Teneurin-3 controls topographic circuit assembly in the hippocampus

Abstract

Brain functions rely on specific patterns of connectivity. Teneurins are evolutionarily conserved transmembrane proteins that instruct synaptic partner matching in Drosophila and are required for vertebrate visual system development. The roles of vertebrate teneurins in connectivity beyond the visual system remain largely unknown and their mechanisms of action have not been demonstrated. Here we show that mouse teneurin-3 is expressed in multiple topographically interconnected areas of the hippocampal region, including proximal CA1, distal subiculum, and medial entorhinal cortex. Viral-genetic analyses reveal that teneurin-3 is required in both CA1 and subicular neurons for the precise targeting of proximal CA1 axons to distal subiculum. Furthermore, teneurin-3 promotes homophilic adhesion in vitro in a splicing isoform-dependent manner. These findings demonstrate striking genetic heterogeneity across multiple hippocampal areas and suggest that teneurin-3 may orchestrate the assembly of a complex distributed circuit in the mammalian brain via matching expression and homophilic attraction.

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Figure 1: Ten3 expression in the developing hippocampal region.
Figure 2: Ten3 is required for the precise CA1→subiculum projection.
Figure 3: Ten3 conditional knockout in CA1.
Figure 4: Ten3 conditional knockout in subiculum.
Figure 5: Ten3 promotes homophilic adhesion in a splicing isoform-dependent manner.

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References

  1. 1

    Scoville, W. B. & Milner, B. Loss of recent memory after bilateral hippocampal lesions. J. Neurol. Neurosurg. Psychiatry 20, 11–21 (1957)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Squire, L. R., Stark, C. E. L. & Clark, R. E. The medial temporal lobe. Annu. Rev. Neurosci. 27, 279–306 (2004)

    Article  CAS  PubMed  Google Scholar 

  3. 3

    O’Keefe, J. & Dostrovsky, J. The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res. 34, 171–175 (1971)

    Article  PubMed  Google Scholar 

  4. 4

    Hafting, T., Fyhn, M., Molden, S., Moser, M. B. & Moser, E. I. Microstructure of a spatial map in the entorhinal cortex. Nature 436, 801–806 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. 5

    van Strien, N. M., Cappaert, N. L. M. & Witter, M. P. The anatomy of memory: an interactive overview of the parahippocampal–hippocampal network. Nat. Rev. Neurosci. 10, 272–282 (2009)

    Article  CAS  PubMed  Google Scholar 

  6. 6

    Tamamaki, N. & Nojyo, Y. Preservation of topography in the connections between the subiculum, field CA1, and the entorhinal cortex in rats. J. Comp. Neurol. 353, 379–390 (1995)

    Article  CAS  PubMed  Google Scholar 

  7. 7

    Naber, P. A., Lopes da Silva, F. H. & Witter, M. P. Reciprocal connections between the entorhinal cortex and hippocampal fields CA1 and the subiculum are in register with the projections from CA1 to the subiculum. Hippocampus 11, 99–104 (2001)

    Article  CAS  PubMed  Google Scholar 

  8. 8

    Igarashi, K. M., Ito, H. T., Moser, E. I. & Moser, M. B. Functional diversity along the transverse axis of hippocampal area CA1. FEBS Lett. 588, 2470–2476 (2014)

    Article  CAS  PubMed  Google Scholar 

  9. 9

    Cembrowski, M. S. et al. Spatial gene-expression gradients underlie prominent heterogeneity of CA1 pyramidal neurons. Neuron 89, 351–368 (2016)

    Article  CAS  PubMed  Google Scholar 

  10. 10

    Sperry, R. W. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl Acad. Sci. USA 50, 703–710 (1963)

    Article  ADS  CAS  Google Scholar 

  11. 11

    Sanes, J. R. & Yamagata, M. Many paths to synaptic specificity. Annu. Rev. Cell Dev. Biol. 25, 161–195 (2009)

    Article  CAS  Google Scholar 

  12. 12

    Kolodkin, A. L. & Tessier-Lavigne, M. Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb. Perspect. Biol. 3, 1–14 (2011)

