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Summary Differences in the growth and maturation of diverse forebrain tissues depend on region-specific transcriptional regulation. Individual transcription factors act simultaneously in multiple regions that develop very differently, raising questions about the extent to which their actions vary regionally. We found that the transcription factor Pax6 affects the transcriptomes and the balance between proliferation and differentiation in opposite directions in the diencephalon versus cerebral cortex. We tested several possible mechanisms to explain Pax6's tissue-specific actions and found that the presence of the transcription factor Foxg1 in the cortex but not in the diencephalon was most influential. We found that Foxg1 is responsible for many of the differences in cell cycle gene expression between the diencephalon and cortex and, in cortex lacking Foxg1, Pax6's action on the balance of proliferation versus differentiation becomes diencephalon like. Our findings reveal a mechanism for generating regional forebrain diversity in which one transcription factor completely reverses the actions of another., Graphical Abstract, Highlights • Pax6 loss affects transcriptome profiles oppositely in cortex versus diencephalon • Pax6 promotes neuron differentiation in cortex, whereas proliferation in the diencephalon • Foxg1-null cortex develops a diencephalon-like profile of cell cycle gene expression • Foxg1 presence in cortex but not in diencephalon explains Pax6 tissue-specific actions, Neuroscience; Molecular Neuroscience; Developmental Neuroscience

BACKGROUND: We studied whether regulation of Cdca7 (Cell division cycle associated 7) expression by transcription factor Pax6 contributes to Pax6's cellular actions during corticogenesis. The function of Cdca7 in mediating Pax6's effects during corticogenesis has not been explored. Pax6 is expressed by radial glial progenitors in the ventricular zone of the embryonic cortical neuroepithelium, where it is required for the development of a normal complement of Tbr2-expressing intermediate progenitor cells in the subventricular zone. Pax6's expression levels are graded across the ventricular zone, with highest levels laterally where Tbr2-expressing progenitors are generated in greatest numbers at early stages of corticogenesis.METHODS: We used in situ hybridization and immunohistochemistry to analyse patterns of Cdca7 and Pax6 expression in cortical tissue from wild-type and Pax6 (-/-) embryos. In each genotype we compared the graded expression of the two genes quantitatively at several ages. To test whether defects in Cdca7 expression in lateral cortical cells might contribute to the cellular defects in this region caused by Pax6 loss, we electroporated a Cdca7 expression vector into wild-type lateral cortex and examined the effect on the production of Tbr2-expressing cells.RESULTS: We found that Cdca7 is co-expressed with Pax6 in cortical progenitors, at levels opposite to those of Pax6. Lowest levels of Cdca7 are found in the radial glial progenitors of lateral cortex, where Pax6 levels are highest. Higher levels of Cdca7 are found in ventral telencephalon, where Pax6 levels are low. Loss of Pax6 causes Cdca7 expression to increase in the lateral cortex. Elevating Cdca7 in normal lateral cortical progenitors to levels close to those normally found in ventral telencephalon reduces their production of Tbr2-expressing cells early in lateral cortical formation.CONCLUSION: Our results suggest that Pax6 normally represses Cdca7 expression in the lateral cortex and that repression of Cdca7 in cells of this region is required for their production of a normal complement of Tbr2-expressing intermediate progenitors.

The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of inhibitor of differentiation (Id) factors. We have previously identified the major binding partner of Id proteins in pluripotent cells as the basic helix-loop-helix (bHLH) transcription factor (TF) E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells., Summary: E2A is usually a permissive partner for instructive transcription factors. Expression and genome editing assays revealed that E2A goes it alone as a homodimer to dominantly impose neural identity on pluripotent cells.

