The transcription factor Foxg1 regulates telencephalic progenitor proliferation cell autonomously, in part by controlling Pax6 expression levels

<p>Abstract</p> <p>Background</p> <p>The transcription factor Foxg1 is an important regulator of telencephalic cell cycles. Its inactivation causes premature lengthening of telencephalic progenitor cell cycles and increased neurogenic divisions, leading to severe hypoplasia of the telencephalon. These proliferation defects could be a secondary consequence of the loss of Foxg1 caused by the abnormal expression of several morphogens (Fibroblast growth factor 8, bone morphogenetic proteins) in the telencephalon of <it>Foxg1 </it>null mutants. Here we investigated whether Foxg1 has a cell autonomous role in the regulation of telencephalic progenitor proliferation. We analysed <it>Foxg1^+/+</it>↔<it>Foxg1^-/-</it>chimeras, in which mutant telencephalic cells have the potential to interact with, and to have any cell non-autonomous defects rescued by, normal wild-type cells.</p> <p>Results</p> <p>Our analysis showed that the <it>Foxg1^-/-</it>cells are under-represented in the chimeric telencephalon and the proportion of them in S-phase is significantly smaller than that of their wild-type neighbours, indicating that their under-representation is caused by a cell autonomous reduction in their proliferation. We then analysed the expression of the cell-cycle regulator Pax6 and found that it is cell-autonomously downregulated in <it>Foxg1^-/-</it>dorsal telencephalic cells. We went on to show that the introduction into <it>Foxg1^-/-</it>embryos of a transgene designed to reverse Pax6 expression defects resulted in a partial rescue of the telencephalic progenitor proliferation defects.</p> <p>Conclusions</p> <p>We conclude that Foxg1 exerts control over telencephalic progenitor proliferation by cell autonomous mechanisms that include the regulation of Pax6, which itself is known to regulate proliferation cell autonomously in a regional manner.</p

Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance

Background: The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6(Sey/Sey)) and are abnormally small in Pax6(Sey/+) mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. Results: Eyes form in PAX77(+/+) embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77(+/+) retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77(+/+) embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6(Sey/+), Pax6(+/+), PAX77(+) and PAX77(+/+)) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the optic chiasm vary differently with gene dosage. Increasing dosage increases the proportion projecting ipsilaterally regardless of the size of the total projection. Conclusion: Pax6 overexpression does not obviously impair the initial formation of the eye and its major cell-types but prevents normal development of the retina from about E14.5, leading eventually to severe retinal degeneration in postnatal life. This sequence is different to that underlying microphthalmia in Pax6(+/-) heterozygotes, which is due primarily to defects in the initial stages of lens formation. Before the onset of severe retinal dysplasia, Pax6 overexpression causes defects of retinal axons, preventing their normal growth and navigation through the optic chiasm

Regulatory role of Pax6 on cell division cycle associated 7 and cortical progenitor cell proliferation

Forebrain development is controlled by a set of transcription factors which are expressed in dynamic spatiotemporal patterns in the embryonic forebrain and are known to regulate complex gene networks. Pax6 is a transcription factor that regulates corticogenesis and mutations affecting Pax6 protein levels cause neurodevelopmental defects in the eyes and forebrain in both humans and mice. In previous studies, it was shown that the graded expression pattern of Pax6 protein, which is high rostro-laterally to low caudo-medially in the cerebral cortex, is critical for its control of cell cycle progression and proliferation of cortical progenitors. However the underlying mechanisms are still unclear. Based on a microarray analysis carried out in our laboratory, a number of cell cycle-related candidate genes that may be affected by Pax6 have been identified. One such gene, Cell division cycle associated 7 (Cdca7) is expressed in a counter-gradient against that of Pax6. In my current study, I found that Cdca7 mRNA expression in the telencephalon is upregulated in Pax6 null (Small eye) mutants and downregulated in mice that overexpress PAX6 (PAX77) across developing time points from E12.5 to E15.5. There are several potential Pax6 binding motifs located in the genomic locus upstream of Cdca7. However, by chromatin immunoprecipitation, it is showed that none of the predicted binding sites are physically bound by Pax6. Promoter luciferase assays using fragments combining five suspected binding motifs show that Pax6 is functionally critical. Cdca7 is also identified as a Myc and E2F1 direct target and is upregulated in some tumours but its biological role is not fully understood. Current work using in utero electroporation to overexpress Cdca7 around the lateral telencephalon, where Cdca7 expression levels are normally low, tested the effects on the proliferation and differentiation of cortical progenitor cells in this region. In E12.5 mice embryos, overexpression of Cdca7 protein causes fewer intermediate progenitor cells and post-mitotic neurons to be produced but these effects were not found in E14.5 embryos. This result implies that Cdca7 may affect cell fate decision during cortical development

