Human Microcephaly Research Articles

Cerebral organoids model human brain development and microcephaly.

The complexity of the human brain has made it difficult to study many brain disorders in model organisms, and highlights the need for an in vitro model of human brain development. We have developed a human pluripotent stem cell-derived 3D organoid culture system, termed cerebral organoid, which develops various discrete though interdependent brain regions. These include cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNAi and patient-specific iPS cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could explain the disease phenotype. Our data demonstrate that 3D organoids can recapitulate development and disease of even this most complex human tissue.

Genetic variation in MKL2 and decreased downstream PCTAIRE1 expression in extreme, fatal primary human microcephaly

The genetic mechanisms driving normal brain development remain largely unknown. We performed genomic and immunohistochemical characterization of a novel, fatal human phenotype including extreme microcephaly with cerebral growth arrest at 14–18 weeks gestation in three full sisters born to healthy, non-consanguineous parents. Analysis of index cases and parents included familial exome sequencing, karyotyping, and genome-wide single nucleotide polymorphism (SNP) array. From proband, control and unrelated microcephalic fetal cortical tissue, we compared gene expression of RNA and targeted immunohistochemistry. Each daughter was homozygous for a rare, non-synonymous, deleterious variant in the MKL2 gene and heterozygous for a private 185 kb deletion on the paternal allele, upstream and in cis with his MKL2 variant allele, eliminating 24 CArG transcription factor binding sites and MIR4718. MKL1 was underexpressed in probands. Dysfunction of MKL2 and its transcriptional coactivation partner, serum response factor (SRF), was supported by a decrease in gene and protein expression of PCTAIRE1, a downstream target of MKL2:SRF heterodimer transcriptional activation, previously shown to result in severe microcephaly in murine models. While disruption of the MKL2:SRF axis has been associated with severe microcephaly and disordered brain development in multiple model systems, the role of this transcription factor complex has not been previously demonstrated in human brain development.

Meier–Gorlin syndrome and Wolf–Hirschhorn syndrome: Two developmental disorders highlighting the importance of efficient DNA replication for normal development and neurogenesis

Microcephaly represents one of the most obvious clinical manifestations of impaired neurogenesis. Defects in the DNA damage response, in DNA repair, and structural abnormalities in centrosomes, centrioles and the spindle microtubule network have all been demonstrated to cause microcephaly in humans. Work describing novel functional defects in cell lines from individuals with either Meier–Gorlin syndrome or Wolf–Hirschhorn syndrome highlight the significance of optimal DNA replication and S phase progression for normal human development, including neurogenesis. These findings illustrate how different primary defects in processes impacting upon DNA replication potentially influence similar phenotypic outcomes, including growth retardation and microcephaly. Herein, we will describe the nature of the S phase defects uncovered for each of these conditions and highlight some of the overlapping cellular features.

Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly

Centrioles are cylindrical structures that are usually composed of nine triplets of microtubules (MTs) organized around a cartwheel-shaped structure. Recent studies have proposed a structural model of the SAS-6-based cartwheel, yet we do not know the molecular detail of how the cartwheel participates in centriolar MT assembly. In this study, we demonstrate that the human microcephaly protein, CEP135, directly interacts with hSAS-6 via its carboxyl-terminus and with MTs via its amino-terminus. Unexpectedly, CEP135 also interacts with another microcephaly protein CPAP via its amino terminal domain. Depletion of CEP135 not only perturbed the centriolar localization of CPAP, but also blocked CPAP-induced centriole elongation. Furthermore, CEP135 depletion led to abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. Overexpression of a CEP135 mutant lacking the proper interaction with hSAS-6 had a dominant-negative effect on centriole assembly. We propose that CEP135 may serve as a linker protein that directly connects the central hub protein, hSAS-6, to the outer MTs, and suggest that this interaction stabilizes the proper cartwheel structure for further CPAP-mediated centriole elongation.

