Linda J. Richards

NFIB or TCF4 haploinsufficiency results in distinct neurodevelopmental disorders that share several characteristic overlapping features. Recent studies suggest that these transcription factors converge by binding to the same cis-regulatory elements and thereby regulate a common set of target genes. Here, we sought to determine whether in mice, NFIB and TCF4 converge during neocortical development. NFIB and TCF4 are co-expressed in radial glial cells and postmitotic neurons. Transcriptomic analyses of Nfib and Tcf4 knockout mice uncovered an overlapping set of downregulated genes involved in neuronal differentiation. Analyses of genome-wide binding further demonstrate that NFIB and TCF4 largely bind to the same regulatory elements. In luciferase assays, NFIB and TCF4 elicit distinct effects on transcriptional activity that differ between the regulatory elements and cell types that we tested. We propose that this heterogeneity is a means through which NFIB and TCF4 function cooperatively to fine-tune target gene expression. Our study provides critical evidence for the molecular convergence of NFIB and TCF4. This indicates that the human syndromes resulting from mutations in these genes may have overlapping underlying etiologies.

DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. To accomplish this, non-mammalian vertebrates such as zebrafish and medaka subtly reprogramme maternal 5mC profiles while maintaining high methylation levels throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. However, while embryonic 5mC is rapidly re-established to high levels upon implantation, the trophectoderm, which gives rise to the placenta, displays sustained and conserved DNA hypomethylation, suggesting that this drastic 5mC erasure may be functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylation dynamics in marsupials, a lineage of therian mammals with a short-lived placenta. We produced a near complete telomere-to-telomere (T2T) genome and generated detailed epigenome maps of embryonic development for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). We found the dunnart embryo exhibits genome wide DNA demethylation at the blastocyst stage, but these changes occur in the trophectoderm only, suggesting that 5mC erasure in the placenta is an ancestral state in therian mammals. Furthermore, the T2T-level dunnart genome assembly enabled identification of sex chromosomes, uncovering extensive hypomethylation of the paternally-inherited inactive X chromosome in females and revealing the previously unannotated master regulator of X chromosome inactivation, lncRNA RSX. Our data indicate that while the use of genome-wide 5mC erasure differs between eutherian and marsupial lineages, 5mC erasure in extraembryonic tissue is ancestral to therian mammals and may be necessary to support placental development.

Cell type specification in the embryonic brain and spinal cord is thought to begin within molecularly defined progenitor domains that do not intermix. Our data provide an alternative model that is spatially and temporally dynamic within a basal ganglia anlage, the medial ganglionic eminence (MGE). MGE progenitor cells are progressively displaced ventrally and caudally from a rostral growth zone (the MGE/LGE sulcus). Progenitors that leave the MGE/LGE sulcus early occupy caudoventral MGE regions, while ones that leave later reside in rostrodorsal MGE regions. As they change position, their transcriptional states and cell type output change. Transcriptional analyses showed an upregulation of the Nfi TFs during the period of progenitor movement. Nfia and Nfib double mutants alter the repertoire of cortical interneuron subtypes. Overall, we present a mechanism that synchronizes regional patterning with tissue growth and links spatial and temporal specification in producing diverse neuronal subtypes.

Mammals have evolved a more complex brain, exemplified by the transformation of the single-layer dorsal cortex of excitatory projection neurons (ExNs) in ancestors into a multilayered cerebral neocortex^1-4 enriched with diverse intratelencephalic and extratelencephalic ExN subtypes^5-7, thereby establishing specialized projection systems that enhance brain connectivity and functionality^5-8. This is in contrast to modern reptiles and birds with single-layered or pseudolayered columnar organization of ExNs^4,9-12. However, the mechanisms underlying these mammalian-specific adaptations remain elusive. By comparing the landscape of gene expression and putative cis-regulatory elements (CREs) in mouse ExN subtypes and through cross-species examination, we identified mammalian-specific CREs, including a subset bound by the transcription factor ZBTB18 (also RP58, ZFP238 or ZNF238) and associated with genes defining intratelencephalic and extratelencephalic subtypes and connectivity, which have been implicated in intellectual disability and autism. Deletion of Zbtb18 in mouse ExNs dysregulated target gene expression, reduced molecular diversity, diminished cortico-spinal and callosal projections and increased intrahemispheric cortico-cortical association projections to the prefrontal cortex, thereby resembling non-mammalian brain. ZBTB18 binding motifs are highly enriched in callosally projecting intratelencephalic-biased putative CREs and show higher conservation specifically in mammals. This study uncovers critical components and mammalian-specific evolutionary adaptations within a regulatory node essential for neocortical ExN identity and connectivity.

The corpus callosum is the largest commissure in the mammalian brain and plays a major role in supporting cognitive processes required for adapting to complex environments. Individuals born with Corpus Callosum Dysgenesis (CCD), characterized by malformations of the corpus callosum, commonly exhibit deficits in social navigation, abstract problem-solving, decision-making, and self-awareness. Metacognition is a key cognitive process that supports these functions; however, it has yet to be tested comprehensively in individuals with CCD. Over three experiments, and three CCD cohorts, we tested the impact of this neurodevelopmental disorder on perceptual accuracy, confidence judgements, and metacognitive efficiency using two variants of a Random Dot Kinematogram task within lab, online, and VR conditions. We found that individuals with CCD typically displayed normal perceptual accuracy but failed to adjust their confidence judgements in line with task difficulty. Computational modelling revealed that this difference was explained by lower metacognitive efficiency driven by consistently lower metacognitive sensitivity. Together, these results provide evidence that the corpus callosum plays a crucial role in supporting metacognition.