Frontotemporal dementia and its subtypes: a genome-wide association study

SummaryBackground Frontotemporal dementia (FTD) is a complex disorder characterised by a broad range of clinical manifestations, differential pathological signatures, and genetic variability. Mutations in three genes—MAPT, GRN, and C9orf72—have been associated with FTD. We sought to identify novel genetic risk loci associated with the disorder. Methods We did a two-stage genome-wide association study on clinical FTD, analysing samples from 3526 patients with {FTD} and 9402 healthy controls. To reduce genetic heterogeneity, all participants were of European ancestry. In the discovery phase (samples from 2154 patients with {FTD} and 4308 controls), we did separate association analyses for each {FTD} subtype (behavioural variant FTD, semantic dementia, progressive non-fluent aphasia, and {FTD} overlapping with motor neuron disease FTD-MND), followed by a meta-analysis of the entire dataset. We carried forward replication of the novel suggestive loci in an independent sample series (samples from 1372 patients and 5094 controls) and then did joint phase and brain expression and methylation quantitative trait loci analyses for the associated (p&lt;5 × 10−8) single-nucleotide polymorphisms. Findings We identified novel associations exceeding the genome-wide significance threshold (p&lt;5 × 10−8). Combined (joint) analyses of discovery and replication phases showed genome-wide significant association at 6p21.3, \{HLA\} locus (immune system), for rs9268877 (p=1·05 × 10−8; odds ratio=1·204 95% \{CI\} 1·11–1·30), rs9268856 (p=5·51 × 10−9; 0·809 0·76–0·86) and rs1980493 (p value=1·57 × 10−8, 0·775 0·69–0·86) in the entire cohort. We also identified a potential novel locus at 11q14, encompassing RAB38/CTSC (the transcripts of which are related to lysosomal biology), for the behavioural \{FTD\} subtype for which joint analyses showed suggestive association for rs302668 (p=2·44 × 10−7; 0·814 0·71–0·92). Analysis of expression and methylation quantitative trait loci data suggested that these loci might affect expression and methylation in cis. Interpretation Our findings suggest that immune system processes (link to 6p21.3) and possibly lysosomal and autophagy pathways (link to 11q14) are potentially involved in FTD. Our findings need to be replicated to better define the association of the newly identified loci with disease and to shed light on the pathomechanisms contributing to FTD. Funding The National Institute of Neurological Disorders and Stroke and National Institute on Aging, the Wellcome/MRC Centre on Parkinson's disease, Alzheimer's Research UK, and Texas Tech University Health Sciences Center

Novel genetic loci associated with hippocampal volume

The hippocampal formation is a brain structure integrally involved in episodic memory, spatial navigation, cognition and stress responsiveness. Structural abnormalities in hippocampal volume and shape are found in several common neuropsychiatric disorders. To identify the genetic underpinnings of hippocampal structure here we perform a genome-wide association study (GWAS) of 33,536 individuals and discover six independent loci significantly associated with hippocampal volume, four of them novel. Of the novel loci, three lie within genes (ASTN2, DPP4 and MAST4) and one is found 200 kb upstream of SHH. A hippocampal subfield analysis shows that a locus within the MSRB3 gene shows evidence of a localized effect along the dentate gyrus, subiculum, CA1 and fissure. Further, we show that genetic variants associated with decreased hippocampal volume are also associated with increased risk for Alzheimer's disease (rg =-0.155). Our findings suggest novel biological pathways through which human genetic variation influences hippocampal volume and risk for neuropsychiatric illness

