CHD8单倍体功能不全导致自闭症和大头畸形,在人群中具有高外显率。Chd8杂合小鼠表现出相对微妙的大脑过度生长,并且在胚胎新皮层中几乎没有基因表达变化。这项研究的目的是产生新的,亚单倍体不足的Chd8小鼠模型使我们能够识别和研究CHD8在胚胎皮质发育过程中的功能。
为了检查某些表型可能仅在小鼠的亚杂合Chd8水平出现的可能性,我们创建了一系列Chd8缺陷小鼠的等位基因,以将CHD8蛋白水平降低到约35%(轻度次形态),野生型水平的10%(重度下形态)和0%(神经特异性条件性敲除)。我们使用RNA测序来比较转录失调,结构MRI和脑重量,以研究对大脑大小的影响,和细胞增殖,免疫染色测定中的分化和凋亡标记,以量化神经祖细胞命运的变化。
轻度的Chd8副形态在出生后表现出显著的致死率,存活的动物比杂合子表现出更明显的脑增生。超过2000个基因在轻度副形态中失调,包括自闭症相关的神经发育和细胞周期基因。我们确定非心室区TBR2中间祖细胞的增殖增加是这些突变体中脑增生的一个潜在原因。严重的Chd8低态表现出甚至更大的转录失调,包括p53通路上调的证据。与轻度的次生相比较,这些小鼠在胚胎大脑皮层显示出减小的大脑大小和增加的细胞凋亡。纯合子,早期神经元祖细胞中Chd8的条件性缺失导致明显的脑发育不全,部分由胚胎新皮质中p53靶基因抑制和凋亡引起。局限性我们的发现确定了自闭症相关因子CHD8在控制小鼠新皮质中中间祖细胞增殖中的重要作用。我们认为CHD8在人脑发育中具有类似的功能,但是需要对人类细胞的研究来证实这一点。因为我们许多CHD8功能降低的小鼠突变体在出生后不久死亡,尚不能完全确定CHD8功能降低在多大程度上导致小鼠自闭症相关行为.
一起,这些发现表明,CHD8在p53通路抑制中的剂量敏感功能,胚胎新皮层中神经发育基因的表达和神经祖细胞的命运。我们得出的结论是,大脑发育对CHD8表达降低非常敏感,并且不同祖细胞群和细胞过程对CHD8剂量的不同敏感性会导致对基因转录和大脑生长的非线性影响。ShaunHurley,ConorMohan和PhilippSuetterlin对这项工作做出了同样的贡献。
CHD8 haploinsufficiency causes autism and macrocephaly with high penetrance in the human population. Chd8 heterozygous mice exhibit relatively subtle brain overgrowth and little gene expression changes in the embryonic neocortex. The purpose of this study was to generate new, sub-haploinsufficient Chd8 mouse models to allow us to identify and study the functions of CHD8 during embryonic cortical development.
To examine the possibility that certain phenotypes may only appear at sub-heterozygous Chd8 levels in the mouse, we created an allelic series of Chd8-deficient mice to reduce CHD8 protein levels to approximately 35% (mild hypomorph), 10% (severe hypomorph) and 0% (neural-specific conditional knockout) of wildtype levels. We used RNA sequencing to compare transcriptional dysregulation, structural MRI and brain weight to investigate effects on brain size, and cell proliferation, differentiation and apoptosis markers in immunostaining assays to quantify changes in neural progenitor fate.
Mild Chd8 hypomorphs displayed significant postnatal lethality, with surviving animals exhibiting more pronounced brain hyperplasia than heterozygotes. Over 2000 genes were dysregulated in mild hypomorphs, including autism-associated neurodevelopmental and cell cycle genes. We identify increased proliferation of non-ventricular zone
TBR2+ intermediate progenitors as one potential cause of brain hyperplasia in these mutants. Severe Chd8 hypomorphs displayed even greater transcriptional dysregulation, including evidence for p53 pathway upregulation. In contrast to mild hypomorphs, these mice displayed reduced brain size and increased apoptosis in the embryonic neocortex. Homozygous, conditional deletion of Chd8 in early neuronal progenitors resulted in pronounced brain hypoplasia, partly caused by p53 target gene derepression and apoptosis in the embryonic neocortex. Limitations Our findings identify an important role for the autism-associated factor CHD8 in controlling the proliferation of intermediate progenitors in the mouse neocortex. We propose that CHD8 has a similar function in human brain development, but studies on human cells are required to confirm this. Because many of our mouse mutants with reduced CHD8 function die shortly after birth, it is not possible to fully determine to what extent reduced CHD8 function results in autism-associated behaviours in mice.
Together, these findings identify important, dosage-sensitive functions for CHD8 in p53 pathway repression, neurodevelopmental gene expression and neural progenitor fate in the embryonic neocortex. We conclude that brain development is acutely sensitive to reduced CHD8 expression and that the varying sensitivities of different progenitor populations and cellular processes to CHD8 dosage result in non-linear effects on gene transcription and brain growth. Shaun Hurley, Conor Mohan and Philipp Suetterlin have contributed equally to this work.