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TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons

Nature Genetics volume 48, pages 1359–1369 (2016)Cite this article

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Abstract

TSHZ3, which encodes a zinc-finger transcription factor, was recently positioned as a hub gene in a module of the genes with the highest expression in the developing human neocortex, but its functions remained unknown. Here we identify TSHZ3 as the critical region for a syndrome associated with heterozygous deletions at 19q12-q13.11, which includes autism spectrum disorder (ASD). In Tshz3-null mice, differentially expressed genes include layer-specific markers of cerebral cortical projection neurons (CPNs), and the human orthologs of these genes are strongly associated with ASD. Furthermore, mice heterozygous for Tshz3 show functional changes at synapses established by CPNs and exhibit core ASD-like behavioral abnormalities. These findings highlight essential roles for Tshz3 in CPN development and function, whose alterations can account for ASD in the newly defined TSHZ3 deletion syndrome.

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Figure 1: Schematic of deletions of the TSHZ3 locus and TSHZ3 expression in the human and mouse fetal neocortex.
Figure 2: Tshz3lacZ/lacZ mice show altered gene expression of cortical layer markers at E18.5.
Figure 3: Cortical layering and major axonal tracts are preserved in Tshz3lacZ/lacZ brains at E18.5.
Figure 4: Tshz3lacZ/+ mice exhibit altered expression of genes in the neocortex from embryonic to postnatal stages, but the main projection systems from deep CPNs are preserved.
Figure 5: Altered corticostriatal synaptic transmission and plasticity in Tshz3lacZ/+ mice.
Figure 6: Tshz3lacZ/+ mice display autism-like behavioral deficits.

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Acknowledgements

We thank M. Galdi, V. Vanoosten and C. Scajola who assisted with behavioral testing. This work was supported by funding from CNRS and Aix-Marseille University to L.K.-L.G., L.F. and M.C.; INSERM and Aix-Marseille University to P.L.R.; Fédération pour la Recherche sur le Cerveau (FRC) to L.F.; National Institutes of Health grants MH103339, MH106934 and MH106874, the Simons Foundation and the Kavli Foundation to N.S.; and the Medical Research Council (MR/L002744/1), the Manchester Biomedical Research Centre and the NIHR Greater Manchester Clinical Research Network to A.S.W. Funding for A.N.G. was provided by a grant from the German Federal Ministry for Education and Research (BMBF, NGFN-plus, 'Alzheimer Disease Integrative Genomics', PNA-01GS08127-3a). Microscopy was performed at the imaging platform of IBDM, supported by the French National Research Agency through the 'Investments for the Future' program (France-BioImaging, ANR-10-INSB-04-01).

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Author notes

  1. Xavier Caubit and Paolo Gubellini: These authors contributed equally to this work.

Authors and Affiliations

  1. Aix-Marseille Université, CNRS, IBDM, Marseille, France

    Xavier Caubit, Paolo Gubellini, Mehdi Metwaly, Bernard Jacq, Ahmed Fatmi, Laurence Had-Aissouni, Pascal Salin, Lydia Kerkerian-Le Goff & Laurent Fasano

  2. Institut de Génétique Médicale, Hôpital Jeanne de Flandre, CHRU Lille, Lille, France

    Joris Andrieux & Fatimetou Abderrehamane

  3. Aix-Marseille Université, INSERM, GMGF, Marseille, France

    Pierre L Roubertoux

  4. Department of Neuroscience, Yale School of Medicine, New Haven, Connecticut, USA

    Kenneth Y Kwan, Ying Zhu & Nenad Sestan

  5. Department of Human Genetics, Molecular and Behavioral Neuroscience Institute (MBNI), University of Michigan, Ann Arbor, Michigan, USA

    Kenneth Y Kwan

  6. Aix-Marseille Université, CNRS, LPC, Marseille, France

    Michèle Carlier

  7. Clinical Genetics Unit, Karolinska University Hospital Solna, Stockholm, Sweden

    Agne Liedén & Eva Rudd

  8. Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington University School of Medicine, St. Louis, Missouri, USA

