Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition Fengyan Zhoua, Guozheng Caoa, Songjun Daia, Guo Lia, Hao Lia, Zhu Dinga, Shouqing Houa, Bingbing Xua, Wendong Youb, Gil Wiseglassc, Feng Shia, Xiaofeng Yangb, Rotem Rubinsteinc, and Yongfeng Jina,b,1 aMOE Laboratory of Biosystems Homeostasis & Protection, Innovation Center for Cell Signaling Network, College of Life Sciences, Zhejiang University, ZJ310058 Hangzhou, Zhejiang, China; bDepartment of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, ZJ310058 Hangzhou, Zhejiang, China; and cSchool of Neurobiology, Biochemistry and Biophysics, Sagol School of Neuroscience, George S. Wise Faculty of Life Science, Tel Aviv University, 69978 Ramat Aviv, Israel Edited by Barry Honig, Howard Hughes Medical Institute, Columbia University, New York, NY, and approved August 20, 2020 (received for review December 15, 2019) Thousands of Down syndrome cell adhesion molecule (Dscam1) (13–16). Different isoforms share the same domain organization isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are re- with 10 Ig domains, 6 fibronectin type III (FNIII) domains, a quired for establishing neural circuits in insects and vertebrates, single transmembrane (TM) region, and a cytoplasmic domain, respectively. The strict homophilic specificity exhibited by these but differ in the primary sequences of at least 1 of 3 Ig domains. proteins has been extensively studied and is thought to be critical Individual neuronal identities are determined by the stochastic for their function in neuronal self-avoidance. In contrast, signifi- expression of a small set of 10 to 50 distinct Dscam1 isoforms of cantly less is known about the Dscam1-related family of ∼100 short- the tens of thousands of possible isoforms that can be generated ened Dscam (sDscam) proteins in Chelicerata. We report that via alternative splicing (8, 14, 17, 18). In contrast to insect Chelicerata sDscamα and some sDscamβ protein trans interactions Dscam1, vertebrate Dscam genes do not produce extensive iso- are strictly homophilic, and that the trans interaction is meditated form diversity (19). In vertebrates, a different set of cell surface via the first Ig domain through an antiparallel interface. Addition- adhesion receptors, the cPcdhs, performs an analogous function ally, different sDscam isoforms interact promiscuously in cis via (20–23). In human and mouse, 53 and 58 cPcdh proteins, re- membrane proximate fibronectin-type III domains. We report that spectively, are encoded by three tandemly arranged gene clusters – cell cell interactions depend on the combined identity of all sDscam of Pcdhα, Pcdhβ, and Pcdhγ (24, 25). Single neuronal surface BIOCHEMISTRY isoforms expressed. A single mismatched sDscam isoform can inter- identity is achieved by a combination of stochastic promoter fere with the interactions of cells that otherwise express an identical selection and alternative splicing (26–28). In addition to engag- set of isoforms. Thus, our data support a model by which sDscam ing in trans (cell-to-cell) through strict homophilic interactions association in cis and trans generates a vast repertoire of combina- (29, 30), cPcdhs also exhibit an additional independent cis (same torial homophilic recognition specificities. We propose that in Che- cell) interaction that is isoform promiscuous. It is surprising that licerata, sDscam combinatorial specificity is sufficient to provide fewer than 60 proteins are able to mediate the process of neu- each neuron with a unique identity for self–nonself discrimination. ronal self-avoidance in the complex mammalian brain, as op- Surprisingly, while sDscams are related to Drosophila Dscam1, our posed to thousands of isoforms required for an analogous results mirror the findings reported for the structurally unrelated function in Drosophila. Studies using cell aggregation assays have vertebrate cPcdh. Thus, our findings suggest a remarkable example found a possible explanation for this challenge. Specifically, in of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of meta- Significance zoan self-avoidance and self–nonself discrimination. Neuronal self-avoidance is a conserved process in vertebrates Down syndrome cell adhesion molecule | homophilic binding | and invertebrates. In Drosophila, self-avoidance is mediated by combinatorial specificity | self-recognition | Chelicerata the Down syndrome cell adhesion molecule (Dscam1) gene that encodes tens of thousands of proteins through alternative atterning of the developing brain is critically affected by the splicing. In vertebrates, an analogous function is performed by Pprecision of selective recognition and the strength of the ∼60 