FOXG1 targets BMP repressors and cell cycle inhibitors in human neural progenitor cells.

FOXG1 is a critical transcription factor in human brain where loss-of-function mutations cause a severe neurodevelopmental disorder, while increased FOXG1 expression is frequently observed in glioblastoma. FOXG1 is an inhibitor of cell patterning and an activator of cell proliferation in chordate model organisms but different mechanisms have been proposed as to how this occurs. To identify genomic targets of FOXG1 in human neural progenitor cells (NPCs), we engineered a cleavable reporter construct in endogenous FOXG1 and performed chromatin immunoprecipitation (ChIP) sequencing. We also performed deep RNA sequencing of NPCs from two females with loss-of-function mutations in FOXG1 and their healthy biological mothers. Integrative analyses of RNA and ChIP sequencing data showed that cell cycle regulation and Bone Morphogenic Protein (BMP) repression gene ontology categories were over-represented as FOXG1 targets. Using engineered brain cell lines, we show that FOXG1 specifically activates SMAD7 and represses CDKN1B. Activation of SMAD7 which inhibits BMP signaling may be one way that FOXG1 patterns the forebrain, while repression of cell cycle regulators such as CDKN1B may be one way that FOXG1 expands the NPC pool to ensure proper brain size. Our data reveal novel mechanisms on how FOXG1 may control forebrain patterning and cell proliferation in human brain development.

New Understanding of Diagnosis, Treatment and Prevention of Endometriosis.

For 100 years, pelvic endometriosis has been considered to originate from the implantation of endometrial cells following retrograde menstruation or metaplasia. Since some observations, such as the clonal aspect, the biochemical variability of lesions and endometriosis in women without endometrium, the genetic-epigenetic (G-E) theory describes that endometriosis only begins after a series of cumulative G-E cellular changes. This explains that the endometriotic may originate from any pluripotent cell apart from the endometrium, that 'endometrium-like cells' can harbour important G-E differences, and that the risk is higher in predisposed women with more inherited incidents. A consequence is a high risk after puberty which decreases progressively thereafter. Considering a 10-year delay between initiation and performing a laparoscopy, this was observed in the United Arab Emirates, Belgium, France and USA. The subsequent growth varies with the G-E changes and the environment but is self-limiting probably because of the immunologic reaction and fibrosis. That each lesion has a different set of G-E incidents explains the variability of pain and the response to hormonal treatment. New lesions may develop, but recurrences after surgical excision are rare. The fibrosis around endometriosis belongs to the body and does not need to be removed. This suggests conservative excision or minimal bowel without safety margins and superficial treatment of ovarian endometriosis. This G-E concept also suggests prevention by decreasing oxidative stress from retrograde menstruation or the peritoneal microbiome. This suggests the prevention of vaginal infections and changes in the gastrointestinal microbiota through food intake and exercise. In conclusion, a higher risk of initiating endometriosis during adolescence was observed in UAE, France, Belgium and USA. This new understanding and the limited growth opens perspectives for earlier diagnosis and better treatment.

Kabuki syndrome stem cell models reveal locus specificity of histone methyltransferase 2D (KMT2D/MLL4).

Kabuki syndrome is frequently caused by loss-of-function mutations in one allele of histone 3 lysine 4 (H3K4) methyltransferase KMT2D and is associated with problems in neurological, immunological and skeletal system development. We generated heterozygous KMT2D knockout and Kabuki patient-derived cell models to investigate the role of reduced dosage of KMT2D in stem cells. We discovered chromosomal locus-specific alterations in gene expression, specifically a 110 Kb region containing Synaptotagmin 3 (SYT3), C-Type Lectin Domain Containing 11A (CLEC11A), Chromosome 19 Open Reading Frame 81 (C19ORF81) and SH3 And Multiple Ankyrin Repeat Domains 1 (SHANK1), suggesting locus-specific targeting of KMT2D. Using whole genome histone methylation mapping, we confirmed locus-specific changes in H3K4 methylation patterning coincident with regional decreases in gene expression in Kabuki cell models. Significantly reduced H3K4 peaks aligned with regions of stem cell maps of H3K27 and H3K4 methylation suggesting KMT2D haploinsufficiency impact bivalent enhancers in stem cells. Preparing the genome for subsequent differentiation cues may be of significant importance for Kabuki-related genes. This work provides a new insight into the mechanism of action of an important gene in bone and brain development and may increase our understanding of a specific function of a human disease-relevant H3K4 methyltransferase family member.

