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Diabetic retinopathy is a common complication of diabetes and remains the leading cause of blindness among the working-age population. For decades, diabetic retinopathy was considered only a microvascular complication, but the retinal microvasculature is intimately associated with and governed by neurons and glia, which are affected even prior to clinically detectable vascular lesions. While progress has been made to improve the vascular alterations, there is still no treatment to counteract the early neuro-glial perturbations in diabetic retinopathy. Diabetes is a complex metabolic disorder, characterized by chronic hyperglycemia along with dyslipidemia, hypoinsulinemia and hypertension. Increasing evidence points to inflammation as one key player in diabetes-associated retinal perturbations, however, the exact underlying molecular mechanisms are not yet fully understood. Interlinked molecular pathways, such as oxidative stress, formation of advanced glycation end-products and increased expression of vascular endothelial growth factor have received a lot of attention as they all contribute to the inflammatory response. In the current review, we focus on the involvement of inflammation in the pathophysiology of diabetic retinopathy with special emphasis on the functional relationships between glial cells and neurons. Finally, we summarize recent advances using novel targets to inhibit inflammation in diabetic retinopathy.

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases—the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear. Although Hippo signaling restricts regeneration in many mammalian organs, the pharmaceutical tools available to modulate the pathway have been limited. Here, the authors report a small molecule that may inhibit a key element in the Hippo cascade and may activate regenerative responses in several mammalian tissues.

The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development. Eze et al. use single-cell sequencing and immunohistochemical validation to create an atlas of early human brain development. In the telencephalon, they discover a diversity of progenitor subtypes, including two that are enriched in humans.

Outer radial glial (oRG) cells are a population of neural stem cells prevalent in the developing human cortex that contribute to its cellular diversity and evolutionary expansion. The mammalian Target of Rapamycin (mTOR) signaling pathway is active in human oRG cells. Mutations in mTOR pathway genes are linked to a variety of neurodevelopmental disorders and malformations of cortical development. We find that dysregulation of mTOR signaling specifically affects oRG cells, but not other progenitor types, by changing the actin cytoskeleton through the activity of the Rho-GTPase, CDC42. These effects change oRG cellular morphology, migration, and mitotic behavior, but do not affect proliferation or cell fate. Thus, mTOR signaling can regulate the architecture of the developing human cortex by maintaining the cytoskeletal organization of oRG cells and the radial glia scaffold. Our study provides insight into how mTOR dysregulation may contribute to neurodevelopmental disease.

Abstract The human neocortex has undergone strong evolutionary expansion, largely due to an increased progenitor population, the basal radial glial cells. These cells are responsible for the production of a diversity of cell types, but the successive cell fate decisions taken by individual progenitors remain unknown. Here we developed a semi-automated live/fixed correlative imaging method to map basal radial glial cell division modes in early fetal tissue and cerebral organoids. Through the live analysis of hundreds of dividing progenitors, we show that basal radial glial cells undergo abundant symmetric amplifying divisions, and frequent self-consuming direct neurogenic divisions, bypassing intermediate progenitors. These direct neurogenic divisions are more abundant in the upper part of the subventricular zone. We furthermore demonstrate asymmetric Notch activation in the self-renewing daughter cells, independently of basal fibre inheritance. Our results reveal a remarkable conservation of fate decisions in cerebral organoids, supporting their value as models of early human neurogenesis.

Background Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that can regulate molecular and cellular processes in brain development and disease. LncRNAs exhibit cell type- and tissue-specific expression, but little is known about the expression and function of lncRNAs in the developing human brain. Furthermore, it has been unclear whether lncRNAs are highly expressed in subsets of cells within tissues, despite appearing lowly expressed in bulk populations. Results We use strand-specific RNA-seq to deeply profile lncRNAs from polyadenylated and total RNA obtained from human neocortex at different stages of development, and we apply this reference to analyze the transcriptomes of single cells. While lncRNAs are generally detected at low levels in bulk tissues, single-cell transcriptomics of hundreds of neocortex cells reveal that many lncRNAs are abundantly expressed in individual cells and are cell type-specific. Notably, LOC646329 is a lncRNA enriched in single radial glia cells but is detected at low abundance in tissues. CRISPRi knockdown of LOC646329 indicates that this lncRNA regulates cell proliferation. Conclusion The discrete and abundant expression of lncRNAs among individual cells has important implications for both their biological function and utility for distinguishing neural cell types.

