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Spotlight

Different flavors of Toll guide olfaction Claudine Neyen1 and Bruno Lemaitre2 1 2

Centre for Integrative Genomics, University of Lausanne, Unil-Sorge, Lausanne, CH-1015 Switzerland Global Health Institute, School of Life Sciences, EPFL, Station 19, Lausanne, CH-1015 Switzerland

Toll-like receptors are historically linked to immunity across animal phyla, but accumulating evidence suggests they play additional roles in neuronal networks and in cell-cell interactions. Ward and colleagues now identify Toll-6 and Toll-7 as instructive guidance cues during Drosophila olfactory development. Toll-like receptors (TLRs) are transmembrane molecules that combine a Toll-IL-1 Receptor (TIR) intracellular signalling domain with an ectodomain of multiple LeucineRich Repeats (LRRs). TLRs probably arose in early Metazoans and are conserved throughout animal evolution [1]. Numbers of TLR family members vary widely, from a single Toll in the nematode Caenorhabditis elegans and around a dozen in mammals, to over 200 in sea urchins. Mammalian TLRs are well-characterized pattern recognition receptors (PRRs) with essential functions in controlling innate and adaptive immunity. PRRs are defined as host molecules able to detect infectious non-self via the recognition of conserved microbial molecules. As such, the role of TLRs in host defence is ancient: Toll signalling is part of the immune arsenal in Drosophila and in the Cnidarian Hydra [2]. Despite the common view that TLRs are exclusively immune receptors, invertebrate TLRs have additional functions in development. Indeed, the historical ancestor of all TLRs, Drosophila Toll-1, was initially discovered for its primordial function in dorsoventral polarity during embryonic development, before being linked to antimicrobial responses. In early development as in immunity, Toll-1 is activated by its endogenous ligand Spa¨tzle (spz) and regulates gene transcription via the canonical Toll-MyD88-NF-kB cassette. Spa¨tzle is a secreted ligand related structurally and functionally to neurotrophins. The Drosophila genome encodes eight additional Toll genes whose functions remained poorly characterized, a situation that is currently changing. A recent study by Ward et al. now sheds light on roles for Toll-6 and Toll-7 in neuronal guidance during Drosophila olfactory development [3]. In Drosophila, as the olfactory circuit assembles, each olfactory receptor neuron (ORN) originating from the fly’s sensory organs needs to find its cognate projection neuron (PN) in a region of the brain called the antennal lobe. Olfactory input converges in tightly packed neuropils called glomeruli, of which there are 43 in Drosophila antennal lobes. Within the glomeruli Corresponding authors: Neyen, C. ([email protected]); Lemaitre, B. ([email protected]). Keywords: Toll; olfactory neurons; development. 1471-4906/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2015.06.008

of the antennal lobe, cell surface molecules guide ORN axons towards the correct dendrites of projector neurons. The cell adhesion and guidance molecules - Semaphorins and Teneurin - were recently identified as ORN-ORN repulsion signals and ORN-PN matchmakers, respectively. While these transmembrane proteins are classically associated with neurons, Ward and colleagues now show that the presence of Toll-7 on pre-synaptic ORNs and the presence of Toll-6 on post-synaptic PNs are required cellautonomously for correct olfactory wiring. Toll-6 and Toll-7 were identified in a candidate-based RNAi screen to identify cell surface receptors and secreted ligands involved in ORN-PN matching during antennal lobe development. Confocal imaging of antennal lobes identified mis-targeting of ORNs and PNs in specific glomeruli when two Toll-family members, Toll-6 and Toll-7, were knocked down. Using newly generated antibodies, the authors find Toll-6 and Toll-7 are expressed in overlapping subsets of glomeruli in the antennal lobe. Through cell-type specific ablation and rescue of the two receptors, they narrow down a specific role for Toll-7 in ORN axons and for Toll-6 in PN dendrites. Overexpression of Toll-7 in ORNs or Toll-6 in PNs that do not normally target Toll-7 or Toll-6 positive glomeruli caused them to rewire to the cognate Toll-positive region, proving that Toll expression in neurons is instructive for neuronal wiring. Mis-targeted expression of Toll-6 or Toll-7 in ORNs and PNs were able to re-direct each other in some instances, suggesting a degree of flexibility and robustness in wiring. Other members of the Toll-6 sub-family (consisting of Toll-2, -6, -7 and -8), which share a similar ectodomain organisation, were able to function interchangeably. Ward et al. show that Toll-6/7 function independently of Spa¨tzle/neurotrophin family ligands, and do not require the TIR domain or canonical pathway components (e.g. MyD88, NF-kB), suggesting that only the extracellular domain of Toll-6 and Toll-7 is involved in axon and dendrite guidance. Alternatively, Tolls might pair with co-receptors or engage a separate signalling machinery that is independent of immune signalling. Two previous studies already highlighted functional links between Drosophila Toll receptors and neurons. In 2013, McIlroy et al. uncovered a role for Toll-6 and Toll-7 in axonal routing and neuronal survival in the central nervous system of flies [4]. Toll-6 and Toll-7 appear to function as neurotrophin receptors, but it is somewhat unclear whether NF-kB signalling is required. Another Toll family member, Toll-8 (Tollo) is expressed in motor neurons, and supports development of neuromuscular junctions. Toll-8 senses the neurotrophin Spa¨tzle 3 released from muscle fibres and engages JNK rather than Trends in Immunology xx (2015) 1–3

