Astrocytes are key components in reactive gliosis after brain injury, yet defined in vitro models dissecting the influence of extracellular matrix components enriched after injury, such as fibrin, on human astrocyte behavior and function are still missing. Here, we use fibrinogen-derived fibrin and fibrin-alginate-RGD (FAR) 3D hydrogel substrates to examine their influence on human induced pluripotent stem cell (hiPSC)-derived astrocyte behavior and on their direct conversion into neurons. Astrocytes develop complex morphologies in 3D-FAR hydrogels, while they are more proliferative and migratory in 3D-fibrin hydrogels (3D-fibrin). Interestingly, gene expression analysis revealed different reactive states of astrocytes in 3D-fibrin and 3D-FAR, which persist over time. The highly inflammatory state and stiffness of 3D-FAR are largely incompatible with direct neuronal reprogramming, hampering the direct conversion even at early stages. Conversely, astrocytes in 3D-fibrin hydrogels can convert into neuronal cells, demonstrating a potent influence of how fibrin is presented on distinct astrocyte states, with great relevance for fate conversion.

Direct conversion of reactive glial cells to neurons is a promising avenue for neuronal replacement therapies after brain injury or neurodegeneration. The overexpression of neurogenic fate determinants in glial cells results in conversion to neurons. For repair purposes, the conversion should ideally be induced in the pathology-induced neuroinflammatory environment. However, very little is known regarding the influence of the injury-induced neuroinflammatory environment and released growth factors on the direct conversion process. We establish a new in vitro culture system of postnatal astrocytes without epidermal growth factor that reflects the direct conversion rate in the injured, neuroinflammatory environment in vivo. We demonstrate that the growth factor combination corresponding to the injured environment defines the ability of glia to be directly converted to neurons. Using this culture system, we show that chromatin structural protein high mobility group box 2 (HMGB2) regulates the direct conversion rate downstream of the growth factor combination. We further demonstrate that Hmgb2 cooperates with neurogenic fate determinants, such as Neurog2, in opening chromatin at the loci of genes regulating neuronal maturation and synapse formation. Consequently, early chromatin rearrangements occur during direct fate conversion and are necessary for full fate conversion. Our data demonstrate novel growth factor-controlled regulation of gene expression during direct fate conversion. This regulation is crucial for proper maturation of induced neurons and could be targeted to improve the repair process.

Astrocytes are increasingly recognized as key regulators of neural circuit development and function, with mounting evidence revealing substantial heterogeneity within and across brain regions. Yet, the full extent of this diversity and its developmental mechanisms remain poorly understood. To address this, we leveraged the uniqueness of the mouse cerebellum, which hosts well-defined astrocyte types and established progenitor pools. Through complementary multi-modal omic approaches, including single-cell RNA sequencing, spatial transcriptomics, trajectory inference, clonal lineage reconstruction, and gene expression and regulatory network analyses, we systematically dissected the molecular diversity and ontogenesis of cerebellar astrocytes. We identified known types and uncovered new subtypes with functional specialization, inferring their developmental trajectories from multiple embryonic niches and postnatal progenitor sources with fate divergence, convergence, and restriction. We further predicted a hierarchical transcriptional regulator code governing this diversification, operating at multiple levels: distinct regulatory modules i) reflect embryonic regionalization and lineage; ii) determine broad astroglial identity; specify iii) Bergmann versus non-Bergmann fates; and guide iv) astrocyte type and v) subtype acquisition. Our findings map and temporally organize transcriptional programs that capture key determinants of astrocyte fate, integrating them along defined trajectories toward diverse astrocyte identities. This high-resolution framework for cerebellar glial diversification offers a model to be challenged across other brain regions. ### Competing Interest Statement The authors have declared no competing interest.

