Nipbl Interacts with Zfp609 and the Integrator Complex to Regulate Cortical Neuron Migration

Mutations in NIPBL are the most frequent cause of Cornelia de Lange syndrome (CdLS), a developmental disorder encompassin

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.Welcome Trust grants: (WT095908, WT098051), FEBS Long-Term Fellowship, Medical Research Council Grant-in-Aid (U117570528)

Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateral(high) to caudo-medial(low) gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. We found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), we showed that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77↔wild-type chimeras indicates that the defect is cell autonomous. We analyzed cortical arealization in PAX77 mice and found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not

LRIG1 is a gatekeeper to exit from quiescence in adult neural stem cells

Adult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence

The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis

Oriented cell behaviors likely have a more important role in limb bud elongation during development than previously suggested by the “growth-based morphogenesis” hypothesis

Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human

RNA activation of haploinsufficient Foxg1 gene in murine neocortex

More than one hundred distinct gene hemizygosities are specifically linked to epilepsy, mental retardation, autism, schizophrenia and neuro-degeneration. Radical repair of these gene deficits via genome engineering is hardly feasible. The same applies to therapeutic stimulation of the spared allele by artificial transactivators. Small activating RNAs (saRNAs) offer an alternative, appealing approach. As a proof-of-principle, here we tested this approach on the Rett syndrome-linked, haploinsufficient, Foxg1 brain patterning gene. We selected a set of artificial small activating RNAs (saRNAs) upregulating it in neocortical precursors and their derivatives. Expression of these effectors achieved a robust biological outcome. saRNA-driven activation (RNAa) was limited to neural cells which normally express Foxg1 and did not hide endogenous gene tuning. saRNAs recognized target chromatin through a ncRNA stemming from it. Gene upregulation required Ago1 and was associated to RNApolII enrichment throughout the Foxg1 locus. Finally, saRNA delivery to murine neonatal brain replicated Foxg1-RNAa in vivo

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

Dynamic Coupling of Pattern Formation and Morphogenesis in the Developing Vertebrate Retina

In this Research Article, Picker et al. show how cells in the retina get their spatial coordinates

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