FOXG1 dose tunes cell proliferation dynamics in human forebrain progenitor cells.

Heterozygous loss-of-function mutations in Forkhead box G1 (FOXG1), a uniquely brain-expressed gene, cause microcephaly, seizures, and severe intellectual disability, whereas increased FOXG1 expression is frequently observed in glioblastoma. To investigate the role of FOXG1 in forebrain cell proliferation, we modeled FOXG1 syndrome using cells from three clinically diagnosed cases with two sex-matched healthy parents and one unrelated sex-matched control. Cells with heterozygous FOXG1 loss showed significant reduction in cell proliferation, increased ratio of cells in G0/G1 stage of the cell cycle, and increased frequency of primary cilia. Engineered loss of FOXG1 recapitulated this effect, while isogenic repair of a patient mutation reverted output markers to wild type. An engineered inducible FOXG1 cell line derived from a FOXG1 syndrome case demonstrated that FOXG1 dose-dependently affects all cell proliferation outputs measured. These findings provide strong support for the critical importance of FOXG1 levels in controlling human brain cell growth in health and disease.

Developmental refinement of visual callosal inputs to ferret area 17.

Corticocortical connections link visual cortical areas in both the ipsilateral and contralateral hemispheres. We studied the postnatal refinement of callosal connections linking multiple cortical areas with ferret area 17 during the period from just before eye opening (4 weeks) to 10 weeks of age. We aimed to determine (1) whether callosal projections from multiple visual cortical areas to area 17 refine with a similar rate and (2) whether the refinement of callosal projections parallels that of intrahemispheric cortical circuits. We injected the bidirectional tracer CTb into area 17, and mapped the areal and laminar distribution of labeled cells in visual areas of the contralateral hemisphere. Like intrahemispheric projections, callosal inputs to area 17 before eye opening are dominated by Suprasylvian area Ssy (with lesser and comparable input from areas 17, 18, 19, and 21), but within 2 weeks of eye opening are jointly dominated by area 18 and Ssy inputs; however, there are fewer labeled cells in the contralateral hemisphere. Unlike intrahemispheric projections, there is no laminar reorganization of callosal inputs; in all visual areas and at all ages studied, the greatest proportion of callosal projections arises from the infragranular layers. Also, unlike intrahemispheric projections, the peak density of callosal cells in each area projecting to area 17 declines more modestly. These results reveal important similarities and differences in the postnatal reorganization of inter- and intrahemispheric projections to area 17.

Visual Corticocortical Inputs to Ferret Area 18.

Visual cortical areas in the adult mammalian brain are linked by a network of interareal feedforward and feedback circuits. We investigated the topography of feedback projections to ferret (Mustela putorius furo) area 18 from extrastriate areas 19, 21, and Ssy. Our objective was to characterize the anatomical organization of the extrastriate feedback pool to area 18. We also wished to determine if feedback projections to area 18 share similar features as feedback projections to area 17. We injected the tracer cholera toxin B subunit (CTb) into area 18 of adult ferrets to visualize the distribution and pattern of retrogradely labeled cells in extrastriate cortex. We find several similarities to the feedback projection to area 17: (i) Multiple visual cortical areas provide feedback to area 18: areas 19, 21, Ssy, and weaker inputs from posterior parietal and lateral temporal visual areas. Within each area a greater proportion of feedback projections arises from the infragranular than from the supragranular layers. (ii) The cortical area immediately rostral to area 18 provides the greatest proportion of total cortical feedback, and has the greatest peak density of cells providing feedback to area 18. (iii) The spacing (peak cell density and nearest neighbor distances) of cells in extrastriate cortex providing feedback to areas 17 and 18 are similar. However, peak density of feedback cells to area 18 is comparable in the supra- and infragranular layers, whereas peak density of feedback cells to area 17 is higher in the infragranular layers. Another prominent difference is that dorsal area 18 receives a cortical input that area 17 does not: from ventral cortex representing the upper visual field; this appears to be roughly 25% of the feedback input to area 18. Lastly, area 17 receives a greater proportion of cortical feedback from area 21 than from Ssy, whereas area 18 receives more feedback from Ssy than from area 21. While the organization of feedback projections from extrastriate cortex to areas 17 and 18 is broadly similar, the main difference in input topography might arise due to differences in visual field representations of the two areas.