Mapping cellular organization in the developing brain presents significant challenges due to the multidimensional nature of the data, characterized by complex spatial patterns that are difficult to interpret without high-throughput tools. Here, we present DeepCellMap, a deep-learning-assisted tool that integrates multi-scale image processing with advanced spatial and clustering statistics. This pipeline is designed to map microglial organization during normal and pathological brain development and has the potential to be adapted to any cell type. Using DeepCellMap, we capture the morphological diversity of microglia, identify strong coupling between proliferative and phagocytic phenotypes, and show that distinct spatial clusters rarely overlap as human brain development progresses. Additionally, we uncover an association between microglia and blood vessels in fetal brains exposed to maternal SARS-CoV-2. These findings offer insights into whether various microglial phenotypes form networks in the developing brain to occupy space, and in conditions involving haemorrhages, whether microglia respond to, or influence changes in blood vessel integrity. DeepCellMap is available as an open-source software and is a powerful tool for extracting spatial statistics and analyzing cellular organization in large tissue sections, accommodating various imaging modalities. This platform opens new avenues for studying brain development and related pathologies.

Viral infection and hypocholesterolaemia are two independent risk factors for intracerebral haemorrhage (ICH), but the molecular mechanisms leading to vascular rupture via these risk factors remain unknown. We hypothesised that the enzyme cholesterol 25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25HC), which modulates cholesterol metabolism during infection, may offer a mechanistic link between cholesterol dysregulation and infection during neurovascular dysfunction. We identified an upregulation of CH25H in infection-associated cerebral haemorrhage, in a SARS-CoV-2-induced zebrafish ICH model and foetal human SARS-CoV-2-associated cortical microbleeds. Using human brain endothelial cells and zebrafish ICH models, we show that 25HC promotes endothelial dysfunction and exacerbates brain bleeding. These effects involved cholesterol metabolism modulation, as cholesterol supplementation rescued these effects, while 25HC and statin treatments interacted to exacerbate dysfunction. We propose that the CH25H/25HC pathway represents an important component in the pathophysiology of brain vessel dysfunction associated with infection and cholesterol dysregulation in the context of ICH.### Competing Interest StatementThe authors have declared no competing interest.

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Maternal viral infection and immune response are known to increase the risk of altered development of the foetal brain. Given the ongoing global pandemic of coronavirus disease 2019 (COVID-19), investigating the impact of SARS-CoV-2 on foetal brain health is of critical importance. Here, we report the presence of SARS-CoV-2 in first and second trimester foetal brain tissue in association with cortical haemorrhages. SARS-CoV-2 spike protein was sparsely detected within progenitors and neurons of the cortex itself, but was abundant in the choroid plexus of haemorrhagic samples. SARS-CoV-2 was also sparsely detected in placenta, amnion and umbilical cord tissues. Cortical haemorrhages were linked to a reduction in blood vessel integrity and an increase in immune cell infiltration into the foetal brain. Our findings indicate that SARS-CoV-2 infection may affect the foetal brain during early gestation and highlight the need for further study of its impact on subsequent neurological development.

Microglia are the brain's resident macrophages, which guide various developmental processes crucial for brain maturation, activity, and plasticity. Microglial progenitors enter the telencephalic wall by the 4th postconceptional week and colonise the fetal brain in a manner that spatiotemporally tracks key neurodevelopmental processes in humans. However, much of what we know about how microglia shape neurodevelopment comes from rodent studies. Multiple differences exist between human and rodent microglia warranting further focus on the human condition, particularly as microglia are emerging as critically involved in the pathological signature of various cognitive and neurodevelopmental disorders. In this article, we review the evidence supporting microglial involvement in basic neurodevelopmental processes by focusing on the human species. We next concur on the neuropathological evidence demonstrating whether and how microglia contribute to the aetiology of two neurodevelopmental disorders: autism spectrum conditions and schizophrenia. Next, we highlight how recent technologies have revolutionised our understanding of microglial biology with a focus on how these tools can help us elucidate at unprecedented resolution the links between microglia and neurodevelopmental disorders. We conclude by reviewing which current treatment approaches have shown most promise towards targeting microglia in neurodevelopmental disorders and suggest novel avenues for future consideration.

