© Development 2014 (doi: 10.1242/dev.105544)

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

Abbreviations: AL, antennal lobe; APF, after puparium formation; BD, basic domain; CUB, complement C1r/s homology domain; Cx, cortex; DG, dentate gyrus; DL, dorsolateral; CA1-3, cornu ammonis region 1-3; ECM, extracellular matrix; FV/VIII, homology to coagulation factors V and VIII domains; GAP, GTPase-activating protein; GP, globus pallidus; GPI, glycophosphatidylinositol; Ig, immunoglobulin-like; IPT, Ig-like, plexins, transcription factor; L1, L1 cell adhesion molecule; MAM, Meprin, A5, Mu domain;

ORN, olfactory receptor neuron; PN, projection neuron; PSI, plexin-semaphorin-integrin; RBD, Rho-GTPase binding domain; SAC, starburst amacrine cell; SL, stratum lacunosum; SP, stratum pyramidale; Str, striatum; Sub, subicullum; Tr, thrombospondin;TRN, thalamic reticular nucleus; VM, ventromedial.

Semaphorins and their receptors

Diversification of semaphorin signalling

Intracellular signalling downstream of plexins Spatiotemporal regulation

0 APF 8 APF 16 APF

Semaphorins as semaphorin receptorsCompetitive ligand interactionsModulatory co-receptors

Cis inhibition

Ligand blocks receptor

Cis activation

Cis inhibition and activation

Mouse brain, hippocampal region

+miR-124

Local protein synthesis Endocytosis

Cytoplasm

Key

PSI Tr GAPCUB

†

º

SecretedTransmembrane GPI-linked

*

Semaphorins Principal semaphorin receptors

Transcriptional and post-transcriptional regulation

MAM FV/VIII

‡

‡

Normal signalling (no inhibition)

Semaphorins exist as secreted, transmembrane or GPI-anchored proteins and have been found in invertebrate (subclasses 1, 2 and 5) and vertebrate (subclasses 3-7) species, as well as in DNA viruses (subclass V). They signal predominantly through plexins. Four subclasses of plexins have been identified (A-D) and subclass-specifc interactions exist between semaphorins and plexins. Semaphorins may also use integrins, neuropilins or other semaphorins as receptors. Domains marked with °, †, * or ‡ are not present in the indicated semaphorin subclasses or semaphorin receptor subclass.

Plexin-dependent semaphorin receptors can contain various co-receptors, including neuropilins, receptor tyrosine kinases, immunoglobulin superfamily members and proteoglycans. These co-receptors provide them with unique signalling capacities and 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.

Competitive interactions between different semaphorins also occur and contribute to neural and bone development. During bone development, these interactions control the balance between bone resorption and formation.

In the Drosophila olfactory system, the repulsive effects of secreted Sema-2 proteins are mediated by the transmembrane semaphorin Sema-1a acting as a receptor.

Larval ORNs, which secrete Sema-2a and Sema-2b, project to the AL. These axons slowly degenerate as the fly develops, giving rise to a temporally receding Sema-2 gradient in the AL.

Sema-1a expression in PNs is required to sense this Sema-2 gradient; PN dendrites expressing high levels of Sema-1a are repelled into the dorsolateral AL, whereas weak Sema-1a expression allows ventromedial PN dendrites to extend towards the ventromedial AL.

The binding of semaphorins to plexins leads to plexin GAP domain activation and signalling through cytosolic protein kinases, GTPases and cytoskeleton-associated proteins. Downstream of Drosophila PlexA, Mical and SelR antagonistically regulate F-actin disassembly. Plexins also regulate cell-cell and cell- substrate adhesion by influencing integrin activity, endocytosis and clustering.

Spatiotemporal regulation of semaphorin signalling component expression occurs at different levels. Transcription factors (e.g. REST/CoREST and Nkx2-1) modulate gene expression, whereas microRNAs (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 synthesis or endocytosis).

Reverse signallingTransmembrane 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.

Mossy fibre axons from DG granule cell neurons form a large axon bundle in the SL. Mossy fibres express plexin A4 to detect the repulsive transmembrane Sema6A on CA3 pyramidal neurons. Plexin A2 on the proximal part of the apical dendrite of CA3 pyramidal neurons binds Sema6A in cis to prevent interactions between Sema6A, on CA3 pryamidal neurons, and plexin A4, on mossy fibre axons. This generates a non-repulsive corridor in the SL, which is invaded by mossy fibres.

e.g. During regulation of SAC morphology and laminar stratification in the mouse retina.

e.g. Layer-specifc innervation of the mouse hippocampal CA3 region by mossy fibre axons.

e.g. Inhibition of synapse formation in C. elegans.

Axons from Cx and Str neurons express plexin D1 and are repelled by Sema3E expressed in the GP and TRN.

Axons from Sub neurons express plexin D1, Nrp1 and VEGFR2 and are attracted by Sema3E secreted by neighbouring axons derived from CA1-CA3 pyramidal neurons.