Mcph1-Deficient Mice Reveal a Role for MCPH1 in Otitis Media

Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1tm1a /tm1a) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1tm1a /tm1a mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1tm1a /tm1a mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1tm1a /tm1a mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.

Genetic causes of microcephaly and lessons for neuronal development.

The study of human developmental microcephaly is providing important insights into brain development. It has become clear that developmental microcephalies are associated with abnormalities in cellular production, and that the pathophysiology of microcephaly provides remarkable insights into how the brain generates the proper number of neurons that determine brain size. Most of the genetic causes of 'primary' developmental microcephaly (i.e., not associated with other syndromic features) are associated with centrosomal abnormalities. In addition to other functions, centrosomal proteins control the mitotic spindle, which is essential for normal cell proliferation during mitosis. However, the brain is often uniquely affected when microcephaly genes are mutated implying special centrosomal-related functions in neuronal production. Although models explaining how this could occur have some compelling data, they are not without controversy. Interestingly, some of the microcephaly genes show evidence that they were targets of evolutionary selection in primates and human ancestors, suggesting potential evolutionary roles in controlling neuronal number and brain volume across species. Mutations in DNA repair pathway genes also lead to microcephaly. Double-stranded DNA breaks appear to be a prominent type of damage that needs to be repaired during brain development, yet why defects in DNA repair affect the brain preferentially and if DNA repair relates to centrosome function, are not clearly understood.

CPAP is required for cilia formation in neuronal cells

SummaryThe primary cilium is a microtubule-based structure protruded from the basal body analogous to the centriole. CPAP (centrosomal P4.1-associated protein) has previously been reported to be a cell cycle-regulated protein that controls centriole length. Mutations in CPAP cause primary microcephaly (MCPH) in humans. Here, using a cell-based system that we established to monitor cilia formation in neuronal CAD (Cath.a-differentiated) cells and hippocampal neurons, we found that CPAP is required for cilia biogenesis. Overexpression of wild-type CPAP promoted cilia formation and induced longer cilia. In contrast, an exogenously expressed CPAP-377EE mutant that lacks tubulin-dimer binding significantly inhibited cilia formation and caused cilia shortening. Furthermore, depletion of CPAP inhibited ciliogenesis and such effect was effectively rescued by expression of wild-type CPAP, but not by the CPAP-377EE mutant. Taken together, our results suggest that CPAP is a positive regulator of ciliogenesis whose intrinsic tubulin-dimer binding activity is required for cilia formation in neuronal cells.

Genomic copy number variations analysis in six neonates with Pierre Robin sequence

Objective To screen for genomic copy number variants(CNVs)in six neonates with Pierre Robin sequence(PRS)by Affymetrix 2.7 M chip to identify possible loci related to PRS.Methods Six neonates with PRS admitted into the Department of Neonatology,Children's Hospital of Fudan University from June 2009 to May 2010 were enrolled in this study.CNVs were detected by Cytogenetic Whole Genome 2.7 M array.Rare CNVs with potential clinical significance that deletion segments' size ＞50 kb and duplication segments' size ＞200 kb were selected based on the analysis of Chromosome Analysis Suite(ChAS)software,false positive CNVs and segments of normal population were excluded.The identified CNVs were compared with those in relative published literatures.Results(1)Among 6 PRS patients,two patients had facial deformation,two had congenital heart defects,one had congenital dysplasia of the laryngeal cartilage and one had choroidal space occupying lesion.(2)Seven rare CNVs whose size from 51-11 956 kb were identified in four neonates,including a 739 kb duplication on lp26.23-p36.22,a 6273 kb deletion on lq43-44,a 51 kb and a 55 kb deletions on 14q32.31,a 1022 kb duplication on 14q11.1-11.2,a 11 956 kb duplication on 20p13 and a 105 kb deletion on 4q23.3.(3)Published literatures showed that deletions of 1q43-44 and 14q32.31 might relate to micro/retrognathia and abnormal palate.Region of chromosome 1q43-q44 contained AKT3 and heterogeneous nuclear ribonucleoprotein U(hnRNPU)genes,and the haploinsufficiency of AKT3 and hnRNPU genes might cause developmental human microcephaly and agenesis of the corpus callosum,speech delay and seizures respectively.Region of chromosome 14q32.31 contained some C/D small nucleolar RNA,and the human imprinted 14q32 domain shared common genomic features with the imprinted 15q11-q13 loci.Conclusions This study established a method to discover whole genome CNVs in identifying novel submicroscopic deletions and duplications.Reviewing of published literatures suggested that deletions of chromosome 1q43-q44 and 14q32.31 might cause Pierre Robin sequence. Key words: Pierre Robin syndrome; Gene dosage; Varition(genetics); Genome; Microarrayanalysis

