBONs that innervate multiple zones, such as MBON-β2β'2a and MBON-γ5β'2a, reducing sema1a activity in these neurons selectively impacts their dendrites in the β'2a zone. Since Sema1a is not differentially localized in the dendrites of these MBONs, the guidance cues that Sema1a responds to might primarily be present in the β'2a zone, with additional guidance signals working collaboratively to sort the dendrites from these MBONs into Sema1a-sensitive and -insensitive zones. Not all MBONs innervating these Sema1a-sensitive zones require Sema1a. For MBON-γ2α'1 and MBON-α'1 that both innervate the α'1 zone, loss of sema1a only affects dendritic innervation by MBON-α'1 but not MBON-γ2α'1. Therefore, how Sema1a functions is also cell-type-specific. Taken together, these results imply that multiple positional cues may be present in each MB lobe zone, with each MBON being equipped with multiple sensors that work in concert to respond to those cues. Moreover, given that Sema1a is broadly expressed in many neurons in the developing brain, including the KCs, Sema1a likely acts with other proteins or signaling molecules to determine guidance specificity (Lin, 2022).

Sema1a has been shown to mediate both neurite attraction and avoidance. Currently, it is unclear which of the two mechanisms underlies its guidance of MBON dendrites. For MBONs whose zonal dendritic innervation requires sema1a, their dendrites can still project to areas nearby their target zones when sema1a activity was removed. Hence, the cues that guide these MBON dendrites are likely to be short-ranged. However, overexpression of sema1a in PPL1-α'2α2 DANs can redirect their dendrites to innervate zones far away from their original location, suggesting that the guidance cues may also exert long-distance functionality. The data indicate that Sema1a functions as a receptor in MBONs. Identification of Sema1a ligands and determining their distributions in the MB lobes are critical steps toward understanding how Sema1a instructs the zonal innervation of MBON dendrites. Plexin A (PlexA) or secreted Semaphorin 2a and 2b (Sema2a and Sema2b) are known ligands for the Sema1a receptor. This study has tested if these canonical ligands of Sema1a are required for the dendritic innervations of β'2- and α'3-projecting MBONs. However, dendritic innervations by these MBONs were minimally affected in homozygous sema2a/2b double mutant flies or when PlexA was knocked down either pan-neuronally or in glia. Therefore, the canonical Sema1a ligands do not seem to play a role in MBON dendritic targeting. However, it remains to be determined if PlexA and Sema2a/2b function redundantly in this system or if an unidentified noncanonical ligand is involved (Lin, 2022).

Although the molecular nature of the positional cues in the MB lobes that organize the zonal patterns of DAN and MBON neurites awaits discovery, the data suggest that these cues likely work in a combinatorial manner. Supporting evidence for this notion comes from the observation that MBON-α'1 and MBON-γ2α'1 use sema1a-dependent and -independent mechanisms to innervate the α'1 zone, indicating that this zone may present at least two different guidance cues. Furthermore, sema1a is expressed in multiple MBONs that innervate distinct MB lobe zones. This pattern could potentially be explained if the positional cues attracting Sema1a-positive neurites appear sequentially in these zones (i.e., so that the zone an MBON innervates is determined by the developmental timing of the MBON). However, the finding that the ectopic innervations of MBON-α'2 in the β'2 and α'1-like zones of the ala brain occur simultaneously argues against that possibility. Therefore, the Sema1a-sensitive zones likely harbor additional zone-specific guidance cues that work in combination with Sema1a to diversify guidance specificity (Lin, 2022).

The observation that the mistargeted MBON-α'2 dendrites in ala mutant brains innervate other zones in the α'β' lobe, but not those in the αβ and γ lobes, has also prompted a hypothesis that there might be general attraction cues emanating from the α'β' lobes for all α'β' lobe-projecting MBONs, separating them from MBONs targeting αβ and γ lobes. Therefore, a hypothetical model is proposed whereby multiple hierarchically-organized positional cues are presented in the MB lobe zones, with these cues acting in concert to pattern zonal innervation by DAN axons and MBON dendrites in the MB lobes (Lin, 2022).

semaphorin I: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 22 October 2023

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