signalling during development Bart C. Jongbloets and R. Jeroen Pasterkamp

Semaphorins and their receptors Intracellular signalling downstream of Spatiotemporal regulation

Semaphorins exist as secreted, transmembrane or Semaphorins Principal semaphorin receptors The binding of Semaphorin Sema-1a Semaphorin Spatiotemporal regulation of semaphorin signalling component expression occurs at different levels. Transcription factors (e.g. REST/CoREST and Nkx2-1) modulate expression, whereas microRNAs GPI-anchored proteins and have been found in semaphorins to plexins (e.g. miR-124 and miR-188) are important post-transcriptional regulators that stimulate mRNA degradation. Regulation also occurs locally in specific subcellular compartments (e.g. by local protein invertebrate (subclasses 1, 2 and 5) and vertebrate leads to GAP synthesis or endocytosis). (subclasses 3-7) species, as well as in DNA domain activation and

(subclass V). They signal predominantly through ‡ Transcriptional and post-transcriptional regulation Local protein synthesis Endocytosis plexins. Four subclasses of plexins have been identified º signalling through CoREST mRNA (A-D) and subclass-specifc interactions exist between cytosolic protein Plexin Cytoplasm miR-124 Sema3A * † Sema3A L1 semaphorins and plexins. Semaphorins may also use ‡ kinases, and Plexin PlexA + plexin A4 integrins, or other semaphorins as cytoskeleton-associated Nucleus TAG1 receptors. Domains marked with °, †, * or ‡ are not proteins. Downstream present in the indicated semaphorin subclasses or repulsion of Drosophila PlexA, Endocytosis semaphorin subclass. P PKA plexin A4 Mical and SelR Mical PIPKI REST CoREST Nrp1 Nrp1 -receptor interactions TransmembraneSecreted GPI-linked PlexA/B Neuropilins Integrins antagonistically regulate Rnd 14-3-3 Nrp1 plexin As F- disassembly. NADPH mRNA Semaphorin class Semaphorin binding receptor Sema1s* Sema5s Sema2s† Sema7A FARP2 plexin Bs degradation L1 1 PlexA Sema4s Sema3s Plexins also regulate R-Ras Rap plexin C1‡ R-Ras 2 PlexB, Sema-1a Sema6s* SemaVs º cell-cell and cell- Nkx2-1 RhoA mRNA plexin D1 substrate adhesion by GDP 3 plexin As, plexin D1, Neuropilins PI3K Akt SelR Nrp2 DNA P P influencing Cell mTOR1 FAK CRMP2 4 plexin Bs, plexin Ds, Nrp1, CD72, TIM-2 Clustering Key activity, endocytosis and migration 5 plexin As CRMP2 GSK-3 RhoA Nrp2 miR-188 6 plexin As Sema IgBD GPI anchor IPT RBD clustering. Endocytosis protein Actin dynamics dynamics mRNA + 7A plexin C1, Integrins Growth Growth Sema3A plexin A4 PSICUB TrMAM GAP FV/VIII Microtubule cytoskeleton F-actin disassembly Cell adhesion Axon repulsion V plexin C1 cone cone Axon repulsion Nrp1 TAG1

Diversification of semaphorin signalling

Reverse signalling Cis inhibition and activation Transmembrane semaphorins can function both as ligands and receptors, a process termed bi-directional signalling. Semaphorin reverse signalling, in which semaphorins act as receptors, contributes to neural and cardiac development. Semaphorins and plexins interact in trans but also in cis. These cis interactions can inhibit or activate plexin signalling. Two modes of cis inhibition have been described: (1) semaphorins bind plexins in cis to prevent signalling in trans with semaphorin ligands on adjacent cells; and (2) plexins bind semaphorins in cis to prevent signalling in trans with plexins on adjacent cells. By contrast, cis activation triggers signalling downstream of plexin. Mouse embryonic heart Cis inhibition Cis activation Ena Cell migration Compact myocardial layer CA3 dendritic P Myocardial cells 1 Forward and reverse signalling Ligand blocks receptor Normal signalling Receptor blocks ligand Mouse brain, hippocampal region (plexin A1+ (no inhibition) SL segment In myocardial cells, simultaneous + Sema6D+) forward and reverse signalling via Signalling? SMP-1 DG mossy fibre (Sema6A Ena Cell + Sema6D and plexin A1 triggers + plexin A2 ) P migration (plexin A4 ) Abl Cell circumferential cell migration, migration which facilitates the expansion of 1 this layer. PLX-1 plexin A2 Sema6A Reverse repulsion plexin A1 Trabecular layer 2 signalling DG granule Sema6A Sema6D 2 Reverse signalling cell neuron Forward Myocardial cells expressing plexin A2 Sema6D, but not plexin A1, Sema6A signalling SL cis inhibition Cell migrate out of the compact layer of Sema6A into the trabecular layer triggered Ras Myocardial cells migration by repulsive plexin A1 to Sema6D GDP SP (Sema6D+) plexin A4 reverse signalling. plexin A2 plexin A4 3 Cardiac jelly (ECM) Actin dynamics + 3 Forward signalling Sema6A CA3 pyramidal Endocardial cells Inward migration of plexin A1- neuron (plexin A1+) expressing endocardial cells into No response Axon repulsion No response Synapse (Sema6A+) the myocardial layer is inhibited formation by Sema6D, which is released Mossy fibre from DG granule cell neurons form a large axon bundle in the SL. Mossy fibres express Cell Cell migration into the cardiac jelly by e.g. During regulation of e.g. Layer-specifc innervation e.g. Inhibition of synapse plexin A4 to detect the repulsive transmembrane Sema6A on CA3 pyramidal neurons. Plexin A2 on the migration myocardial cells. SAC morphology and of the mouse hippocampal formation in C. elegans. proximal part of the apical dendrite of CA3 pyramidal neurons binds Sema6A in cis to prevent interactions laminar stratification in CA3 region by mossy fibre between Sema6A, on CA3 pryamidal neurons, and plexin A4, on mossy fibre axons. This generates a the mouse retina. axons. non-repulsive corridor in the SL, which is invaded by mossy fibres.