    Article  CAS  Google Scholar 

  13. 13

    Hong, W., Mosca, T. J. & Luo, L. Teneurins instruct synaptic partner matching in an olfactory map. Nature 484, 201–207 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Mosca, T. J., Hong, W., Dani, V. S., Favaloro, V. & Luo, L. Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice. Nature 484, 237–241 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Tucker, R. P., Beckmann, J., Leachman, N. T., Schöler, J. & Chiquet-Ehrismann, R. Phylogenetic analysis of the teneurins: conserved features and premetazoan ancestry. Mol. Biol. Evol. 29, 1019–1029 (2012)

    Article  CAS  PubMed  Google Scholar 

  16. 16

    Li, H., Bishop, K. M. & O’Leary, D. D. M. Potential target genes of EMX2 include odz/Ten-m and other gene families with implications for cortical patterning. Mol. Cell. Neurosci. 33, 136–149 (2006)

    Article  CAS  PubMed  Google Scholar 

  17. 17

    Psychiatric GWAS Consortium Bipolar Disorder Working Group. Large-scale genome-wide association analysis of bipolar disorder identifies a new susceptibility locus near ODZ4. Nat. Genet. 43, 977–983 (2011)

  18. 18

    Green, E. K. et al. Replication of bipolar disorder susceptibility alleles and identification of two novel genome-wide significant associations in a new bipolar disorder case-control sample. Mol. Psychiatry 18, 1302–1307 (2013)

    Article  CAS  PubMed  Google Scholar 

  19. 19

    Mühleisen, T. W. et al. Genome-wide association study reveals two new risk loci for bipolar disorder. Nat. Commun. 5, 3339 (2014)

    Article  ADS  CAS  PubMed  Google Scholar 

  20. 20

    Croarkin, P. E. et al. Genetic risk score analysis in early-onset bipolar disorder. J. Clin. Psychiatry 78, 1337–1343 (2017)

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Ivorra, J. L. et al. Replication of previous genome-wide association studies of psychiatric diseases in a large schizophrenia case-control sample from Spain. Schizophr. Res. 159, 107–113 (2014)

    Article  PubMed  Google Scholar 

  22. 22

    Aldahmesh, M. A., Mohammed, J. Y., Al-Hazzaa, S. & Alkuraya, F. S. Homozygous null mutation in ODZ3 causes microphthalmia in humans. Genet. Med. 14, 900–904 (2012)

    Article  CAS  PubMed  Google Scholar 

  23. 23

    Alkelai, A. et al. A role for TENM1 mutations in congenital general anosmia. Clin. Genet. 90, 211–219 (2016)

    Article  CAS  PubMed  Google Scholar 

  24. 24

    Leamey, C. A. et al. Ten_m3 regulates eye-specific patterning in the mammalian visual pathway and is required for binocular vision. PLoS Biol. 5, e241 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Dharmaratne, N. et al. Ten-m3 is required for the development of topography in the ipsilateral retinocollicular pathway. PLoS ONE 7, e43083 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Antinucci, P., Nikolaou, N., Meyer, M. P. & Hindges, R. Teneurin-3 specifies morphological and functional connectivity of retinal ganglion cells in the vertebrate visual system. Cell Reports 5, 582–592 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Antinucci, P., Suleyman, O., Monfries, C. & Hindges, R. Neural mechanisms generating orientation selectivity in the retina. Curr. Biol. 26, 1802–1815 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Rubin, B. P., Tucker, R. P., Brown-Luedi, M., Martin, D. & Chiquet-Ehrismann, R. Teneurin 2 is expressed by the neurons of the thalamofugal visual system in situ and promotes homophilic cell-cell adhesion in vitro. Development 129, 4697–4705 (2002)

    CAS  PubMed  Google Scholar 

  29. 29

    Beckmann, J., Schubert, R., Chiquet-Ehrismann, R. & Müller, D. J. Deciphering teneurin domains that facilitate cellular recognition, cell-cell adhesion, and neurite outgrowth using atomic force microscopy-based single-cell force spectroscopy. Nano Lett. 13, 2937–2946 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  30. 30

    Boucard, A. A., Maxeiner, S. & Südhof, T. C. Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing. J. Biol. Chem. 289, 387–402 (2014)

    Article  CAS  PubMed  Google Scholar 

  31. 31

    Silva, J.-P. et al. Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities. Proc. Natl Acad. Sci. USA 108, 12113–12118 (2011)