The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation

Centriole duplication involves the growth of a procentriole next to the parental centriole. Mutations in STIL and CPAP/CENPJ cause primary microcephaly (MCPH). Here, we show that human STIL has an asymmetric localization to the daughter centriole and is required for procentriole formation. STIL levels oscillate during the cell cycle. Interestingly, STIL interacts directly with CPAP and forms a complex with hSAS6. A natural mutation of CPAP (E1235V) that causes MCPH in humans leads to significantly lower binding to STIL. Overexpression of STIL induced the formation of multiple procentrioles around the parental centriole. STIL depletion inhibited normal centriole duplication, Plk4-induced centriole amplification, and CPAP-induced centriole elongation, and resulted in a failure to localize hSAS6 and CPAP to the base of the nascent procentriole. Furthermore, hSAS6 depletion hindered STIL targeting to the procentriole, implying that STIL and hSAS6 are mutually dependent for their centriolar localization. Together, our results indicate that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH.

Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

Human Mutations in NDE1 Cause Extreme Microcephaly with Lissencephaly

Genes disrupted in human microcephaly (meaning "small brain") define key regulators of neural progenitor proliferation and cell-fate specification. In comparison, genes mutated in human lissencephaly (lissos means smooth and cephalos means brain) highlight critical regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure, a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the C-terminal NDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulation of cytoskeletal dynamics in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable, cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.

CDK5RAP2 stimulates microtubule nucleation by the γ-tubulin ring complex

CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)-binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC-tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC-mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC-binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2-6. RNA interference-mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC-mediated microtubule nucleation.

Adaptive Evolution of Four Microcephaly Genes and the Evolution of Brain Size in Anthropoid Primates

The anatomical basis and adaptive function of the expansion in primate brain size have long been studied; however, we are only beginning to understand the genetic basis of these evolutionary changes. Genes linked to human primary microcephaly have received much attention as they have accelerated evolutionary rates along lineages leading to humans. However, these studies focus narrowly on apes, and the link between microcephaly gene evolution and brain evolution is disputed. We analyzed the molecular evolution of four genes associated with microcephaly (ASPM, CDK5RAP2, CENPJ, MCPH1) across 21 species representing all major clades of anthropoid primates. Contrary to prevailing assumptions, positive selection was not limited to or intensified along the lineage leading to humans. In fact we show that all four loci were subject to positive selection across the anthropoid primate phylogeny. We developed clearly defined hypotheses to explicitly test if selection on these loci was associated with the evolution of brain size. We found positive relationships between both CDK5RAP2 and ASPM and neonatal brain mass and somewhat weaker relationships between these genes and adult brain size. In contrast, there is no evidence linking CENPJ and MCPH1 to brain size evolution. The stronger association of ASPM and CDK5RAP2 evolution with neonatal brain size than with adult brain size is consistent with these loci having a direct effect on prenatal neuronal proliferation. These results suggest that primate brain size may have at least a partially conserved genetic basis. Our results contradict a previous study that linked adaptive evolution of ASPM to changes in relative cortex size; however, our analysis indicates that this conclusion is not robust. Our finding that the coding regions of two widely expressed loci has experienced pervasive positive selection in relation to a complex, quantitative developmental phenotype provides a notable counterexample to the commonly asserted hypothesis that cis-regulatory regions play a dominant role in phenotypic evolution.