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Table 1. Evidence for non-immune functions of Toll-like receptors across species. Adapted from [1] and references within.

Vertebrates

Species

Receptor

Function(s)

Ligand(s)

Mammals M. musculus

TLR-2

Positive regulator of adult hippocampal neurogenesis Negative regulator of axonal growth Negative regulator of adult hippocampal neurogenesis Negative regulator of neurite outgrowth Negative regulator of neurite outgrowth, induction of neuronal apoptosis Dorsoventral patterning Heterotypic cell adhesion Heart development (dorsal vessel formation) Muscle development (expression required in nonmuscle cells) Synaptogenesis of motoneurons (expression required in muscle cells) Salivary gland invagination Follicle cell migration Cell adhesion, morphogenesis Ectodermal expression drives induction of neuron-specific glycosylation Synaptic growth at the neuromuscular junction Planar polarity, convergent extension Olfactory neuronal guidance Synaptogenesis of motoneurons Olfaction, development

Toll agonist (endogenous DAMPsa?) Toll agonist (PAMPsb, endogenous DAMPs ?) Toll agonist (endogenous DAMPs?) Toll agonist

TLR-3 TLR-4 TLR-7 TLR-8

Invertebrates

Insects D. melanogaster

Toll-1

Toll-2/18-wheeler

Toll-8/Tollo

Toll-2, -6, -8 Toll-6, Toll-7

Nematodes C. elegans

Tol-1

Signalling module(s) NF-kB

Refs

NF-kB

[1]

NF-kB, IRF

[1]

NF-kB

[3]

Toll agonist

Non-NF-kB

[1]

Spz ? Homophilic?

NF-kB ? ?

[1] [1] [1]

?

NF-kB

[1]

?

?

[1]

? ? ? ?

Non-NF-kB ? ? ?

[1] [1] [1] [1]

Spz3

Non-NF-kB, JNK Non-NF-kB?

[5]

Non-NF-kB NF-kB (?)

[3] [4]

Non-NF-kB

[6]

Heterophilic Tolls Heterophilic Tolls Neurotrophin 1 (spz2), Neurotrophin 2 (spz5) ?

[1]

[8]

a

Danger Associated Molecular Patterns,

b

Pathogen Associated Molecular Patterns.

canonical NF-kB signalling [5]. In contrast to Ward et al., these two studies observe a requirement for ligandactivated Toll signalling. In the nematode C. elegans, expression of the unique Tol-1 gene is restricted to neuronal tissues [6]. Tol-1 mutants lacking the TIR domain have normal neuronal morphology and do not exhibit any immune phenotype, but are impaired in their olfactory capacity to avoid infectious bacteria. While Tol-1 null mutants show severe developmental defects, the lack of developmental phenotype in TIR mutants suggests Tol-1 acts as a cell-cell adhesion molecule rather than a signalling receptor, in concordance with the findings of Ward and colleagues. Interestingly, ligand-independent, heterophilic Toll interactions govern direct cell-cell interaction in other developmental contexts. Mutations in Toll-2 (18-wheeler) cause defective epithelial morphogenesis, and Toll-2 overexpression induces cell-cell adhesion. Toll-2 does not use the canonical pathway for adhesion but rather engages RhoGTPases [7]. In a recent study, Pare´ et al. describe a Toll positional code defining stripes reminiscent of pair-rule genes in the developing Drosophila embryo. 2