    Article  ADS  PubMed  Google Scholar 

  32. 32

    O’Sullivan, M. L. et al. FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development. Neuron 73, 903–910 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Loh, K. H. et al. Proteomic analysis of unbounded cellular compartments: synaptic clefts. Cell 166, 1295–1307 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Steward, O. Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat. J. Comp. Neurol. 167, 285–314 (1976)

    Article  CAS  PubMed  Google Scholar 

  35. 35

    van Groen, T., Miettinen, P. & Kadish, I. The entorhinal cortex of the mouse: organization of the projection to the hippocampal formation. Hippocampus 13, 133–149 (2003)

    Article  PubMed  Google Scholar 

  36. 36

    Muraoka, D., Katsuyama, Y., Kikkawa, S. & Terashima, T. Postnatal development of entorhinodentate projection of the Reeler mutant mouse. Dev. Neurosci. 29, 59–72 (2007)

    Article  CAS  PubMed  Google Scholar 

  37. 37

    Tamamaki, N. & Nojyo, Y. Disposition of the slab-like modules formed by axon branches originating from single CA1 pyramidal neurons in the rat hippocampus. J. Comp. Neurol. 291, 509–519 (1990)

    Article  CAS  PubMed  Google Scholar 

  38. 38

    Amaral, D. G., Dolorfo, C. & Alvarez-Royo, P. Organization of CA1 projections to the subiculum: a PHA-L analysis in the rat. Hippocampus 1, 415–435 (1991)

    Article  CAS  PubMed  Google Scholar 

  39. 39

    Honda, Y. & Ishizuka, N. Topographic distribution of cortical projection cells in the rat subiculum. Neurosci. Res. 92, 1–20 (2015)

    Article  PubMed  Google Scholar 

  40. 40

    Witter, M. P. & Groenewegen, H. J. The subiculum: cytoarchitectonically a simple structure, but hodologically complex. Prog. Brain Res. 83, 47–58 (1990)

    Article  CAS  PubMed  Google Scholar 

  41. 41

    Gerfen, C. R. & Sawchenko, P. E. An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res. 290, 219–238 (1984)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Mosca, T. J. & Luo, L. Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins. eLife 3, e03726 (2014)

    PubMed  PubMed Central  Google Scholar 

  43. 43

    Leamey, C. A . et al. Differential gene expression between sensory neocortical areas: potential roles for Ten_m3 and Bcl6 in patterning visual and somatosensory pathways. Cereb. Cortex 18, 53–66 (2008)

    Article  PubMed  Google Scholar 

  44. 44

    Oohashi, T. et al. Mouse Ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues. J. Cell Biol. 145, 563–577 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Ben-Zur, T., Feige, E., Motro, B. & Wides, R. The mammalian Odz gene family: homologs of a Drosophila pair-rule gene with expression implying distinct yet overlapping developmental roles. Dev. Biol. 217, 107–120 (2000)

    Article  CAS  PubMed  Google Scholar 

  46. 46

    O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44 (D1), D733–D745 (2016)

    Article  CAS  Google Scholar 

  47. 47

    Luo, L. & Flanagan, J. G. Development of continuous and discrete neural maps. Neuron 56, 284–300 (2007)

    Article  CAS  PubMed  Google Scholar 

  48. 48

    Bonanomi, D. & Pfaff, S. L. Motor axon pathfinding. Cold Spring Harb. Perspect. Biol. 2, a001735 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Feng, K. et al. All four members of the Ten-m/Odz family of transmembrane proteins form dimers. J. Biol. Chem. 277, 26128–26135 (2002)

    Article  CAS  PubMed  Google Scholar 

  50. 50

    Triplett, J. W. et al. Competition is a driving force in topographic mapping. Proc. Natl Acad. Sci. USA 108, 19060–19065 (2011)

    Article  ADS  PubMed  Google Scholar 

  51. 51

    Tasic, B. et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc. Natl Acad. Sci. USA 108, 7902–7907 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  52. 52

    Weissbourd, B. et al. Presynaptic partners of dorsal raphe serotonergic and GABAergic neurons. Neuron 83, 645–662 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Beier, K. T. et al. Circuit architecture of VTA dopamine neurons revealed by systematic input-output mapping. Cell 162, 622–634 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Lu, Y. C. et al. Structural basis of latrophilin-FLRT-UNC5 interaction in cell adhesion. Structure 23, 1678–1691 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Sisino, G., Bellavia, D., Corallo, M., Geraci, F. & Barbieri, R. A homemade cytospin apparatus. Anal. Biochem. 359, 283–284 (2006)