WDR62 is associated with the spindle pole and is mutated in human microcephaly

Autosomal recessive primary microcephaly (MCPH) is a disorder of neurodevelopment resulting in a small brain. We identified WDR62 as the second most common cause of MCPH after finding homozygous missense and frame-shifting mutations in seven MCPH families. In human cell lines, we found that WDR62 is a spindle pole protein, as are ASPM and STIL, the MCPH7 and MCHP7 proteins. Mutant WDR62 proteins failed to localize to the mitotic spindle pole. In human and mouse embryonic brain, we found that WDR62 expression was restricted to neural precursors undergoing mitosis. These data lend support to the hypothesis that the exquisite control of the cleavage furrow orientation in mammalian neural precursor cell mitosis, controlled in great part by the centrosomes and spindle poles, is critical both in causing MCPH when perturbed and, when modulated, generating the evolutionarily enlarged human brain.

Mutations in WDR62, encoding a centrosome-associated protein, cause microcephaly with simplified gyri and abnormal cortical architecture.

Genes associated with human microcephaly, a condition characterized by a small brain, include critical regulators of proliferation, cell fate, and DNA repair. We describe a syndrome of congenital microcephaly and diverse defects in cerebral cortical architecture. Genome-wide linkage analysis in two families identified a 7.5 Mb locus on chromosome 19q13.12 containing 148 genes. Targeted high throughput sequence analysis of linked genes in each family yielded > 4000 DNA variants and implicated a single gene, WDR62, as harboring potentially deleterious changes. We subsequently identified additional WDR62 mutations in four other families. MRI and postmortem brain analysis supports important roles for WDR62 in proliferation and migration of neuronal precursors. WDR62 is a WD40 repeat-containing protein expressed in neuronal precursors as well as postmitotic neurons in the developing brain and localizes to the spindle poles of dividing cells. The diverse phenotypes of WDR62 suggest central roles in many aspects of cerebral cortical development.

Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline

Mutations in ASPM (abnormal spindle-like microcephaly associated) cause primary microcephaly in humans, a disorder characterized by a major reduction in brain size in the apparent absence of nonneurological anomalies. The function of the Aspm protein in neural progenitor cell expansion, as well as its localization to the mitotic spindle and midbody, suggest that it regulates brain development by a cell division-related mechanism. Furthermore, evidence that positive selection affected ASPM during primate evolution has led to suggestions that such a function changed during primate evolution. Here, we report that in Aspm mutant mice, truncated Aspm proteins similar to those causing microcephaly in humans fail to localize to the midbody during M-phase and cause mild microcephaly. A human ASPM transgene rescues this phenotype but, interestingly, does not cause a gain of function. Strikingly, truncated Aspm proteins also cause a massive loss of germ cells, resulting in a severe reduction in testis and ovary size accompanied by reduced fertility. These germline effects, too, are fully rescued by the human ASPM transgene, indicating that ASPM is functionally similar in mice and humans. Our findings broaden the spectrum of phenotypic effects of ASPM mutations and raise the possibility that positive selection of ASPM during primate evolution reflects its function in the germline.

C-mycand N-mycpromote active stem cell metabolism and cycling as architects of the developing brain

myc genes are associated with a wide variety of human cancers including most types of nervous system tumors. While the mechanisms by which myc overexpression causes tumorigenesis are multifaceted and have yet to be clearly elucidated, they are at least in part related to endogenous myc function in normal cells. Knockout (KO) of either c-myc or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre impairs mouse brain growth and mutation of N-myc also causes microcephaly in humans in Feingold Syndrome. To further define myc function in NSC and nervous system development, we created a double KO (DKO) for c- and N-myc using nestin-cre. The DKO mice display profoundly impaired overall brain growth associated with decreased cell cycling and migration of NSC, which are strikingly decreased in number. The DKO brain also exhibits specific changes in gene expression including downregulation of genes involved in protein and nucleotide metabolism, mitosis, and chromatin structure as well as upregulation of genes associated with differentiation. Together these data support a model of nervous system tumorigenesis in which excess myc aberrantly locks in a developmentally active chromatin state characterized by overactive cell cycling, and metabolism as well as blocked differentiation.