The transcription factor Pax6 controls multiple aspects of forebrain development. Conditional deletion of Pax6 in embryonic mouse cortex causes increased proliferation of cortical progenitor cells and a concomitant decrease in neural differentiation. Notch signaling regulates the balance between proliferation and differentiation of cortical progenitor cells, suggesting a possible connection between Pax6 and Notch signaling. We investigated how expression of the Notch ligand delta-like 1 (Dll1) is altered by loss of Pax6. Acute cortex-specific deletion of Pax6 resulted in a widespread decrease in the density of Dll1+ cells at embryonic days 12.5 and 13.5 (E12.5 and E13.5). In constitutive loss-of-function mutants, decreases in the densities of Dll1+ cells were more limited both spatially and temporally. Controlled over-expression of Pax6 had no detectable effect on Dll1 expression. The proneural transcription factor Neurog2 is a target of Pax6 that can activate Dll1 expression and we found clear co-expression of Neurog2 and Dll1 in radial glial progenitors, suggesting that Pax6’s effect on Dll1 could be mediated through Neurog2. However, we found no change in Dll1+ cells in Neurog2−/− cortex suggesting either that Neurog2 is not directly involved, or that its loss of function in embryonic cortex can be compensated for.

The recent development of organoid techniques, in which embryonic brain-like tissue can be grown from human or mouse stem cells in vitro offers the potential to transform the way in which brain development is studied. In this review, we summarize key aspects of the embryonic development of mammalian forebrains, focussing in particular on the cerebral cortex and highlight significant differences between mouse and primates, including human. We discuss recent work using cerebral organoids that has revealed key similarities and differences between their development and that of the brain in vivo. Finally, we outline the ways in which cerebral organoids can be used in combination with CRISPR/Cas9 genome editing to unravel genetic mechanisms that control embryonic development of the cerebral cortex, how this can help us understand the causes of neurodevelopmental disorders and some of the key challenges which will have to be resolved before organoids can become a mainstream tool to study brain development.

The cerebral cortex contains an enormous number of neurons, allowing it to perform highly complex neural tasks. Understanding how these neurons develop at the correct time and place and in accurate numbers constitutes a major challenge. Here, we demonstrate a novel role for Gli3, a key regulator of cortical development, in cortical neurogenesis. We show that the onset of neuron formation is delayed in Gli3 conditional mouse mutants. Gene expression profiling and cell cycle measurements indicate that shortening of the G1 and S phases in radial glial cells precedes this delay. Reduced G1 length correlates with an upregulation of the cyclin-dependent kinase gene Cdk6, which is directly regulated by Gli3. Moreover, pharmacological interference with Cdk6 function rescues the delayed neurogenesis in Gli3 mutant embryos. Overall, our data indicate that Gli3 controls the onset of cortical neurogenesis by determining the levels of Cdk6 expression, thereby regulating neuronal output and cortical size.

Understanding brain development remains a major challenge at the heart of understanding what makes us human. The neocortex, in evolutionary terms the newest part of the cerebral cortex, is the seat of higher cognitive functions. Its normal development requires the production, positioning and appropriate interconnection of very large numbers of both excitatory and inhibitory neurons. Pax6 is one of a relatively small group of transcription factors that exert high-level control of cortical development, and whose mutation or deletion from developing embryos causes major brain defects and a wide range of neurodevelopmental disorders. Pax6 is very highly conserved between primate and non-primate species, is expressed in a gradient throughout the developing cortex and is essential for normal corticogenesis. Our understanding of Pax6’s functions and the cellular processes that it regulates during mammalian cortical development has significantly advanced in the last decade, owing to the combined application of genetic and biochemical analyses. Here we review the functional importance of Pax6 in regulating cortical progenitor proliferation, neurogenesis, and formation of cortical layers and highlight important differences between rodents and primates. We also review the pathological effects of PAX6 mutations in human neurodevelopmental disorders. Finally, we discuss some aspects of Pax6’s molecular actions including its own complex transcriptional regulation, the distinct molecular functions of its splice variants and some of Pax6’s known direct targets which mediate its actions during cortical development.