Brain function requires exquisitely adapted plasticity at multiple scales, from synapses to whole-brain networks. Evidence for large-scale plasticity in functional brain networks comes from neuroimaging data across a variety of species, particularly during development and following injury. However, how large-scale network remodelling is achieved at the microscopic level is unknown as the growth of entirely new long-distance axons is unlikely to occur. Recent insights from electron microscopic connectome studies and single-cell projectomes of neurons in the brains of multiple model organisms have provided new evidence for the incredible structural complexity of axons and their branches that traverse the brain. This evidence shows highly arborized axonal projections, differentially myelinated branches of the same axon, and axonal regions devoid of synaptic contacts but with the potential to form synaptic connections in new or additional areas. Recent electron microscopic data suggest that these axonal features may be evolutionarily conserved. Here we consider whether these features could enable long-range and large-scale neuroplastic changes at a functional level, particularly following focal brain injury. These insights contribute to our emerging understanding of how the brain undergoes large-scale reorganization to adapt to changing circumstances.

Nuclear Factor I (NFI) misexpressions in humans are associated with severe brain malformations, yet the underlying mechanisms remain poorly understood. Here we show that NFIs regulate the broad lineage progression and lifespan of radial glial progenitors (RGPs), and consequently neocortical development in a dose-dependent manner. Human cerebral organoids carrying patient-mimicking NFI mutations exhibit expression level-dependent bidirectional impairments in RGP temporal development, coinciding with patient phenotypes. In mouse models, selective removal of NFIs leads to a dramatic protraction of RGP lineage progression and lifespan, excessive progeny output, and neocortical overgrowth and abnormal folding, whereas overexpression of NFIs accelerates RGP lineage progression, resulting in developmental stage-dependent precocious productions of diverse neural progenies. Moreover, NFIs exhibit a positive auto-regulation and progressive increase in expression, and regulate distinct temporal-specific targets underlying RGP lineage progression. These results suggest that NFIs act as evolutionarily conserved key global temporal regulators of RGP lineage progression and neocortical development.

DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. To accomplish this, non-mammalian vertebrates such as zebrafish and medaka subtly reprogram maternal 5mC profiles while maintaining high methylation levels throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. However, while embryonic 5mC is rapidly re-established to high levels upon implantation, the trophectoderm, which gives rise to the placenta, displays sustained and conserved DNA hypomethylation, suggesting that this drastic 5mC erasure may be functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylation dynamics in marsupials, a lineage of therian mammals with a short-lived placenta. We produced a near complete telomere-to-telomere (T2T) genome and generated detailed epigenome maps of embryonic development for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). We found the dunnart embryo exhibits genome wide DNA demethylation at the blastocyst stage, but these changes occur in the trophectoderm only, suggesting that 5mC erasure in the placenta is an ancestral state in therian mammals. Furthermore, the T2T-level dunnart genome assembly enabled identification of sex chromosomes, uncovering extensive hypomethylation of the paternally-inherited inactive X chromosome in females and revealing the previously unannotated master regulator of X chromosome inactivation, lncRNA Rsx. Our data indicate that while the use of genome-wide 5mC erasure differs between eutherian and marsupial lineages, 5mC erasure in extraembryonic tissue is ancestral to therian mammals and may be necessary to support placental development.

DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. To accomplish this, non-mammalian vertebrates such as zebrafish and medaka subtly reprogram maternal 5mC profiles while maintaining high methylation levels throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. However, while embryonic 5mC is rapidly re-established to high levels upon implantation, the trophectoderm, which gives rise to the placenta, displays sustained and conserved DNA hypomethylation, suggesting that this drastic 5mC erasure may be functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylation dynamics in marsupials, a lineage of therian mammals with a short-lived placenta. We produced a near complete telomere-to-telomere (T2T) genome and generated detailed epigenome maps of embryonic development for an Australian marsupial, the fat-tailed dunnart ( Sminthopsis crassicaudata ). We found the dunnart embryo exhibits genome wide DNA demethylation at the blastocyst stage, but these changes occur in the trophectoderm only, suggesting that 5mC erasure in the placenta is an ancestral state in therian mammals. Furthermore, the T2T-level dunnart genome assembly enabled identification of sex chromosomes, uncovering extensive hypomethylation of the paternally-inherited inactive X chromosome in females and revealing the previously unannotated master regulator of X chromosome inactivation, lncRNA Rsx . Our data indicate that while the use of genome-wide 5mC erasure differs between eutherian and marsupial lineages, 5mC erasure in extraembryonic tissue is ancestral to therian mammals and may be necessary to support placental development. HIGHLIGHTS: First embryonic DNA methylation maps in an Australian marsupial Extensive global erasure of DNA methylation in the trophectoderm Maintenance of high DNA methylation in the embryonic lineage Hypomethylated paternal X chromosome with methylated escapee genes