    Article  CAS  PubMed  Google Scholar 

  56. 56

    Witter, M. in The Mouse Nervous System (eds Watson, C., Paxinos, G. & Puelles, L. ) 112–139 (Elsevier, 2012)

  57. 57

    Kohara, K. et al. Cell type-specific genetic and optogenetic tools reveal hippocampal CA2 circuits. Nat. Neurosci. 17, 269–279 (2014)

    Article  CAS  PubMed  Google Scholar 

  58. 58

    Caballero-Bleda, M. & Witter, M. P. Regional and laminar organization of projections from the presubiculum and parasubiculum to the entorhinal cortex: an anterograde tracing study in the rat. J. Comp. Neurol. 328, 115–129 (1993)

    Article  CAS  PubMed  Google Scholar 

  59. 59

    Honda, Y. & Ishizuka, N. Organization of connectivity of the rat presubiculum: I. efferent projections to the medial entorhinal cortex. J. Comp. Neurol. 473, 463–484 (2004)

    Article  PubMed  Google Scholar 

  60. 60

    O’Reilly, K. C., Gulden Dahl, A., Ulsaker Kruge, I. & Witter, M. P. Subicular–parahippocampal projections revisited: development of a complex topography in the rat. J. Comp. Neurol. 521, 4284–4299 (2013)

    Article  PubMed  Google Scholar 

  61. 61

    Wright, N. F., Vann, S. D., Erichsen, J. T., O’Mara, S. M. & Aggleton, J. P. Segregation of parallel inputs to the anteromedial and anteroventral thalamic nuclei of the rat. J. Comp. Neurol. 521, 2966–2986 (2013)

    Article  PubMed  PubMed Central  Google Scholar 

  62. 62

    Meibach, R. C. & Siegel, A. Efferent connections of the hippocampal formation in the rat. Brain Res. 124, 197–224 (1977)

    Article  CAS  PubMed  Google Scholar 

  63. 63

    Ishizuka, N. Laminar organization of the pyramidal cell layer of the subiculum in the rat. J. Comp. Neurol. 435, 89–110 (2001)

    Article  CAS  PubMed  Google Scholar 

  64. 64

    Wright, N. F., Erichsen, J. T., Vann, S. D., O’Mara, S. M. & Aggleton, J. P. Parallel but separate inputs from limbic cortices to the mammillary bodies and anterior thalamic nuclei in the rat. J. Comp. Neurol. 518, 2334–2354 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Guo and the Howard Hughes Medical Institute/Janelia Research Campus for producing the Ten3cre and Ten3fl alleles, M. Sur for providing the Ten3Δ4 allele, the Neuroscience Gene Vector and Virus Core at Stanford for producing viruses, K. DeLoach for technical assistance, T. Mosca and W. Hong for advice and inspiration, members of the Luo laboratory for discussion and support, P. Thomas for the dual transcription unit vector, T. Südhof for advice and the latrophilin-3 construct, and K. Shen, L. Giocomo, T. Mosca, H. Li, J. Li, J. Lui, E. Richman and A. Shuster for critiques of the manuscript. D.S.B. was supported by a National Institute on Deafness and Other Communication Disorders predoctoral fellowship (F31DC013240), L.A.D. was supported by an National Institute of Neurological Disorders and Stroke postdoctoral fellowship (F32NS087860) and L.L. is an investigator of Howard Hughes Medical Institute. This work was supported by a National Institutes of Health grant (R01-NS050580 to L.L.).

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Contributions

D.S.B. performed all the experiments and analysed the data except the electrophysiology experiments. L.A.D. performed the electrophysiology experiments and analysed the data. D.T.P. assisted in aggregation assays. L.L. supervised the project. D.S.B. and L.L. wrote the paper.