Tissues undergo complex shape changes during development that are crucial for their later architecture and function. Understanding how these changes in shape, the morphodynamics, occur is important not only to understand developmental processes, but also to reveal how dysregulation of these processes leads to developmental defects. The precise spatiotemporal events and morphological dynamics of human brain development have remained relatively poorly understood, partly due to limited access to early human neural tissues. In the last decade, cerebral organoids have provided a novel approach to address this open question, enabling early human neural development to be observed and manipulated in vitro (Pasca et al., 2015; Camp et al., 2015; Lancaster et al., 2013).A recent preprint by Jain and colleagues (Jain et al., 2023 preprint) used a combination of single-cell RNA-sequencing (scRNA-seq) with a novel imaging and cell-labelling approach to track the changes in patterning and morphology of early organoids over several weeks. In order to visualise the morphodynamics in intact organoids, Jain and colleagues used an elegant labelling system, combining induced pluripotent stem cells (iPSCs) lines, each carrying a GFP- or RFP-tagged protein to label one of five cellular structures: the plasma membrane (CAAX), actin cytoskeleton (actin), microtubules (tubulin), nucleus (histone) and nuclear envelope (lamin). These cell lines were then combined together with a non-labelled iPSC line to generate mosaic and sparsely labelled organoids, which enabled single cells to be tracked. The morphology of the organoids was then analysed using measurements of overall size, as well as lumen number and volume.This long-term imaging of whole organoids provided a window into the cellular- and tissue-level changes in shape that occur in early neural development. Combining this with the scRNA-seq data, the authors started to pick apart the genetic changes linked to the morphodynamics observed. One key family of genes that was upregulated at times of morphological change were extracellular matrix (ECM) and ECM-associated genes. ECM has long been reported to play a role in development and morphogenesis, due to its known functions in cell migration, proliferation and organisation of the developing tissues (Barros et al., 2011; Long and Huttner, 2019; Hynes, 2009).Jain and colleagues then explored the role of exogenously supplied ECM, using Matrigel, in regulating this morphogenesis and patterning. Organoids were generated using an unguided protocol, with or without Matrigel (or 0.6% agarose) added at the onset of live imaging at day 4 of culture. The authors showed that, without Matrigel, organoids developed different morphologies, with slower growth of both organoid and lumen volume and disrupted cell polarity. Leveraging the multi-mosaic labelling of cellular structures, the authors next analysed the morphology at the cellular level. Using partition-based graph abstraction (PAGA) trajectory analysis, they observed that membrane elongation and compression of nuclei occur during a key morphological change: the transition from neuroectodermal to neuroepithelial cells.This change in cellular morphology was also accompanied by the perpendicular alignment of the cells to the organoid surface, which was increased in the presence of Matrigel. Matrigel also increased the homogeneity of cell morphologies, with cells displaying a higher degree of alignment and a more elongated morphology at earlier time points. This morphological change at the cellular level was associated with a change in cell identity, with Matrigel-exposed organoids containing significantly more telencephalic progenitors and fewer neural crest cells at day 13 than those grown in the no-Matrigel or agarose conditions.In the absence of Matrigel, scRNA-seq showed an increase in Wnt and Hippo pathway components, suggesting that Matrigel might be altering both the rostral-caudal and dorso-ventral patterning of the organoids. When the authors activated the YAP1 pathway in the presence of Matrigel, the organoids displayed altered overall and lumen morphologies and expressed genes associated with the caudalisation of the developing neuroepithelium. They suggest that Matrigel is, therefore, promoting lumen expansion and patterning of mainly rostral prosencephalic regions, which instead pattern into more caudal regions in the absence of Matrigel.Overall, Jain and colleagues have shown a significant role of Matrigel in the morphodynamics of organoid development across the scales, from gene expression to cell shape and alignment to organoid architecture. How Matrigel drives morphodynamics is an interesting question and one that others are also trying to address. Two recent papers (Chiaradia et al., 2023; Martins-Costa et al., 2023) also investigated the effect of Matrigel on guided telencephalic organoids and guided or unguided cerebral organoid development. Both papers report some similar effects of Matrigel on organoid size and structure as that reported by Jain and colleagues. Chiaradia and colleagues confirm that Matrigel improved the neuroepithelial-like structure, although they also reported that this can sometimes be observed in no Matrigel organoids too. Martins-Costa and colleagues go one step further, reporting that from day 20 onwards telencephalic organoids grown in the absence of Matrigel eventually arranged in the correct apico-basal axis without displaying any morphological differences.There are several possibilities for this contrast in results, one of which is currently hotly debated in the field; the heterogeneity of organoids (Velasco et al., 2019; Lancaster and Knoblich, 2014; Di Lullo and Kriegstein, 2017). There are many variations of protocols used to generate organoids, which can also yield intra-batch variability and variability between different cell lines. For example, the addition of WNT inhibitors by Chiaradia and colleagues and Martins-Costa and colleagues (Chiaradia et al., 2023; Martins-Costa et al., 2023) and the timing of the addition of Matrigel, could result in differences in organoid development when compared with the unguided organoid protocol used by Jain and colleagues. Navigating the effect of this when interpreting results is not easy, but there are some consistent changes that have been observed. For example, Chiaradia and colleagues report that organoids with an initial proper morphology will then display the correct cellular identity regardless of culture conditions, but that Matrigel may help to set up this initial morphology. This report is consistent with the findings of Jain and colleagues that Matrigel helped to pattern the structure of early organoids, and with the findings of Martins-Costa and colleagues that Matrigel can accelerate the formation of correct tissue polarity.However, the exact role of the ECM compared with the other components of Matrigel is still an open question. Martins-Costa and colleagues report that the addition of purified mouse laminin 1 or collagen IV was not sufficient to replicate the effects of Matrigel, which is perhaps unsurprising given the complex mixture of components it contains. Jain and colleagues show that encapsulation of organoids in 0.6% agarose promotes neuroepithelium and lumen formation and affects organoid patterning by promoting more telencephalic fates. This organoid encapsulation can induce a similar phenotype to organoids grown with Matrigel, indicating that capture of molecules secreted by the organoid or the extracellular mechanical environment might also be one of the roles of the ECM. Matrigel is often used as a source of ECM, but is known to contain signalling factors that could also affect morphodynamics, many of which may interact with the ECM components. Untangling the role of ECM is further complicated by our incomplete understanding of what ECM is present in the fetal brain, hindering our ability to replicate it in organoids. Once this information is known, developing an ECM composition with a more spatial and temporal approach could help recreate a more in vivo-like situation and enable the role of ECM to be explored further. Synthetic, tuneable ECM may be a way to achieve this and could also be used to generate basement-membrane-like microenvironments in which to grow organoids.How the ECM shapes the developing neuroepithelium and how it is directly or indirectly linked to some of the major signalling pathways remains to be defined, but Jain and colleagues add exciting new data to the growing evidence that it contributes to the morphodynamics of brain development, in both organoids and tissue-based models (Jain et al., 2023 preprint; Chiaradia et al., 2023; Martins-Costa et al., 2023; Long et al., 2018; Long and Huttner, 2019).