clustered protocadherins (cPcdh) through promoter choice. interactions between cell adhesion receptors (1, 2). Two large Here we use cell aggregation assays to study the binding cell adhesion receptor families, Down syndrome cell adhesion preferences of ∼100 sDscam protein in scorpion. We report molecule (Dscam1) of the immunoglobulin superfamily and that while related in sequence to the fly Dscam, the scorpion clustered protocadherins (cPcdhs) of the cadherin superfamily, sDscam adopts a strategy that is similar to that of vertebrate play a central role in neural circuit assembly in insects and ver- cPcdhs, of combined specificity when coexpressed. Our find- tebrates, respectively. These proteins mediate highly selective ings identify sDscams as likely candidates to mediate neuronal homophilic interactions and generate a unique molecular iden- self-avoidance in Chelicerata, as well as provide a remarkable tity at the surface of individual neurons, thereby enabling them example of convergent evolution. to distinguish self from nonself and ultimately to self-avoid. Genetic studies using fly and mouse neurons have described a Author contributions: Y.J. conceived this project; F.Z., G.C., S.D., G.L., H.L., B.X., and Y.J. remarkably similar molecular strategy of self-avoidance (3–12). designed research; F.Z., G.C., S.D., G.L., and H.L. performed research; F.Z., Z.D., and S.H. Homophilic interactions between identical repertoires of Dscam/ performed homology modeling and protein–protein docking; F.Z., Z.D., S.H., B.X., W.Y., cPcdh proteins on the surface of the same neuron lead to self- G.W., F.S., X.Y., R.R., and Y.J. analyzed data; and F.Z., R.R., and Y.J. wrote the paper. recognition and result in neurite repulsion. In contrast, contact The authors declare no competing interest. by two arbors from distinct neurons, with differing isoform This article is a PNAS Direct Submission. compositions, does not result in homophilic binding and does not Published under the PNAS license. trigger an avoidance mechanism. 1To whom correspondence may be addressed. Email: [email protected]. In Drosophila, neuronal self-avoidance is mediated by sto- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ chastic alternative splicing of a single gene, Dscam1, that can doi:10.1073/pnas.1921983117/-/DCSupplemental. encode as many as 19,008 isoforms with distinct ectodomains www.pnas.org/cgi/doi/10.1073/pnas.1921983117 PNAS Latest Articles | 1of12 Downloaded by guest on September 23, 2021 these assays recognition of cells that express multiple distinct cells using an insect baculovirus expression system (Fig. 1B). This cPcdh isoforms was observed to be dependent on the combined system is a powerful tool for investigating homophilic interac- identity of all expressed isoforms (29, 30). That is, two cells that tions between expressed cell surface adhesion molecules (35). express a mismatched isoform will not bind to each other even if An analogous approach, using different cells, was used in studies all other expressed cPcdh isoforms are identical. of trans binding properties of mouse cPcdh and Drosophila The Chelicerata subphylum is a basal branch of arthropods Dscam (7, 29, 30). Sf9 cells that expressed constructs encoding that includes animals, such as spiders and scorpions, with rela- sDscamβ6v2, either full-length (β6v2FL-mCherry) or lacking the tively complex brains that are similar in magnitude to that of the cytoplasmic domain (β6v2Δcyto-mCherry), exhibited strong ag- Drosophila brain (31). In contrast to Drosophila Dscam1, Cheli- gregation (SI Appendix, Fig. S1A). This finding indicates that the cerata Dscam genes do not generate highly diverse proteins and do homophilic interaction is mediated by sDscamβ6v2 in trans in- not have cPcdh genes. Recently, we discovered a “hybrid” gene dependent of the cytoplasmic region. We therefore used Δcyto family in the subphylum Chelicerata that is particularly relevant to constructs for all sDscam proteins in the cell aggregation assay. the remarkable functional convergence of Drosophila Dscam1 and We performed a systematic analysis of the homophilic inter- vertebrate cPcdhs. This gene family is composed of Dscam-related actions for 86 of the 95 sDscam proteins (34 of 40 sDscamα, and genes with tandemly arrayed 5′ cassettes, which encode ∼50 to 100 52 of 55 sDscamβ1–β6), as 9 sDscam cDNAs failed to be cloned isoforms each with alternative promoters for the number of iso- (SI Appendix, Table S1). We found that all of the 34 sDscamαs, forms varying across Chelicerata species (32, 33). Although these which were individually expressed, formed homophilic aggre- Chelicerata Dscams are evolutionarily related to Drosophila