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Teneurin-3 controls topographic circuit assembly in the hippocampus

Nature volume 554, pages 328–333 (2018)Cite this article

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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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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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Authors and Affiliations

  1. Howard Hughes Medical Institute, Stanford University, Stanford, 94305, California, USA

    Dominic S. Berns, Laura A. DeNardo, Daniel T. Pederick & Liqun Luo

  2. Department of Biology, Stanford University, Stanford, 94305, California, USA

    Dominic S. Berns, Laura A. DeNardo, Daniel T. Pederick & Liqun Luo

  3. Neurosciences Graduate Program, Stanford University, Stanford, 94305, California, USA

    Dominic S. Berns

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  1. Dominic S. Berns
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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.

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Correspondence to Liqun Luo.

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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 g–i. 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 b–g, 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. b–d, 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 c–g 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. b–d, 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.

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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.

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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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