    Marwan Shinawi

  9. Service de Génétique Clinique, Hôpital Jeanne de Flandre, CHRU Lille, Lille, France

    Catherine Vincent-Delorme

  10. Service de Neuropédiatrie, Hôpital Salengro, CHRU Lille, Lille, France

    Jean-Marie Cuisset & Marie-Pierre Lemaitre

  11. Centre de Cytogénétique, Hôpital Saint Vincent de Paul, GHICL, UCL, Lille, France

    Bénédicte Duban & Jean-François Lemaitre

  12. Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester Academic Health Science Centre and the Royal Manchester Children's and St Mary's Hospitals, Manchester, UK

    Adrian S Woolf

  13. UCL Institute of Child Health, London, UK

    Detlef Bockenhauer

  14. MGX-Montpellier GenomiX, Institut de Génomique Fonctionnelle, Montpellier, France

    Dany Severac & Emeric Dubois

  15. Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité University Hospital Berlin, Berlin, Germany

    Alistair N Garratt

Authors
  1. Xavier Caubit
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  2. Paolo Gubellini
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Contributions

X.C., P.G., P.L.R., L.H.-A., N.S., A.N.G., L.K.-L.G. and L.F. designed the study. X.C., P.G., P.L.R., J.A., A.N.G., B.J., M.M., L.H.-A., K.Y.K., P.S. and Y.Z. performed experiments. J.A., A.L., E.R., M.S., C.V.-D., J.-M.C., M.-P.L., F.A., B.D., J.-F.L., A.S.W. and D.B. contributed clinical samples and clinical data. A.F., X.C. and L.F. prepared RNA samples, A.F. performed qRT-PCR, and D.S. and E.D. produced RNA–seq data and performed bioinformatics analysis of them (MGX-Montpellier GenomiX). X.C., P.G., P.L.R., L.H.-A., P.L.R., M.C., A.N.G., B.J., K.Y.K., N.S., L.K.-L.G. and L.F. analyzed data. X.C., P.G., P.L.R., L.H.-A., N.S., A.N.G., A.S.W., L.K.-L.G. and L.F. wrote the manuscript.

Corresponding author

Correspondence to Laurent Fasano.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Genes coexpressed with TSHZ3 in the human neocortex and associated human pathologies.

(a) Module of genes coexpressed with TSHZ3 in the human neocortex at mid-gestation. The TSHZ3 module contains 50 genes including TSHZ3. Blue ‘balls’ correspond to genes that have been associated with ASD. Larger font size is used for genes encoding transcription factors known to influence cortical projection neuron differentiation, which all have been associated with ASD. (b) Histogram showing the most represented human pathologies associated with genes present in the TSHZ3 coexpression module (sorted in order of decreasing representation). Each gene is scored on the basis of the number of relevant publications that associate it with a pathology. Scores were given as follows: 1, 1 publication; 2, 2 publications; 3, >2 publications. ASD, autism spectrum disorder; ALS, amyotrophic lateral sclerosis; ADHD, attention deficit hyperactivity disorder.

Supplementary Figure 2 Tshz3lacZ/lacZ mice show altered gene expression at E18.5.

(a) Variation in mRNA levels analyzed by qRT–PCR in the cortex of Tshz3lacZ/lacZ versus wild-type mice. (b) Detection by in situ hybridization of differentially expressed genes and qRT–PCR analyses in the cortex of Tshz3lacZ/lacZ versus wild-type mice. Sagittal brain sections at E18.5 are shown (rostral, left; dorsal, top). Fgf10 expression in L5, which is caudally restricted in wild-type mice, is increased in the rostro-caudal extent of the cortex in Tshz3lacZ/lacZ mutant. The caudal expression of Ngfr in the subplate and L6 is strongly decreased in the mutant. The caudal expression of Col5a1 in the suplate is strongly decreased. Igfbp3 expression is strongly decreased caudally in the subplate and L6. (c) Tshz3 expression in a wild-type sagittal section. Data in a and b are shown as means ± s.e.m. (n = 3). *P < 0.05, **P < 0.02, unpaired two-tailed t test). For the large views in b and c, scale bar = 1 mm; for close-up views, scale bar = 100 μm.

