Autosomal dominant omodysplasia is a rare skeletal dysplasia characterized by short humeri, radial head dislocation, short first metacarpals, facial dysmorphism and genitourinary anomalies. We performed next-generation whole-exome sequencing and comparative analysis of a proband with omodysplasia, her unaffected parents and her affected daughter. We identified a de novo mutation in FRIZZLED2 (FZD2) in the proband and her daughter that was not found in unaffected family members. The FZD2 mutation (c.1644G>A) changes a tryptophan residue at amino acid 548 to a premature stop (p.Trp548*). This altered protein is still produced in vitro, but we show reduced ability of this mutant form of FZD2 to interact with its downstream target DISHEVELLED. Furthermore, expressing the mutant form of FZD2 in vitro is not able to facilitate the cellular response to canonical Wnt signaling like wild-type FZD2. We therefore conclude that the FRIZZLED2 mutation is a de novo, novel cause for autosomal dominant omodysplasia.

Primary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2 R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2 R254C/Znf ) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2 R254C/Znf brain revealed a reduction in layer V (CTIP2 + ) neurons, while the overall cell density of the cortex is unchanged. Moreover, disruption of Copb2 in mouse neurospheres resulted in reduced proliferation. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest.peer-reviewed)

During postnatal cerebellar development, differentiating Purkinje cell (PC) dendrites extend towards the pial surface and progressively contact immature granule cell parallel fiber (PF) axons in the deep external granule layer (EGL), thus forming a zone of synaptic contact called the molecular layer (ML). The neuronal cell adhesion molecule, TAG-1, is transiently expressed on PF axons in the deep EGL (Yamamoto et al. [1986] J. Neurosci. 12:3576-3594). To determine the spatiotemporal relationship between Purkinje cell dendritic differentiation and the cessation of TAG-1 expression, sagittal sections from developing rat cerebellum were double-labeled for TAG-1 and the Purkinje cell-specific marker, calbindin, by using indirect immunofluorescence. At postnatal day 2 (P2) and P5, confocal microscopy revealed that TAG-1 immunoreactivity began above the furthest superficial extent of the Purkinje cell apical dendritic cap. By P10, PC dendrites penetrated partially into the TAG-1-positive deep EGL, creating a narrow region of overlap in TAG-1/calbindin staining at the deep EGL/ML border. In contrast, at P15 and P20, TAG-1 staining began directly above the furthest superficial extent of the Purkinje cell dendrites, with little or no overlap in TAG-1/calbindin staining. Staining for the synaptic vesicle glycoprotein, synaptophysin, was dim throughout most of the TAG-1-positive deep EGL, although bright synaptophysin immunoreactivity was observed throughout the ML. Overlap in TAG-1/synaptophysin staining was observed primarily at the deep EGL/ML border, suggesting that robust expression of synaptophysin in granule cells does not begin until after contact with Purkinje cell dendrites has been initiated. Our results suggest that factors present in the developing ML may influence the cessation of TAG-1 expression and the initiation of synaptophysin expression at the border region between the ML and the deep EGL.

In this study we investigate the roles of the organizer factors chordin and noggin, which are dedicated antagonists of the bone morphogenetic proteins(BMPs), in formation of the mammalian head. The mouse chordin and noggin genes(Chrd and Nog) are expressed in the organizer (the node) and its mesendodermal derivatives, including the prechordal plate, an organizing center for rostral development. They are also expressed at lower levels in and around the anterior neural ridge, another rostral organizing center. To elucidate roles of Chrd and Nog that are masked by the severe phenotype and early lethality of the double null, we have characterized embryos of the genotype Chrd-/-;Nog+/-. These animals display partially penetrant neonatal lethality, with defects restricted to the head. The variable phenotypes include cyclopia,holoprosencephaly, and rostral truncations of the brain and craniofacial skeleton. In situ hybridization reveals a loss of SHH expression and signaling by the prechordal plate, and a decrease in FGF8 expression and signaling by the anterior neural ridge at the five-somite stage. DefectiveChrd-/-;Nog+/- embryos exhibit reduced cell proliferation in the rostral neuroepithelium at 10 somites, followed by increased cell death 1 day later. Because these phenotypes result from reduced levels of BMP antagonists, we hypothesized that they are due to increased BMP activity. Ectopic application of BMP2 to wild-type cephalic explants results in decreased FGF8 and SHH expression in rostral tissue, suggesting that the decreased expression of FGF8 and SHH observed in vivo is due to ectopic BMP activity. Cephalic explants isolated from Chrd;Nog double mutant embryos show an increased sensitivity to ectopic BMP protein, further supporting the hypothesis that these mutants are deficient in BMP antagonism. These results indicate that the BMP antagonists chordin and noggin promote the inductive and trophic activities of rostral organizing centers in early development of the mammalian head.

KEY WORDSTubulin human forebrain development ABSTRACT Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations results from de novo heterozygous mutations in tubulin genes. However, the absolute requirement for most of these genes in development and disease has not been previously tested in genetic, loss of function models. Here we present two novel pathogenic tubulin alleles: a human TUBA1A missense variant with a phenotype more severe than most tubulinopathies and a mouse ENU allele of Tuba1a. Furthermore, we directly test the requirement for Tuba1a, Tuba8, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tuba8, Tubb2a or Tubb2b does not lead to cortical malformation phenotypes or impair survival. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. Thus, despite their functional similarity, the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.