Astrocyte heterogeneity has been well explored, but our understanding of white matter (WM) astrocytes and their distinctions from gray matter (GM) astrocytes remains limited. Here, we compared astrocytes from cortical GM and WM/corpus callosum (WM/CC) using single-cell RNA sequencing and spatial transcriptomics of the murine forebrain. The comparison revealed similarities but also significant differences between WM and GM astrocytes, including cytoskeletal and metabolic hallmarks specific to WM astrocytes with molecular properties also shared with human WM astrocytes. When we compared murine astrocytes from two different WM regions, the cortex and cerebellum, we found that they exhibited distinct, region-specific molecular properties, with the cerebellum lacking, for example, a specific cluster of WM astrocytes expressing progenitor and proliferation genes. Functional experiments confirmed astrocyte proliferation in the WM/CC, but not in the cerebellar WM, suggesting that the WM/CC may be a source of continued astrogenesis.

Traumatic brain injury leads to a highly orchestrated immune- and glial cell response partially responsible for long-lasting disability and the development of secondary neurodegenerative diseases. A holistic understanding of the mechanisms controlling the responses of specific cell types and their crosstalk is required to develop an efficient strategy for better regeneration. Here, we combine spatial and single-cell transcriptomics to chart the transcriptomic signature of the injured male murine cerebral cortex, and identify specific states of different glial cells contributing to this signature. Interestingly, distinct glial cells share a large fraction of injury-regulated genes, including inflammatory programs downstream of the innate immune-associated pathways Cxcr3 and Tlr1/2. Systemic manipulation of these pathways decreases the reactivity state of glial cells associated with poor regeneration. The functional relevance of the discovered shared signature of glial cells highlights the importance of our resource enabling comprehensive analysis of early events after brain injury.

Astrocytes exhibit dual roles in central nervous system (CNS) recovery, offering both beneficial and detrimental effects. Following CNS injury, a subset of astrocytes undergoes proliferation, de-differentiation, and acquires self-renewal and neurosphere-forming capabilities in vitro. This subset of astrocytes represents a promising target for initiating brain repair processes and holds potential for neural recovery. However, studying these rare plastic astrocytes is challenging due to the absence of distinct markers. In our study, we characterized these astrocytic subpopulations using comparative single-cell transcriptome analysis. By leveraging the regenerative properties observed in radial glia of zebrafish, we identified and characterized injury-induced plastic astrocytes in mice. These injury-induced astrocytic subpopulations were predominantly proliferative and demonstrated the capacity for self-renewal and neurosphere formation, ultimately differentiating exclusively into astrocytes. Integration with scRNAseq data of the subependymal zone (SEZ) allowed us to trace the origins of these injury-induced plastic astrocytic subpopulations to parenchymal astrocytes. Our analysis revealed that a subset of these injury-induced astrocytes shares transcriptional similarities with endogenous transient amplifying progenitors (TAPs) within the SEZ, rather than with neural stem cells (NSCs). Notably, these injury-induced TAP-like cells exhibit distinct differentiation trajectories, favoring gliogenic over neurogenic differentiation. In summary, our study identifies a rare subset of injury-induced, proliferative plastic astrocytes with neurosphere-forming capacities. These cells originate from reactive astrocytes and resemble TAPs in their transcriptional profile. This study enhances our understanding of astrocyte plasticity post-injury. ### Competing Interest Statement The authors have declared no competing interest.

The glial environment influences neurological disease progression, yet much of our knowledge still relies on preclinical animal studies, especially regarding astrocyte heterogeneity. In murine models of traumatic brain injury, beneficial functions of proliferating reactive astrocytes on disease outcome have been unraveled, but little is known regarding if and when they are present in human brain pathology. Here we examined a broad spectrum of pathologies with and without intracerebral hemorrhage and found a striking correlation between lesions involving blood-brain barrier rupture and astrocyte proliferation that was further corroborated in an assay probing for neural stem cell potential. Most importantly, proteomic analysis unraveled a crucial signaling pathway regulating this astrocyte plasticity with GALECTIN3 as a novel marker for proliferating astrocytes and the GALECTIN3-binding protein LGALS3BP as a functional hub mediating astrocyte proliferation and neurosphere formation. Taken together, this work identifies a therapeutically relevant astrocyte response and their molecular regulators in different pathologies affecting the human cerebral cortex.