CDK5RAP2 Regulates Centriole Engagement and Cohesion in Mice

Centriole duplication occurs once per cell cycle, ensuring that each cell contains two centrosomes, each containing a mother-daughter pair of tightly engaged centrioles at mitotic entry. Loss of the tight engagement between mother and daughter centrioles appears to license the next round of centriole duplication. However, the molecular mechanisms regulating this process remain largely unknown. Mutations in CDK5RAP2, which encodes a centrosomal protein, cause autosomal recessive primary microcephaly in humans. Here we show that CDK5RAP2 loss of function in mice causes centriole amplification with a preponderance of single, unpaired centrioles and increased numbers of daughter-daughter centriole pairs. These results indicate that CDK5RAP2 is required to maintain centriole engagement and cohesion, thereby restricting centriole replication. Early in mitosis, amplified centrosomes assemble multipolar spindles in CDK5RAP2 mutant cells. Moreover, both mother and daughter centrioles are amplified and the excess mother centrioles template multiple primary cilia in CDK5RAP2 mutant cells.

Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors

Microcephaly affects approximately 1% of the population and is associated with mental retardation, motor defects and, in some cases, seizures. We analyzed the mechanisms underlying brain size determination in a mouse model of human microcephaly. The Hertwig's anemia (an) mutant shows peripheral blood cytopenias, spontaneous aneuploidy and a predisposition to hematopoietic tumors. We found that the an mutation is a genomic inversion of exon 4 of Cdk5rap2, resulting in an in-frame deletion of exon 4 from the mRNA. The finding that CDK5RAP2 human mutations cause microcephaly prompted further analysis of Cdk5rap2(an/an) mice and we demonstrated that these mice exhibit microcephaly comparable to that of the human disease, resulting from striking neurogenic defects that include proliferative and survival defects in neuronal progenitors. Cdk5rap2(an/an) neuronal precursors exit the cell cycle prematurely and many undergo apoptosis. These defects are associated with impaired mitotic progression coupled with abnormal mitotic spindle pole number and mitotic orientation. Our findings suggest that the reduction in brain size observed in humans with mutations in CDK5RAP2 is associated with impaired centrosomal function and with changes in mitotic spindle orientation during progenitor proliferation.

Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors.

Microcephaly affects approximately 1% of the population and is associated with mental retardation, motor defects and, in some cases, seizures. We analyzed the mechanisms underlying brain size determination in a mouse model of human microcephaly. The Hertwig's anemia (an) mutant shows peripheral blood cytopenias, spontaneous aneuploidy and a predisposition to hematopoietic tumors. We found that the an mutation is a genomic inversion of exon 4 of Cdk5rap2, resulting in an in-frame deletion of exon 4 from the mRNA. The finding that CDK5RAP2 human mutations cause microcephaly prompted further analysis of Cdk5rap2(an/an) mice and we demonstrated that these mice exhibit microcephaly comparable to that of the human disease, resulting from striking neurogenic defects that include proliferative and survival defects in neuronal progenitors. Cdk5rap2(an/an) neuronal precursors exit the cell cycle prematurely and many undergo apoptosis. These defects are associated with impaired mitotic progression coupled with abnormal mitotic spindle pole number and mitotic orientation. Our findings suggest that the reduction in brain size observed in humans with mutations in CDK5RAP2 is associated with impaired centrosomal function and with changes in mitotic spindle orientation during progenitor proliferation.