The corpus callosum (CC) connects the left and right cerebral hemispheres in mammals and its development requires intercellular communication at the telencephalic midline mediated by signaling proteins. Heparan sulfate (HS) is a sulfated polysaccharide that decorates cell surface and extracellular matrix proteins and regulates the biological activity of numerous signaling proteins via sugar-protein interactions. HS is subject to regulated enzymatic sulfation and desulfation and an attractive, although not proven, hypothesis is that the biological activity of HS is regulated by a sugar sulfate code. Mutant mouse embryos lacking the heparan sulfotransferases Hs2st or Hs6st1 have severe CC phenotypes and form Probst bundles of noncrossing axons flanking large tangles of midline glial processes. Here, we identify a precocious accumulation of Sox9-expressing glial cells in the indusium griseum region and a corresponding depletion at the glial wedge associated with the formation of Probst bundles along the rostrocaudal axis in both mutants. Molecularly, we found a surprising hyperactivation of Erk signaling in Hs2st(-/-) (2-fold) and Hs6st1(-/-) (6-fold) embryonic telencephalon that was most striking at the midline, where Erk signaling is lowest in wild-types, and a 2-fold increase in Fgf8 protein levels in Hs6st1(-/-) embryos that could underpin Erk hyperactivation and excessive glial movement to the indusium griseum. The tightly linked Hs6st1(-/-) CC glial and axonal phenotypes can be rescued by genetic or pharmacological suppression of Fgf8/Erk axis components. Overall, our data fit a model in which Hs2st and Hs6st1 normally generate conditions conducive to CC development by generating an HS-containing environment that keeps Erk signaling in check.

Early events in the formation of the thalamocortical tract remain poorly understood. Recent work has suggested that thalamocortical axons follow a path pioneered by transient thalamic afferents originating from the medial part of the ventral telencephalon. We studied the development of these transient afferents and the thalamocortical tract in mutant mice lacking transcription factors normally expressed in the dorsal thalamus or ventral telencephalon. Pax6 is expressed in the dorsal thalamus, but not in the medial part of the ventral telencephalon, and the thalamocortical tract fails to form in Pax6(-/-) embryos. We found that transient thalamic afferents from the ventral telencephalon do not form in Pax6(-/-) embryos; this may contribute to the failure of their thalamocortical development. The distribution of Pax6(-/-) cells in Pax6(-/-) Pax6(+/+) chimeras supports conclusions drawn from forebrain marker gene expression that Pax6 is not required for the normal development of the medial part of the ventral telencephalon but is required in the dorsal thalamus. Failure of the transient afferent pathway to develop is therefore likely a cell nonautonomous defect reflecting primary defects in the thalamus. We then examined the formation of thalamic afferents and efferents in Foxg1(-/-) embryos, which lack recognizable ventral telencephalic structures. In these embryos thalamic efferents navigate correctly through the thalamus but fail to turn laterally into the telencephalon, whereas other axons are able to cross the diencephalic/telencephalic boundary. Our results support a role for the ventral telencephalon in guiding the early development of the thalamocortical tract and identify a new role for the transcription factor Pax6 in regulating the ability of the thalamus to attract ventral telencephalic afferents.

A collection of folklore and local history stories from Cuileannach (B.) (school) (Cullenagh Upper, Co. Kerry), collected as part of the Schools' Folklore Scheme, 1937-1938 under the supervision of teacher Peadar Ó Cochláin., Seanchaisleáin na Dúthaí seo - Dúnló / #missing, -- Seanchaisleáin na Dúthaí seo - Caisleán an Chorraigh / #missing, -- Seanchaisleáin na Dúthaí seo - Palas / #missing, -- Teampall an Chnocáin / #missing, -- Droichead na Spideoige / #missing, -- Cill Lócháin / Sullivan, Michael O' -- Cill Ghobnait / Sullivan, Michael O' -- Glaisín na Chorrgaidh / #missing, -- Leabaidh na Bó / #missing, -- Filí na Dúthaí / Curran, Teddy -- Gabha agus an Cheárta / Curran, Teddy -- Lios na Leacht / Sheehy, Brendan -- Tailors / Moriarty, Denis -- Religious Stories / Gillicuddy, Denis Mc -- We hear several religious stories told by the people. They say that when Our Lord was crowned with thorns a robin happened to be passing by and... -- Tailor / Mason, John O' -- Cures / Sullivan, Michael O' -- Old Toys / Sullivan, Michael O' -- Funny Story / Kelliher, John -- Benburb -- Late Mr C. Martin - Funeral of Man who Planned Course for Atlantic Flight -- Circular to Teachers of National Schools - Questionnaire Regarding Holy Wells -- Signs of the Weather / Gillicuddy, Denis Mc -- What Your County Has Done, Supported by funding from the Department of Arts, Heritage and the Gaeltacht (Ireland), University College Dublin, and the National Folklore Foundation (Fondúireacht Bhéaloideas Éireann), 2014-2016.