Emerging evidence suggests that new cells, including neurons, can be generated within the adult hypothalamus, suggesting the existence of a local neural stem/progenitor cell niche. Here, we identify α-tanycytes as key components of a hypothalamic niche in the adult mouse. Long-term lineage tracing in vivo using a GLAST::CreER T2 conditional driver indicates that α-tanycytes are self-renewing cells that constitutively give rise to new tanycytes, astrocytes and sparse numbers of neurons. In vitro studies demonstrate that α-tanycytes, but not β-tanycytes or parenchymal cells, are neurospherogenic. Distinct subpopulations of α-tanycytes exist, amongst which only GFAP-positive dorsal α2-tanycytes possess stem-like neurospherogenic activity. Fgf-10 and Fgf-18 are expressed specifically within ventral tanycyte subpopulations; α-tanycytes require fibroblast growth factor signalling to maintain their proliferation ex vivo and elevated fibroblast growth factor levels lead to enhanced proliferation of α-tanycytes in vivo. Our results suggest that α-tanycytes form the critical component of a hypothalamic stem cell niche, and that local fibroblast growth factor signalling governs their proliferation. De novo neurogenesis occurs within the adult hypothalamus, suggesting the existence of a local neural stem cell niche. Here, the authors show that α-tanycytes lining the third ventricle are self-renewing multipotent neural stem cells, whose proliferation is dependent upon local FGF signalling.

The mechanisms governing the expansion of neuron number in specific brain regions are still poorly understood. Enlarged neuron numbers in different species are often anticipated by increased numbers of progenitors dividing in the subventricular zone. Here we present live imaging analysis of radial glial cells and their progeny in the ventral telencephalon, the region with the largest subventricular zone in the murine brain during neurogenesis. We observe lineage amplification by a new type of progenitor, including bipolar radial glial cells dividing at subapical positions and generating further proliferating progeny. The frequency of this new type of progenitor is increased not only in larger clones of the mouse lateral ganglionic eminence but also in cerebral cortices of gyrated species, and upon inducing gyrification in the murine cerebral cortex. This implies key roles of this new type of radial glia in ontogeny and phylogeny. Amplification of neural progenitor cells mediates expansion of brain regions. Using imaging of progenitor cell amplification in the mouse ventral forebrain, the authors identify a new progenitor type with high frequency, which they also show to be present in expanded brain regions of other species.

Humans have evolved an extraordinarily expanded and complex cerebral cortex associated with developmental and gene regulatory modifications1, 2–3. Human accelerated regions (HARs) are highly conserved DNA sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to species-specific cortical development remains largely unknown4,5. HARE5 is a HAR transcriptional enhancer of the WNT signalling receptor Frizzled8 that is active during brain development6. Here, using genome-edited mouse (Mus musculus, Mm) and primate models, we demonstrated that human (Homo sapiens, Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacities of neural progenitor cells. Hs-HARE5 knock-in mice have significantly enlarged neocortices, containing more excitatory neurons. By measuring neural dynamics in vivo, we showed that these anatomical features result in increased functional independence between cortical regions. We assessed underlying developmental mechanisms using fixed and live imaging, lineage analysis and single-cell RNA sequencing. We discovered that Hs-HARE5 modifies radial glial cell behaviour, with increased self-renewal at early developmental stages, followed by expanded neurogenic potential. Using genome-edited human and chimpanzee (Pan troglodytes, Pt) neural progenitor cells and cortical organoids, we showed that four human-specific variants of Hs-HARE5 drive increased enhancer activity that promotes progenitor proliferation. Finally, we showed that Hs-HARE5 increased progenitor proliferation by amplifying canonical WNT signalling. These findings illustrate how small changes in regulatory DNA can directly affect critical signalling pathways to modulate brain development. Our study uncovered new functions of HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.HARE5, a human accelerated region enhancer, modulates cortical development by influencing neural progenitor cell behaviour, leading to an enlarged neocortex with increased functional independence between cortical regions through amplified WNT signalling.

Developing precise targeted cell ablation in the axolotl ( Ambystoma mexicanum ) is crucial for elucidating the roles or interactions of specific cell types in regeneration and modeling diseases. Here we establish a Nitroreductase (NTR)-based inducible cell ablation system in axolotls. Through generation of Sox2:Cherry-NTR knock-in axolotls, we achieve efficient ablation of ependymoglial cells (EGCs) in the central nervous system. Combined spinal cord and brain transplantation and injury models demonstrate regeneration failure upon EGC depletion, suggesting that EGCs are sole source of central nervous system regeneration. Additionally, EGC ablation in the spinal cord resulted in delayed tail regeneration. Moreover, we establish NeuroD6:Cherry-NTR and NeuroD6:Cherry-NTR2.0 knock-in lines to ablate postmitotic cortical neurons, enable the investigation of brain regeneration after large-scale neuronal depletion. We found that NTR2.0 (but not NTR) leads to elimination of >95% of targeted neurons in the dorsal pallium. All lost neuronal subtypes are chronologically regenerated with laminar-like distribution mirroring developmental patterning. Finally, we create Cre-LoxP-based conditional NTR2.0 transgenic axolotls using a constitutive CAGGs promoter, enabling tissue-specific ablation of specific cell types when crossed to existing Cre lines. In summary, our study establishes an efficient and versatile targeted cell ablation system in axolotls, providing a valuable tool for deep dissection of tissue regeneration in axolotls. Here they establish an NTR-based genetic ablation tool in axolotls to investigate the function of specific cells. They use this system to demonstrate the capacity of ependymoglial cells for central nervous system regeneration.