SUMMARY Neocortex expansion during evolution is linked to higher numbers of neurons thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells (BPs) during development. Here we show that EREG , encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of BPs via EGFR-mediated signaling, whereas ablation of EREG in human cortical organoids reduces BP proliferation. Addition of EPIREGULIN to cortical organoids promotes a further increase in proliferation of gorilla but not human BPs. Finally, we identify putative cis-regulatory elements that may contribute to inter-species differences in EREG expression. Overall, our results suggest that species-specific expression of EPIREGULIN may contribute to increased neocortex size in primates by providing a pro-proliferative signal to BPs in the subventricular zone progenitor niche.

>The mammalian central nervous system (CNS) is highly complex,with a vast array of processes and interactions occurring in a dynamic and often transient manner.How these processes are combined to regulate our behavior remains poorly understood.This has in turn led to a lack of understanding of how these processes have gone awry in the many disorders of the nervous system.In order to address this,researchers need a controlled way to manipulate the nervous system in in vitro and ex vivo cultures,in both a specific area and for a specific period of time to start to pick apart these interactions.To date,this has been technically challenging,especially when modeling focal injury to the CNS or when working with human brain tissue.

Extracellular matrix (ECM) has long been known to regulate many aspects of neural development in many different species. However, the role of the ECM in the development of the human neocortex is not yet fully understood. In this review we discuss the role of the ECM in human neocortex development and the different model systems that can be used to investigate this. In particular, we will focus on how the ECM regulates human neural stem and progenitor cell proliferation and differentiation, how the ECM regulates the architecture of the developing human neocortex and the effect of mutations in ECM and ECM-associated genes in neurodevelopmental disorders.

How our brains have developed to perform the many complex functions that make us human has long remained a question of great interest. Over the last few decades, many scientists from a wide range of fields have tried to answer this question by aiming to uncover the mechanisms that regulate the development of the human neocortex. They have approached this on different scales, focusing microscopically on individual cells all the way up to macroscopically imaging entire brains within living patients. In this review we will summarise these key findings and how they fit together.

