KCTD13 is a major driver of mirrored neuroanatomical phenotypes of the 16p11.2 copy number variant

Abstract

Copy number variants (CNVs) are major contributors to genetic disorders. We have dissected a region of the 16p11.2 chromosome--which encompasses 29 genes--that confers susceptibility to neurocognitive defects when deleted or duplicated. Overexpression of each human transcript in zebrafish embryos identified KCTD13 as the sole message capable of inducing the microcephaly phenotype associated with the 16p11.2 duplication, whereas suppression of the same locus yielded the macrocephalic phenotype associated with the 16p11.2 deletion, capturing the mirror phenotypes of humans. Analyses of zebrafish and mouse embryos suggest that microcephaly is caused by decreased proliferation of neuronal progenitors with concomitant increase in apoptosis in the developing brain, whereas macrocephaly arises by increased proliferation and no changes in apoptosis. A role for KCTD13 dosage changes is consistent with autism in both a recently reported family with a reduced 16p11.2 deletion and a subject reported here with a complex 16p11.2 rearrangement involving de novo structural alteration of KCTD13. Our data suggest that KCTD13 is a major driver for the neurodevelopmental phenotypes associated with the 16p11.2 CNV, reinforce the idea that one or a small number of transcripts within a CNV can underpin clinical phenotypes, and offer an efficient route to identifying dosage-sensitive loci.

Figure 1. Systematic analysis of 16p11.2 del/dup genes in vivo induces defects in head size

(a) Schematic of chromosome 16, zoomed in the 16p11.2 CNV, showing gene content (not to scale) above the black line. (b) Plot of head size measurements (in µm) of human mRNA overexpression combinations measured across ~50 embryos/injection cocktail. In all but one injection, both controls and human overexpressed genes result in indistinguishable median head size, with minimal variance. By contrast, embryos injected with the KCTD13/CDIPT mRNAs cocktail show consistent and significant reduction of head size.

Figure 2. KCTD13 dosage changes lead to head size, proliferation and apoptosis defects

(a) From top to bottom, dorsal (left) and lateral (right) views of representative embryos injected with Kctd13 MO, control, or KCTD13 mRNA. (b) Graph of the ratio between control and injected embryos head size measures at 4.5 dpf (n=45). (c) Phospho-histone H3 (top panel) and TUNEL (bottom panel) staining for proliferating or apoptotic cells in zebrafish brain at 2 dpf and 3 dpf respectively. From left to right are representative examples of MO-, control- and mRNA-injected embryos. (d) Graph of phospho-histone H3 and TUNEL quantifications from 20 MO-, control and mRNA-injected embryos. Data from three independent experiments are represented as mean ± s.d. *** p<0.00001; two-tailed t-test comparisons between control and either MO-and mRNA-injected embryos.

Figure 3. KCTD13 dosage changes lead to neuroanatomical defects

DAPI staining on transverse sections of the telencephalon (a, b, c), the diencephalon (d, e, f), and the mesencephalon (g, h, i) of embryos injected with kctd13 MO, control or KCTD13 mRNA at 4.5 dpf. (j) The planes of section are illustrated with red lines on dorsal views of kctd13 MO-, control-, and KCTD13 mRNA-injected embryos (left to right). Higher magnifications of the telencephalon (a–c) and the diencephalon (d–f) are in (a’–f’). The Meckel pharyngeal cartilage (a–c) and the palatoquadrate pharyngeal cartilage (d–f) are in insets. Scale bar, 100µm. (k) Bar graph of the total number of nuclei for the three classes of embryos in the telencephalon, diencephalon and mesencephalon at 4.5 dpf (3 adjacent sections, n=4). (l) Bar graph of the number of HuC/D positive cells in the telencephalon for kctd13 MO-, control- and KCTD13 mRNA-injected embryos at 4.5 dpf (3 adjacent sections, n=4). (m) Ventral and (n) dorsal views of kctd13 MO-, control-, and KCTD13 mRNA-injected embryos at 2 dpf (left to right). Data are represented as mean ± s.d. * p<0.01; two-tailed t-test comparisons between control and either MO- and mRNA-injected embryos.

Figure 4. Kctd13 regulates mammalian cell proliferation in vitro and in vivo

(a) Knockdown of Kctd13 in Neuro-2a cells results in an increase in the number of BrdU+/GFP+ cells relative to control cells. Error bars represent the standard error from two independent experiments. (b) Analysis of E15.5 mouse cortices injected with either Kctd13 or control shRNA reveal a similar increase in BrdU+/GFP+ cells in knockdown tissue (n=3, error bars represent the standard error). ** p<0.01; ***p<0.001.