Binding of Sema6D to a plexin A1/TREM2/DAP12 receptor complex stimulates osteoclast differentiation and bone resorption.

Sema3A, derived from sensor axons innervating the bone, and its receptor Nrp1 sequester plexin A1 from the Sema6D receptor complex. This inhibits Sema6D-mediated osteoclast differentiation.

Binding of Sema3A to Nrp1/plexin A1 promotes osteoblast differentiation and inhibits the migration of osteoclast precursor cells.

Ligand-receptor interactions

Semaphorin class 1 2 3 4 5 6 7A V

Semaphorin binding receptor PlexAPlexB, Sema-1aplexin As, plexin D1, Neuropilins plexin Bs, plexin Ds, Nrp1, CD72, TIM-2plexin Asplexin Asplexin C1, Integrinsplexin C1

Neuropilins IntegrinsPlexA/Bplexin Asplexin Bsplexin C1‡plexin D1

Sema Ig IPT RBDGPI anchorBD

Plexin

Microtubule cytoskeleton F-actin disassembly

PlexA

Cell adhesion

Plexin Integrins

Clustering

Endocytosis

Semaphorin Sema-1a Semaphorin

P PKA

14-3-3JRnd

R-Ras Rap

PI3K Akt

CRMP2 GSK-3G

Mical

SelR

NADPH

PIPKIL

FARP2R-Ras

mRNAdegradation

Nucleus

Nrp2mRNA

DNA

CoREST mRNA

Nrp1

Nrp2

CoRESTREST

Nkx2-1

+miR-188

RhoA mRNA

RhoAprotein

Sema3A

Nrp1

Growthcone

Growthcone

Axon repulsion

Axonrepulsion

Cellmigration

plexin A4

Sema3A

mTOR1

plexin A4

Nrp1

L1

TAG1

FAK

Actin dynamics

CRMP2

Microtubule dynamicsSema3ANrp1

plexin A4TAG1Axon repulsion

Endocytosis

L1

Sema-2s

Cell migration

Sema6D

plexin A1

Cell migration

EnaAbl

Ena

P

P

Reversesignalling

Forwardsignalling

Mouse embryonic heart

Endocardial cells(plexin A1+)

Cardiac jelly(ECM)

Trabecular layer

Compact myocardial layerMyocardial cells(plexin A1+

Sema6D+)

Myocardial cells(Sema6D+)

1

1

2

3

2

3

Cellmigration Cell

migration

Cellmigration

Cellmigration

In myocardial cells, simultaneous forward and reverse signalling via Sema6D and plexin A1 triggers circumferential cell migration, which facilitates the expansion of this layer.

Myocardial cells expressing Sema6D, but not plexin A1, migrate out of the compact layer into the trabecular layer triggered by repulsive plexin A1 to Sema6D reverse signalling.

Inward migration of plexin A1- expressing endocardial cells into the myocardial layer is inhibited by Sema6D, which is released into the cardiac jelly by myocardial cells.

Sema6A

plexin A4

Receptor blocks ligand

plexin A2

plexin A4

Sema6A

plexin A2

No response No responseAxon repulsion

Signalling?

PLX-1

SMP-1

Actin dynamics

Ras

Synapseformation

Sema6Arepulsion

plexin A2cis inhibitionof Sema6A

SL

SP

CA3 pyramidalneuron(Sema6A+)

Sema6A+

CA3 dendriticSL segment(Sema6A+

plexin A2+)

DG granulecell neuron

DG mossy fibre(plexin A4+)

Sema3E

Axon repulsion

Axon repulsion

plexin D1

Nrp1VEGFR2

Axon attraction

Axon attraction+

Sub CA1-3CA1-3

Str Cx

Fornix

TRNGP

Midbrain

Mammillarybody

Sema3E+ plexin D1+

Nrp1+

VEGFR2+

=

=

Septum

Contralateralhippocampus Axons

Dorsal

Ventral

RostralCaudal

Coronal mousebrain sections

Mouse brain

Sema3E+ plexin D1++

plexin A1

Sema6D

TREM2DAP12

Nrp1

Sema3A

Osteoclastdifferentiation

Osteoclastdifferentiation

Nrp1

plexin A1

Sema3A

Osteoclastdifferentiation

Osteoclastprecursormigration

Bone

Osteoclastdifferentiation

Osteoblastdifferentiation

Sema6D

Sema3A

Resorption

Formation

?

Sema-1a

Axonrepulsion

LateralMedial

Dorsal

Ventral

Larval ORN

Adult ALconfiguration

DL-PNSema-1a

Sema-2a/b

VM-PN

Larval ALconfiguration

Reverse signalling

Forward signalling

Forward and reverse signalling

Sema5sSema1s*Sema4sSema6s*

Sema2s†Sema3sSemaVs º

Sema7A

GDP

GDP

P P