Corresponding author

Correspondence to Liqun Luo.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Witter and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Ten3 staining details and controls.

a, Diagram of Ten3 protein showing location of antibody epitopes, specific domains, and region deleted in the Ten3Δ4 mutant. In Ten3Δ4, a neomycin resistance cassette (Neo) replaces 110 base pairs of sequence directly N-terminal to the transmembrane domain. The Ten3IC antibody was used for all Ten3 stainings in the paper except d and e of this figure. Scale bar, 200 amino acids. b, c, Ten3IC staining (red) on P9 horizontal sections of Ten3WT (b) and mutant Ten3Δ4/Δ4 (c) brains, showing loss of staining in Ten3 mutants. Note that the Ten3IC epitope is located N-terminal to Δ4, suggesting that in Ten3Δ4/Δ4 either the mRNA undergoes nonsense-mediated decay or the truncated protein is not stable. d, e, Ten3EC staining on P10 horizontal sections of Ten3WT (d) and mutant Ten3Δ4/Δ4 (e) brains. Staining has a higher background than Ten3IC antibody, but signal is present in proximal CA1 (arrow) and distal subiculum (arrowheads), similar to Ten3IC, which is absent in the knockout (open arrow/arrowheads). f, Ten3 staining (red) on P10 horizontal section with boxes around regions magnified in gi. g, Ten3 staining in dentate gyrus from f, CA3, and CA1. Intensity was increased to highlight Ten3 signal in axons and dendrites. Ten3 in stratum radiatum of proximal CA1 (arrowhead) is most probably from CA1 dendrites, since CA3 cells, the major source of axons in this layer, did not express Ten3 mRNA (Fig. 1b). Ten3 in the molecular layers of dentate gyrus and CA3 (arrows) is probably contributed by the axons of MEC layer II neurons56, since dentate gyrus and CA3 neurons did not express Ten3 mRNA (Fig. 1b). h, Proximal CA1 pyramidal cell layer from f, showing Ten3 signal in cell bodies. i, Proximal CA1 stratum lacunosum-moleculare from f, showing Ten3 signal in the region where MEC axons synapse onto CA1 pyramidal neuron dendrites. j, In situ hybridization on P9 horizontal section for Ten3 mRNA (magenta) combined with immunostaining for PCP4 (green), a marker of CA2 neurons57. No overlap between Ten3 and PCP4 was observed. Scale bars, 200 μm in bg, j, and 100 μm in h, i.

Extended Data Figure 2 Distribution of Ten3 mRNA in sagittal sections.

a, In situ hybridization for Ten3 mRNA on sagittal section of P10 brain. Top, merged image with Ten3 mRNA signal in red and DAPI in blue; middle, Ten3 mRNA signal alone; bottom: DAPI signal alone. b, Top: magnified image of Ten3 in situ hybridization in CA1; bottom: quantification of Ten3 mRNA along the proximal–distal axis of CA1 (n = 12 sections, four mice), showing a graded signal that peaks in proximal CA1 and decreases to a minimum in distal CA1. Proximal–distal axis is divided into 100 bins, with 1 being most proximal and 100 most distal. Shaded curves represent mean ± s.e.m. c, Top: magnified image of Ten3 in situ hybridization in subiculum; bottom: quantification of Ten3 mRNA along the proximal–distal axis of subiculum (n = 14 sections, four mice) showing a graded signal that peaks in distal subiculum and decreases to a minimum in proximal subiculum. The distributions in CA1 and subiculum are similarly shaped but reversed along the proximal–distal axis, reflecting the graded topographic connections along this axis (see Fig. 2 and Extended Data Fig. 5c). Scale bars, 200 μm in all panels. Source data

Extended Data Figure 3 Ten3 expression and topography details.