Modulatory co-receptors Competitive ligand interactions Semaphorins as semaphorin receptors Plexin-dependent semaphorin receptors can contain various co-receptors, including neuropilins, receptor tyrosine kinases, Competitive interactions between different semaphorins also occur and contribute to neural and bone development. During bone development, these interactions control the balance In the Drosophila olfactory system, the repulsive effects of secreted Sema-2 proteins are mediated by the transmembrane immunoglobulin superfamily members and proteoglycans. These co-receptors provide them with unique signalling capacities and between bone resorption and formation. semaphorin Sema-1a acting as a receptor. often determine the response to a specifc semaphorin. This is exemplified by Sema3E and its receptor plexin D1 during the development of long axon tracts in the mouse brain. 0 APF 8 APF 16 APF Sema3A Bone Dorsal Sema6D Sema3E Mouse brain Sema3A Sema-2s Sema-1a Caudal DL-PN Rostral plexin A1 Adult AL configuration Ventral Nrp1 Coronal mouse plexin A1 VM-PN VEGFR2 brain sections TREM2 plexin D1 DAP12 Sema6D Larval AL Osteoblast Sub CA1-3 configuration Nrp1 Nrp1 Sema3A differentiation ? Sema-2a/b Fornix Mammillary Contralateral body Larval ORN Axons hippocampus Midbrain Sema-1a Dorsal Str Cx Septum Formation Osteoclast Medial Lateral differentiation Axon repulsion Axon attraction GP Osteoclast Osteoclast Osteoclast Osteoclast Resorption differentiation differentiation differentiation precursor Ventral Axons from Cx and Axons from Sub neurons TRN + + migration Str neurons express express plexin D1, Nrp1 and Sema3E + plexin D1 = Axon repulsion Binding of Sema6D to a Sema3A, derived from sensor axons Larval ORNs, which secrete Sema-2a and Sema-1a expression in PNs is required to sense this Sema-2 plexin D1 and are VEGFR2 and are attracted plexin A1/TREM2/DAP12 innervating the bone, and its receptor Sema-2b, project to the AL. These axons gradient; PN dendrites expressing high levels of Sema-1a are repelled by Sema3E by Sema3E secreted by + receptor complex Nrp1 sequester plexin A1 from the Binding of Sema3A to Nrp1/plexin A1 Axon Sema3E + plexin D1+ = Axon attraction slowly degenerate as the fly develops, repelled into the dorsolateral AL, whereas weak Sema-1a expression expressed in the GP neighbouring axons derived stimulates osteoclast Sema6D receptor complex. This promotes osteoblast differentiation repulsion Nrp1+ giving rise to a temporally receding allows ventromedial PN dendrites to extend towards the and TRN. from CA1-CA3 pyramidal differentiation and bone inhibits Sema6D-mediated osteoclast and inhibits the migration of VEGFR2+ Sema-2 gradient in the AL. ventromedial AL. neurons. resorption. differentiation. osteoclast precursor cells.

Abbreviations: AL, antennal lobe; APF, after puparium formation; BD, basic domain; CUB, complement C1r/s homology domain; ORN, olfactory receptor neuron; PN, projection neuron; PSI, plexin-semaphorin-integrin; RBD, Rho-GTPase binding domain; Cx, cortex; DG, dentate gyrus; DL, dorsolateral; CA1-3, cornu ammonis region 1-3; ECM, extracellular matrix; FV/VIII, homology to SAC, starburst amacrine cell; SL, stratum lacunosum; SP, stratum pyramidale; Str, striatum; Sub, subicullum; Tr, thrombospondin; coagulation factors V and VIII domains; GAP, GTPase-activating protein; GP, globus pallidus; GPI, glycophosphatidylinositol; TRN, thalamic reticular nucleus; VM, ventromedial. Ig, immunoglobulin-like; IPT, Ig-like, plexins, ; L1, L1 cell adhesion molecule; MAM, Meprin, A5, Mu domain; © Development 2014 (doi: 10.1242/dev.105544)