Protection or repair of the nigrostriatal pathway represents a principal disease-modifying therapeutic strategy for Parkinson's disease (PD). Glial cell line-derived neurotrophic factor (GDNF) holds great therapeutic potential for PD, but its efficacious delivery remains difficult. The aim of this study was to evaluate the potential of different biomaterials (hydrogels, microspheres, cryogels and microcontact printed surfaces) for reconstructing the nigrostriatal pathway in organotypic co-culture of ventral mesencephalon and dorsal striatum. The biomaterials (either alone or loaded with GDNF) were locally applied onto the brain co-slices and fiber growth between the co-slices was evaluated after three weeks in culture based on staining for tyrosine hydroxylase (TH). Collagen hydrogels loaded with GDNF slightly promoted the TH+ nerve fiber growth towards the dorsal striatum, while GDNF loaded microspheres embedded within the hydrogels did not provide an improvement. Cryogels alone or loaded with GDNF also enhanced TH+ fiber growth. Lines of GDNF immobilized onto the membrane inserts via microcontact printing also significantly improved TH+ fiber growth. In conclusion, this study shows that various biomaterials and tissue engineering techniques can be employed to regenerate the nigrostriatal pathway in organotypic brain slices. This comparison of techniques highlights the relative merits of different technologies that researchers can use/develop for neuronal regeneration strategies.

The neurodevelopmental phenotype in Down Syndrome (DS), or Trisomy 21, is variable including a wide spectrum of cognitive impairment and a high risk of early-onset Alzheimer's disease (AD). A key metabolite of interest within the brain in DS is Myo-inositol (mIns). The NA+/mIns co-transporter is located on human chromosome 21 and is overexpressed in DS. In adults with DS, elevated brain mIns was previously associated with cognitive impairment and proposed as a risk marker for progression to AD. However, it is unknown if brain mIns is increased earlier in development.The aim of this study was to estimate mIns concentration levels and key brain metabolites [N-acetylaspartate (NAA), Choline (Cho) and Creatine (Cr)] in the developing brain in DS and aged-matched controls. We used in vivo magnetic resonance spectroscopy (MRS) in neonates with DS (n = 12) and age-matched controls (n = 26) scanned just after birth (36–45 weeks postmenstrual age). Moreover, we used Mass Spectrometry in early (10–20 weeks post conception) ex vivo fetal brain tissue samples from DS (n = 14) and control (n = 30) cases.Relative to [Cho] and [Cr], we report elevated ratios of [mIns] in vivo in the basal ganglia/thalamus, in neonates with DS, when compared to age-matched typically developing controls. Glycine concentration ratios [Gly]/[Cr] and [Cho]/[Cr] also appear elevated. We observed elevated [mIns] in the ex vivo fetal cortical brain tissue in DS compared with controls.In conclusion, a higher level of brain mIns was evident as early as 10 weeks post conception and was measurable in vivo from 36 weeks post-menstrual age. Future work will determine if this early difference in metabolites is linked to cognitive outcomes in childhood or has utility as a potential treatment biomarker for early intervention.

The human brain has unique features that are difficult to study in animal models, including the mechanisms underlying neurodevelopmental and psychiatric disorders. Despite recent advances in human primary brain tissue culture systems, the use of these models to elucidate cellular disease mechanisms remains limited. A major reason for this is the lack of tools available to precisely manipulate a specific area of the tissue in a reproducible manner. Here we report an easy-to-use tool for site-specific manipulation of human brain tissue in culture. We show that line-shaped cryogel scaffolds synthesized with precise microscale dimensions allow the targeted delivery of a reagent to a specific region of human brain tissue in culture. 3-sulfopropyl acrylate (SPA) was incorporated into the cryogel network to yield a negative surface charge for the reversible binding of molecular cargo. The fluorescent dyes BODIPY and DiI were used as model cargos to show that placement of dye loaded scaffolds onto brain tissue in culture resulted in controlled delivery without a burst release, and labelling of specific regions without tissue damage. We further show that cryogels can deliver tetrodotoxin to tissue, inhibiting neuronal function in a reversible manner. The robust nature and precise dimensions of the cryogel resulted in a user-friendly and reproducible tool to manipulate primary human tissue cultures. These easy-to-use cryogels offer an innovate approach for more complex manipulations of ex-vivo tissue.