Supplementary Figure 3 Membrane properties of striatal MSNs are similar in wild-type and Tshz3lacZ/+ mice.

(a–e) Resting membrane potential (RMP; P = 0.069, Student’s t test) (a), action potential (AP) threshold (P = 0.746, Mann–Whitney test) (b), pattern of AP discharge (traces depict the responses of two MSNs to current steps of –100, +100 and +200 pA) (c), current–voltage relationship (d) and resulting input resistance (Ri) (e) (linear regression and slope comparison between wild-type and Tshz3lacZ/+ mice, F(1,127) = 1.486, P = 0.225). All data are expressed as means ± s.e.m.

Supplementary Figure 4 Visual, auditory and olfactory screening in Tshz3lacZ/+ and wild-type mice.

(a–c) No significant differences were found between the two genotypes (n = 11 male mice per group) for visual performance (t < 1; d.f. = 20; P = 0.55) (a), auditory performance (t = 0.46; d.f. = 20; P = 0.65) (b) and olfactory exploration in the habituation/dishabituation test (c). Data are shown as means ± s.e.m. In c, time sniffing non-social (water, violet, vanilla) and social (B6, SWR) odors was analyzed with mixed ANOVA (genotype factor with two levels, Tshz3lacZ/+ and wild type, and 15 odors as repeated measures). The genotype factor was not significant (F < 1; d.f. = 1, 20).

Supplementary Figure 5 Sociability and preference in social novelty of Tshz3lacZ/+ male mice in the three-chamber apparatus.

(a) Time spent in the empty lateral compartments did not differ between the genotypes (F(1, 19) = 2.17; P = 0.16), but, with the effect size η2 being 0.10, we included the total activity in an ANCOVA. (b,c) Mean exploration times (±s.e.m.) that measured sociability (b) and preference for social novelty (c), after controlling for the effect of total activity. A double interaction between the genotype factor and the two condition factors (sociability and preference for social novelty) reached P = 0.005 (F(1, 19) = 10.08), with a large effect size (partial η2 = 0.35). We performed two partial ANOVAs to break out the double interaction. The first partial ANOVA showed an interaction with a large effect size (partial η2 = 0.31) between the genotype factor and the sociability factor (F(1, 19) = 8.51; P = 0.009). Tshz3lacZ/+ males did not explore the CD1 partner and empty box differently. Wild-type males explored the CD1 partner more than the empty box (dependent t(10) = 4.72; P < 0.001) with a large effect size of 0.76. The second partial ANOVA showed that the interaction was not significant between the genotype factor and the social novelty factor (F(1, 19) = 2.08; P = 0.16; partial η2 = 0.10). The two genotypes explored the new CD1 partner more than the already known CD1 partner. However, the difference was significant only for the wild-type male group (dependent t(10) = 3.08; P = 0.01) with an effect size of 0.61. n = 11 male mice per group. *P < 0.01, **P < 0.001. All data are expressed as means ± s.e.m.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 2, 6, 7 and 8. (PDF 1301 kb)

Supplementary Table 1

Human brain and nervous system pathologies associated with genes present in the TSHZ3 module. (XLSX 43 kb)

Supplementary Table 3

DEX gene markers in E18.5 Tshz3lacZ/lacZ cortex. (XLSX 21 kb)

Supplementary Table 4

Analysis of pathways represented in and GO terms associated with DEX genes. (XLSX 84 kb)

Supplementary Table 5

Human brain and nervous system pathologies associated with orthologs of mouse Tshz3-regulated genes. (XLSX 57 kb)

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Caubit, X., Gubellini, P., Andrieux, J. et al. TSHZ3 deletion causes an autism syndrome and defects in cortical projection neurons. Nat Genet 48, 1359–1369 (2016). https://doi.org/10.1038/ng.3681

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