a, Ten3 staining (red) on P10 horizontal section. Dotted rectangles highlight staining in the hippocampal region and anteroventral thalamic nucleus, which are magnified in b and c. b, The parahippocampal region from a, showing expression of Ten3 relative to the proximal–distal (P–D) axes (arrows) in the presubiculum and parasubiculum. The connectivity of these regions is complex58,59, but seems to be consistent with preferential connectivity between Ten3-expressing subregions. Ten3 is expressed in distal presubiculum (close to parasubiculum), which projects to MEC near the parasubicular border59, and receives projections from distal subiculum60, both Ten3-high subregions. Ten3 is also expressed in proximal parasubiculum, which projects to MEC58 and receives projections from distal subiculum60, again both Ten3-high subregions. c, The anteroventral thalamus from a, showing intense Ten3 staining in the anterior and lateral division of the anteroventral thalamic nucleus (AVT, outlined). Distal subiculum, another Ten3-high region, projects to the anteroventral thalamic nucleus, whereas proximal subiculum projects to the anteromedial thalamus61. d, In situ hybridization for Ten3 mRNA (green) on P9 horizontal section from similar location as c. e, Ten3 staining (red) on P10 horizontal section, more ventral than a. Dotted rectangle highlights intense staining in the medial mammillary nucleus, which is magnified in f. f, Medial mammillary nucleus from e, showing Ten3 labelling in the lateral division of the medial mammillary nucleus (lMMn). Outlines show location of lateral mammillary nucleus (LM), lateral division of the medial mammillary nucleus, and medial division of the medial mammillary nucleus (mMMn). Proximal subicular neurons project to the medial division of the medial mammillary nucleus, whereas Ten3-high distal subicular neurons project to the Ten3-high lateral division40,62,63,64. The neurons of the lateral division project to the Ten3-high anteroventral thalamic nucleus, while medial division neurons project to the anteromedial thalamic nucleus61. g, In situ hybridization for Ten3 mRNA (green) on P9 horizontal section from similar location as f. Scale bars, 500 μm in a and e; 200 μm elsewhere. In summary, the pattern of Ten3-high to Ten3-high connectivity observed for CA1, subiculum, and entorhinal cortex seems to extend to many of the topographic projections formed between these subregions and the presubiculum, parasubiculum, thalamus, and mammillary nucleus.

Extended Data Figure 4 Generation and characterization of Ten3cre.

a, Design of Ten3cre. Top: region of chromosome 8 containing Ten3 exon 1, which contains the start codon (ATG). Middle: targeting construct with cre open reading frame inserted directly after the Ten3 start codon. cre is followed by a synthetic intron, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and bovine growth hormone polyadenylation sequence (bGH pA) (see key on right). Neo includes a phosphoglycerate kinase (PGK) promoter driving the resistance gene. Bottom: genomic region after homologous recombination. The endogenous exon 1 sequence after the start codon is replaced with cre. Neo was not removed by flippase (FLP)-mediated recombination in the mice used in Fig. 2. bd, Cre and Ten3 protein expression in P10 horizontal sections from Ten3cre mice. White dotted lines highlight proximal and distal borders of CA1 and subiculum. b, c, Cre expression (green) mimics the distribution of Ten3 expression (magenta) in Ten3Het mice. d, In Ten3cre/cre mice, Ten3 immunostaining is absent. Scale bars, 200 μm.

Extended Data Figure 5 Analysis of CA1→subiculum projections with various injection sites in Ten3Het control and Ten3KO mice.

a, b, PHA-L (green) injection in distal CA1 (a) and corresponding projection in proximal subiculum (b) in Ten3Het mouse. Scale bars, 200 μm. c, d, Averaged normalized injection (c) and projection (d) traces of all Ten3Het (black) and Ten3KO (red) mice analysed, binned into five groups by the mean position of the injection, and plotted from most proximal (top) to most distal (bottom) injections (bin limits and number of mice per bin listed on the right of d). Proximal–distal axis position is numbered from 1 (most proximal) to 100 (most distal). Shaded error curves represent mean ± s.e.m. at each bin. e, Projection width in subiculum versus injection mean position in CA1 for all mice (Ten3Het: n = 31, black circles; Ten3KO: n = 38, red triangles). f, Projection width data binned by injection mean. Number of mice per bin same as d. Projection width was significantly increased in Ten3KO for the three most proximal bins. ****P < 0.0001; multiplicity-adjusted P values after two-way ANOVA with Šídák’s correction for multiple comparisons. Error bars, mean ± s.e.m. g, Projection mean position in subiculum versus injection mean position in CA1 for all mice used (Ten3Het: n = 31; Ten3KO: n = 38), with superimposed linear regression lines (Ten3Het: R2 = 0.9812; Ten3KO: R2 = 0.9515). The slopes were significantly different (P < 0.0001), indicating a less sharp topography in Ten3KO mice. Bin 1 data (most proximal, injection mean 10–25) in cg are the same data as in Fig. 2e–g.