ABSTRACT Increase in the size of human neocortex, acquired in evolution, accounts for the unique cognitive capacity of humans. This expansion appears to reflect the evolutionarily-enhanced proliferative ability of basal progenitors (BPs) in mammalian cortex, which may have been acquired through epigenetic alterations in BPs. However, whether or how the epigenome in BPs differs across species is not known. Here, we report that histone H3 acetylation is a key epigenetic regulation in BP amplification and cortical expansion. Through epigenetic profiling of sorted BPs, we show that H3K9 acetylation is low in murine BPs and high in human BPs. Elevated H3K9ac preferentially increases BP proliferation, increasing the size and folding of the normally smooth mouse neocortex. Mechanistically, H3K9ac drives BP amplification by increasing expression of the evolutionarily regulated gene, TRNP1, in the developing cortex. Our findings demonstrate a previously unknown mechanism that controls cortical architecture. One Sentence Summary H3K9ac promotes basal progenitor amplification, neocortex expansion and gyrification by activating TRNP1 expression in evolution.

The vulnerability of the mammalian brain is mainly due to its limited ability to generate new neurons once fully matured. Direct conversion of non-neuronal cells to neurons opens up a new avenue for therapeutic intervention and has made great strides also for in vivo applications in the injured brain. These great achievements raise the issue of adequate identity and chromatin hallmarks of the induced neurons. This may be particularly important, as aberrant epigenetic settings may reveal their adverse effects only in certain brain activity states. Therefore, we review here the knowledge about epigenetic memory and partially resetting of chromatin hallmarks from other reprogramming fields, before moving to the knowledge in direct neuronal reprogramming, which is still limited. Most importantly, novel tools are available now to manipulate specific epigenetic marks at specific sites of the genome. Applying these will eventually allow erasing aberrant epigenetic memory and paving the way towards new therapeutic approaches for brain repair.

Background: The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time-and cost-effective methods enabling large-scale screening approaches.Results: Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs.Conclusions: The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.

Neuronal differentiation relies on a set of interconnected molecular events to achieve the differentiation of pan-neuronal hallmarks, together with neuronal subtype-specific features. Here, we propose a conceptual framework for these events, based on recent findings. This framework encompasses a dimension in time during development, progressing from early master regulators to later expressed effector genes and terminal selector genes. As a horizontal intersection, we propose the action of permissive fate determinants that are critical in allowing progression through the above transcriptional phases. Typically, these are widely expressed and often interact with the chromatin remodeling machinery. We conclude by discussing this model in the context of the direct fate conversion of various somatic cells into neurons.

The mature phenotype of CNS neurons is thought to be set at an early progenitor stage. A study now shows that expression of Fezf2 alone can turn striatal GABAergic precursors into glutamatergic corticofugal neurons.

Nat. Neurosci. 12, 1229–1237 (2009); published online 13 September 2009; corrected after print 25 September 2009 In the version of this article initially published, one of the corresponding authors' email addresses was misspelled. It should be luisapinto@ecsaude.uminho.pt. In addition, errors occurred in some of the numbers listed in the last subsection of the Results section.

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Progression of progenitor cells towards neuronal differentiation is tightly linked with cell cycle control and the switch from proliferative to neuron-generating divisions. We have previously shown that the neuronal protein BM88 drives neuroblastoma cells towards exit from the cell cycle and differentiation into a neuronal phenotype in vitro. Here, we explored the role of BM88 during neuronal birth, cell cycle exit and the initiation of differentiation in vivo. By double- and triple-labelling with the S-phase marker BrdU or the late G2 and M-phase marker cyclin B1, antibodies to BM88 and markers of the neuronal or glial cell lineages, we demonstrate that in the rodent forebrain, BM88 is expressed in multipotential progenitor cells before terminal mitosis and in their neuronal progeny during the neurogenic interval, as well as in the adult. Further, we defined at E16 a cohort of proliferative progenitors that exit S phase in synchrony, and by following their fate for 24 h we show that BM88 is associated with the dynamics of neuron-generating divisions. Expression of BM88 was also evident in cycling cortical radial glial cells, which constitute the main neurogenic population in the cerebral cortex. In agreement, BM88 expression was markedly reduced and restricted to a smaller percentage of cells in the cerebral cortex of the Small eye mutant mice, which lack functional Pax6 and exhibit severe neurogenesis defects. Our data show an interesting correlation between BM88 expression and the progression of progenitor cells towards neuronal differentiation during the neurogenic interval.