The etiology and pathophysiology of Tourette Syndrome (TS) remain poorly understood. Multiple lines of evidence suggest that a complex genetic background and the cortico-striato-thalamo-cortical circuit are involved. The role of Lhx6 and Lhx8 in the development of the striatal interneurons, prompted us to investigate them as novel candidate genes for TS. We performed a comparative study of the expression of Lhx6 and Lhx8 and investigated genetic association with TS using two samples of trios (TSGeneSEE and German sample -222 families). We show that Lhx6 and Lhx8 expression in the forebrain is evolutionarily conserved, underlining their possible importance in TS-related pathophysiological pathways. Our tagging-single nucleotide polymorphism (tSNP)-based association analysis was negative for association with LHX8. However, we found positive association with LHX6 in the TSGeneSEE sample (corrected P-value = 0.006 for three-site haplotype around SNP rs3808901) but no association in the sample of German families. Interestingly, the SNP allele that was identified to be significantly associated in the TSGeneSEE dataset, showed an opposite trend of transmission in the German dataset. Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by multiple motor and vocal tics and high comorbidity rates with obsessive compulsive disorder (OCD) and attention deficit and hyperactivity disorder (ADHD) (Swain et al. 2007). The fact that symptoms appear to peak during adolescence and often remit as individuals progress into adulthood, suggests the intriguing hypothesis that neurodevelopmental pathways are involved (Bloch and Leckman 2009). It is currently thought that environmental and genetic factors interact in order to lead to the onset of symptoms. However, the exact role and the contribution of each of these factors have not been yet elucidated.

MicroCephaly Primary Hereditary (MCPH) is a rare congenital neurodevelopmental disorder characterized by a significant reduction of the occipitofrontal head circumference and mild to moderate mental disability. Patients have small brains, though with overall normal architecture; therefore, studying MCPH can reveal not only the pathological mechanisms leading to this condition, but also the mechanisms operating during normal development. MCPH is genetically heterogeneous, with 27 genes listed so far in the Online Mendelian Inheritance in Man (OMIM) database. In this review, we discuss the role of MCPH proteins and delineate the molecular mechanisms and common pathways in which they participate.

BackgroundNon Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels.Methodology/Principal FindingsA stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50–60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.Conclusions/SignificanceOur NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.

Winged helix proteins have critical roles in a variety of developmental processes. During a screening for genes expressed in the developing forebrain, we identified HSPC280, a non-typical winged helix protein, which shares similarity with a protein-protein interaction domain found in the proteins of the actin-binding Rho-activating protein family. In this work, we analyzed HSPC280 expression during mouse development as well as during neuronal differentiation of mouse Neuro2a cells. HSPC280 expression is tightly regulated; during mouse development, it was detected predominantly in the ganglionic eminences of the ventral telencephalon, from their appearance at E11.5 to P0, with the highest levels between E13.5 and E15.5, a period that correlates with the peak of neurogenesis in these structures. Comparative expression analysis of HSPC280 with Dlx2, cyclinD2 and Lhx6 revealed that, within the ganglionic eminences, HSPC280 was restricted in the proliferating cell population of the subventricular zone, in a pattern similar to that of cyclinD2. Finally, we showed that HSPC280 is a nuclear protein which, when overexpressed in Neuro2a cells, it inhibited neuronal differentiation in vitro, suggesting its involvement in the mechanisms controlling neural progenitor cells proliferation.

Nonradioactive colorimetric in situ hybridization (NoRISH) has been widely applied to analyze gene expression at the single-cell level. Zinc fixation is time efficient and provides excellent tissue morphology. Furthermore, it improves the preservation of the RNA, facilitating the detection of rare transcripts or the identification of expressing cells scattered within a tissue. Here we present a rapid, highly sensitive NoRISH method that uses a zinc-salt-based fixative and is especially suitable for the study of genes expressed at low levels and/or in a small number of cells within a structure.

CRISPR-based technologies have revolutionised genome editing and are widely used for knocking out genes in cell lines and organisms. From a practical perspective, a critical factor that largely influences the successful outcome of CRISPR gene knockout experiments is the reliable and fast identification of fully mutated cells carrying exclusively null alleles of the target gene. Here we describe a novel strategy based on the well-documented reliability and simplicity of the classical PCR-Restriction Fragment Length Polymorphism (RFLP), which allows the assessment of the editing efficiency in pools of edited cells and the effective identification of cell clones that carry exclusively mutated alleles. This fast and cost-effective method, named PIM-RFLP (PCR Induced Mutagenesis-RFLP), is executed in two steps. In the first step, the editing target is amplified by a set of mutagenic primers that create a restriction enzyme degenerate cleavage site in the amplification product of the wild type allele. As a proof of principle, we chose the XcmI restriction site because it is especially suitable since it has the particularity of containing nine centrally placed non-specific nucleotides. This gives great flexibility in the mutagenic primers design and allows for efficient execution of the mutagenic PCR. In the second step, the evaluation of the editing efficiency in pools of edited cells or the identification of fully mutated single-cell derived clones is achieved following the standard procedure for any PCR-RFLP assay: digestion of the PCR products and analysis of the restriction fragments in an agarose gel.