Identification of four novel susceptibility loci for oestrogen receptor negative breast cancer

Common variants in 94 loci have been associated with breast cancer including 15 loci with genome-wide significant associations (P<5 × 10−8) with oestrogen receptor (ER)-negative breast cancer and BRCA1-associated breast cancer risk. In this study, to identify new ER-negative susceptibility loci, we performed a meta-analysis of 11 genome-wide association studies (GWAS) consisting of 4,939 ER-negative cases and 14,352 controls, combined with 7,333 ER-negative cases and 42,468 controls and 15,252 BRCA1 mutation carriers genotyped on the iCOGS array. We identify four previously unidentified loci including two loci at 13q22 near KLF5, a 2p23.2 locus near WDR43 and a 2q33 locus near PPIL3 that display genome-wide significant associations with ER-negative breast cancer. In addition, 19 known breast cancer risk loci have genome-wide significant associations and 40 had moderate associations (P<0.05) with ER-negative disease. Using functional and eQTL studies we implicate TRMT61B and WDR43 at 2p23.2 and PPIL3 at 2q33 in ER-negative breast cancer aetiology. All ER-negative loci combined account for ∼11% of familial relative risk for ER-negative disease and may contribute to improved ER-negative and BRCA1 breast cancer risk prediction

Polygenic risk scores and breast and epithelial ovarian cancer risks for carriers of BRCA1 and BRCA2 pathogenic variants

Purpose We assessed the associations between population-based polygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks forBRCA1andBRCA2pathogenic variant carriers. Methods Retrospective cohort data on 18,935BRCA1and 12,339BRCA2female pathogenic variant carriers of European ancestry were available. Three versions of a 313 single-nucleotide polymorphism (SNP) BC PRS were evaluated based on whether they predict overall, estrogen receptor (ER)-negative, or ER-positive BC, and two PRS for overall or high-grade serous EOC. Associations were validated in a prospective cohort. Results The ER-negative PRS showed the strongest association with BC risk forBRCA1carriers (hazard ratio [HR] per standard deviation = 1.29 [95% CI 1.25-1.33],P = 3x10(-72)). ForBRCA2, the strongest association was with overall BC PRS (HR = 1.31 [95% CI 1.27-1.36],P = 7x10(-50)). HR estimates decreased significantly with age and there was evidence for differences in associations by predicted variant effects on protein expression. The HR estimates were smaller than general population estimates. The high-grade serous PRS yielded the strongest associations with EOC risk forBRCA1(HR = 1.32 [95% CI 1.25-1.40],P = 3x10(-22)) andBRCA2(HR = 1.44 [95% CI 1.30-1.60],P = 4x10(-12)) carriers. The associations in the prospective cohort were similar. Conclusion Population-based PRS are strongly associated with BC and EOC risks forBRCA1/2carriers and predict substantial absolute risk differences for women at PRS distribution extremes.Peer reviewe

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium

This work was supported by the Wellcome Trust (grant 088876/Z/09/Z to JDW, REH and JMC). The funding body had no role in the design of the study, the collection, analysis and interpretation of data or the preparation of the manuscriptPeer reviewedPublisher PD

Genetic architecture of subcortical brain structures in 38,851 individuals

Subcortical brain structures are integral to motion, consciousness, emotions and learning. We identified common genetic variation related to the volumes of the nucleus accumbens, amygdala, brainstem, caudate nucleus, globus pallidus, putamen and thalamus, using genome-wide association analyses in almost 40,000 individuals from CHARGE, ENIGMA and UK Biobank. We show that variability in subcortical volumes is heritable, and identify 48 significantly associated loci (40 novel at the time of analysis). Annotation of these loci by utilizing gene expression, methylation and neuropathological data identified 199 genes putatively implicated in neurodevelopment, synaptic signaling, axonal transport, apoptosis, inflammation/infection and susceptibility to neurological disorders. This set of genes is significantly enriched for Drosophila orthologs associated with neurodevelopmental phenotypes, suggesting evolutionarily conserved mechanisms. Our findings uncover novel biology and potential drug targets underlying brain development and disease