Extended Data Figure 6 Generation and characterization of Ten3flox.

a, Design of Ten3fl. Top: region of chromosome 8 containing Ten3 exon 4, which is 239 base pairs long and encodes 19 of the 21 amino acids in the transmembrane domain. Guide RNA (gRNA) targets shown in red (see key at right). Line 2: targeting construct with loxP sites inserted 5′ and 3′ of exon 4. Neo includes a PGK promoter driving the resistance gene. Line 3: genomic region after homology-directed repair. Bottom: deletion of exon 4 after Cre-mediated recombination between loxP sites. Neo was removed by FLP-mediated recombination in some of the mice used in Figs 3 and 4. In addition to deleting exon 4, the reading frame 3′ to exon 4 is frame-shifted with respect to the reading frame 5′ to exon 4. bd, Cre (green) and Ten3 (magenta) protein expression in P10 horizontal sections from Ten3fl/+ (b), Ten3Het (c), and Ten3cre/fl (d) mice. Ten3 staining is absent in Ten3cre/fl mice. White dotted lines highlight proximal and distal borders of CA1 and subiculum. Scale bars, 200 μm.

Extended Data Figure 7 Time course of CA1→subiculum projection development.

Sagittal sections from mice injected with BDA (green) in CA1 at P0, and perfused for staining at P2 (a, b), P4 (c, d), P6 (e, f), or P8 (g, h). Two mice are shown for each time point with a pair of images per mouse. Within each pair, the left image shows the section that contains the centre of the injection site at CA1, whereas the right image shows a magnified image of the section containing the highest density of projection at subiculum. Dashed lines mark proximal and distal CA1 borders in the left panels, and proximal and distal subicular borders in the right panels. CA1 axons are largely absent at subiculum at P2, and increase intensity from P4 to P8. Scale bars, 200 μm.

Extended Data Figure 8 Subiculum conditional knockout plots.

a, b, Plots from Ten3WT (a) and Ten3fl/fl (b) mice with minimal GFP–Cre expression. Heatmaps show normalized PHA-L fluorescence intensity (red, left) and normalized GFP–Cre intensity (green, middle) in subiculum, same mice. Each row is one section, 120 μm between rows, colour bars shown below a, and proximal–distal position is on the x axis. Surface plots are to the right of the corresponding heatmaps, showing PHA-L fluorescence intensity as height and GFP–Cre fluorescence intensity according to the colour map shown below a. P, proximal; D, distal; M, medial; L, lateral. Projections are similar between Ten3WT and Ten3fl/fl. c, d, Plots from Ten3WT (c) and Ten3fl/fl (d) mice with high GFP–Cre expression in subiculum. In Ten3fl/fl mice, PHA-L signal is decreased in GFP–Cre regions. Number of mice shown in Fig. 4. Source data

Extended Data Figure 9 Latrophilin-3 and Ten3 aggregation assay.

a, Images from aggregation assay with cells co-transfected with latrophilin-3 (Lphn3) and mCherry (magenta) mixed with cells co-transfected with GFP and empty vector (left), A0B0 isoform of Ten3 (middle), or A1B1 isoform of Ten3 (right). Scale bar, 200 μm, applies to all images. b, Quantification of aggregate sizes pooled from three biological replicates. Dashed red line shows cutoff at 600 μm2, the size of a large GFP+ cell from the control images. Asterisks denote significance from Dunn’s multiple comparisons test after a Kruskal–Wallis test, comparing all conditions with the Lphn3 and control mix, Lphn3 + control: n = 32 particles above threshold; Lphn3 + A0B0: n = 172; Lphn3 + A1B1: n = 159. NS, not significant; ****P ≤ 0.0001, multiplicity-adjusted P values. Source data

Extended Data Figure 10 Aggregation assays for cells expressing different Ten3 splicing isoforms.

a, Cell aggregation assay with combinations of K562 cells expressing the A1B1, A2B1, or A3B1 Ten3 isoform along with GFP or mCherry. Scale bar in bottom right panel, 200 μm, applies to all images. b, Quantification of aggregates observed in three biological replicates of the aggregation experiment in a. At least 100 aggregates were counted across the three replicates in each of the 10 mixing conditions, except for the GFP-alone and mCherry-alone controls, where no aggregates were observed. One hundred per cent of aggregates were mixed in combinations where both cell populations expressed a Ten3 isoform. No mixed aggregates were observed in combinations of Ten3-expressing cells with cells expressing mCherry alone, confirming that the aggregation is Ten3-dependent and not due to an endogenously expressed interaction partner. Fractions in parentheses indicate aggregates of a particular type out of all aggregates counted in that condition.

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Berns, D., DeNardo, L., Pederick, D. et al. Teneurin-3 controls topographic circuit assembly in the hippocampus. Nature 554, 328–333 (2018). https://doi.org/10.1038/nature25463

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