The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.

Cortical folding is a key feature of the evolutionary expansion of the neocortex. This folding is thought to allow the increase of the cortical surface area within the confinement of the developing skull. Despite its functional importance, the mechanisms that regulate the development of cortical folds have remained elusive. This chapter will discuss several important aspects of folding, including the timing of cortical fold development, the evolution of cortical folding, the cellular and mechanical mechanisms suggested to regulate folding, the model systems being used to study these mechanisms, and, finally, the neurodevelopmental disorders that affect it. While key advances have recently been made in the field, it is clear that several questions remain open, especially those regarding the function of cortical folding and mechanisms that regulate it.

Neocortex expansion is largely based on the proliferative capacity of basal progenitors (BPs), which is increased by extracellular matrix (ECM) components via integrin signaling. Here we show that the transcription factor Sox9 drives expression of ECM components and that laminin 211 increases BP proliferation in embryonic mouse neocortex. We show that Sox9 is expressed in human and ferret BPs and is required for BP proliferation in embryonic ferret neocortex. Conditional Sox9 expression in the mouse BP lineage, where it normally is not expressed, increases BP proliferation, reduces Tbr2 levels and induces Olig2 expression, indicative of premature gliogenesis. Conditional Sox9 expression also results in cell-non-autonomous stimulation of BP proliferation followed by increased upper-layer neuron production. Our findings demonstrate that Sox9 exerts concerted effects on transcription, BP proliferation, neuron production, and neurogenic vs. gliogenic BP cell fate, suggesting that Sox9 may have contributed to promote neocortical expansion.

The evolutionary expansion of the mammalian neocortex (Ncx) is thought to be linked to increased proliferative capacity of basal progenitors (BPs) and their neurogenic capacity. Here, by quantifying BP morphology in the developing Ncx of mouse, ferret, and human, we show that increased BP proliferative capacity is linked to an increase in BP process number. We identify human membrane-bound PALMDELPHIN (PALMD-Caax) as an underlying factor, and we show that it drives BP process growth and proliferation when expressed in developing mouse and ferret Ncx. Conversely, CRISPR/Cas9-mediated disruption of PALMD or its binding partner ADDUCIN-γ in fetal human Ncx reduces BP process numbers and proliferation. We further show that PALMD-induced processes enable BPs to receive pro-proliferative integrin-dependent signals. These findings provide a link between BP morphology and proliferation, suggesting that changes in BP morphology may have contributed to the evolutionary expansion of the Ncx.

During development, both cells and tissues must acquire the correct shape to allow their proper function. This is especially relevant in the nervous system, where the shape of individual cell processes, such as the axons and dendrites, and the shape of entire tissues, such as the folding of the neocortex, are highly specialized. While many aspects of neural development have been uncovered, there are still several open questions concerning the mechanisms governing cell and tissue shape. In this review, we discuss the role of the extracellular matrix (ECM) in these processes. In particular, we consider how the ECM regulates cell shape, proliferation, differentiation and migration, and more recent work highlighting a key role of ECM in the morphogenesis of neural tissues.

The expansion of the neocortex during mammalian evolution has been linked to an enlargement of the subventricular zone during cortical development and an increase in the proliferation of the basal progenitors residing therein. Here, we explored a potential role of YAP, the major downstream effector of the Hippo signaling pathway, in proliferation of basal progenitors. We show that YAP expression and activity are high in ferret and human basal progenitors, which are known to exhibit high proliferative capacity, but low in mouse basal progenitors, which lack such capacity. To induce YAP activity in mouse basal progenitors, we expressed a constitutively active YAP (CA-YAP). This resulted in an increase in proliferation of basal progenitor. In addition, CA-YAP expressing mouse basal progenitors promoted the production of upper-layer neurons. To investigate if YAP is required for the proliferation of basal progenitors, we pharmacologically interfered with the function of YAP in the developing ferret and human neocortex. This resulted in a decrease of cycling basal progenitors. In concert, genetical interference with the function of YAP in ferret developing neocortex resulted in decreased abundance of basal progenitors. Together, our data indicate that YAP promotes the proliferation of basal progenitors and suggest that changes in YAP activity levels contributed to the evolutionary expansion of the neocortex.

The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. Consequently, the postnatal ferret neocortex exhibits increased neuron density in the upper cortical layers and expands in both the radial and tangential dimensions. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in the developing ferret neocortex.