This ‘Toll sat nav’ of spatial cues originating from Toll heterophilic interactions guides planar cell polarity and cell intercalation for convergent extension along the anterior-posterior axis [8]. Both studies point to a connection between Toll receptor signalling and cell-cell communication, possibly involving interplay between Toll and the cellular contractile machinery. From an evolutionary perspective, it is still a puzzle whether TLRs emerged as pattern recognition molecules, detecting non-self, or as cell-adhesion molecules, detecting self. The amplification of the TLR gene family in sea urchins for example is still not understood, and may yet reveal roles in cell adhesion. A fascinating hypothesis proposes that PRRs evolved with the dawn of multicellularity, guiding cells of a same species to form homogenous colonies while excluding others [9]. In such scenarios, the ectodomain of Toll would have been sufficient to support adhesion, and indeed the LRR modules are found in the common ancestor of the Holozoa (i.e. all animals and choanoflagellates, but not fungi) [10]. Table 1 lists evidence of non-immune functions for TLRs across metazoans that may shed light on the emergence and physiological role

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Spotlight of Tolls. Similar to observations in Drosophila, several mammalian TLRs have been implicated in neuronal guidance cues, albeit mostly with inhibitory function. Since mammalian studies rely on synthetic or bacterial TLR agonists, the physiologically relevant ligands remain unclear. DAMPs (Danger Associated molecular patterns) have been proposed as TLR activators, signalling neuronal cell death and the need for repair. Whether mammalian TLRs have bona-fide non-immune functions based purely on adhesive or repulsive receptor interactions remains to be assessed. Our knowledge of TLR biology is evolving beyond canonical roles in immunity, towards self-homeostatic functions encompassing not only defense against invaders, but also shaping normal self. Open questions regard primarily the ligands that activate Toll receptors in neuronal contexts, requirements for co-receptors, alternative signalling modules, and whether Tolls are directly adhesive or mainly instructive in neuronal guidance. Future work in invertebrates and vertebrates will doubtlessly unravel the molecular mechanisms underlying the functions of Tolls in non-immune contexts.

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References 1 Leulier, F. and Lemaitre, B. (2008) Toll-like receptors–taking an evolutionary approach. Nat. Rev. Genet. 9, 165–178 2 Bosch, T.C. (2014) Rethinking the role of immunity: lessons from Hydra. Trends Immunol. 35, 495–502 3 Ward, A. et al. (2015) Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a drosophila olfactory circuit. Neuron 85, 1013–1028 4 McIlroy, G. et al. (2013) Toll-6 and Toll-7 function as neurotrophin receptors in the Drosophila melanogaster CNS. Nat. Neurosci. 16, 1248–1256 5 Ballard, S.L. et al. (2014) Retrograde neurotrophin signaling through Tollo regulates synaptic growth in Drosophila. J. Cell Biol. 204, 1157–1172 6 Pujol, N. et al. (2001) A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans. Curr. Biol. 11, 809–821 7 Kolesnikov, T. and Beckendorf, S.K. (2007) 18 wheeler regulates apical constriction of salivary gland cells via the Rho-GTPase-signaling pathway. Dev. Biol. 307, 53–61 8 Pare, A.C. et al. (2014) A positional Toll receptor code directs convergent extension in Drosophila. Nature 515, 523–527 9 Dionne, M.S. (2013) Comparative immunology: allorecognition and variable surface receptors outside the jawed vertebrates. Curr. Opin. Immunol. 25, 608–612 10 Suga, H. et al. (2013) The Capsaspora genome reveals a complex unicellular prehistory of animals. Nat. Commun. 4, 2325

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Different flavors of Toll guide olfaction.

Toll-like receptors are historically linked to immunity across animal phyla, but accumulating evidence suggests they play additional roles in neuronal...
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