The laminar position, morphology, and gene expression profiles of cortical astrocytes are influenced by time of birth from ventricular/subventricular progenitors.

Astrocytes that reside in superficial (SL) and deep cortical layers have distinct molecular profiles and morphologies, which may underlie specific functions. Here, we demonstrate that the production of SL and deep layer (DL) astrocyte populations from neural progenitor cells in the mouse is temporally regulated. Lineage tracking following in utero and postnatal electroporation with PiggyBac (PB) EGFP and birth dating with EdU and FlashTag, showed that apical progenitors produce astrocytes during late embryogenesis (E16.5) that are biased to the SL, while postnatally labeled (P0) astrocytes are biased to the DL. In contrast, astrocytes born during the predominantly neurogenic window (E14.5) showed a random distribution in the SL and DL. Of interest, E13.5 astrocytes birth dated at E13.5 with EdU showed a lower layer bias, while FT labeling of apical progenitors showed no bias. Finally, examination of the morphologies of "biased" E16.5- and P0-labeled astrocytes demonstrated that E16.5-labeled astrocytes exhibit different morphologies in different layers, while P0-labeled astrocytes do not. Differences based on time of birth are also observed in the molecular profiles of E16.5 versus P0-labeled astrocytes. Altogether, these results suggest that the morphological, molecular, and positional diversity of cortical astrocytes is related to their time of birth from ventricular/subventricular zone progenitors.

Ectopic Expression of Neurod1 Is Sufficient for Functional Recovery following a Sensory-Motor Cortical Stroke.

Stroke is the leading cause of adult disability worldwide. The majority of stroke survivors are left with devastating functional impairments for which few treatment options exist. Recently, a number of studies have used ectopic expression of transcription factors that direct neuronal cell fate with the intention of converting astrocytes to neurons in various models of brain injury and disease. While there have been reports that question whether astrocyte-to-neuron conversion occurs in vivo, here, we have asked if ectopic expression of the transcription factor Neurod1 is sufficient to promote improved functional outcomes when delivered in the subacute phase following endothelin-1-induced sensory-motor cortex stroke. We used an adeno-associated virus to deliver Neurod1 from the short GFAP promoter and demonstrated improved functional outcomes as early as 28 days post-stroke and persisting to at least 63 days post-stroke. Using Cre-based cell fate tracking, we showed that functional recovery correlated with the expression of neuronal markers in transduced cells by 28 days post-stroke. By 63 days post-stroke, the reporter-expressing cells comprised ~20% of all the neurons in the perilesional cortex and expressed markers of cortical neuron subtypes. Overall, our findings indicate that ectopic expression of Neurod1 in the stroke-injured brain is sufficient to enhance neural repair.

Integrating single-cell and spatially resolved transcriptomic strategies to survey the astrocyte response to stroke in male mice.

Astrocytes, a type of glial cell in the central nervous system (CNS), adopt diverse states in response to injury that are influenced by their location relative to the insult. Here, we describe a platform for spatially resolved, single-cell transcriptomics and proteomics, called tDISCO (tissue-digital microfluidic isolation of single cells for -Omics). We use tDISCO alongside two high-throughput platforms for spatial (Visium) and single-cell transcriptomics (10X Chromium) to examine the heterogeneity of the astrocyte response to a cortical ischemic stroke in male mice. We show that integration of Visium and 10X Chromium datasets infers two astrocyte populations, proximal or distal to the injury site, while tDISCO determines the spatial boundaries and molecular profiles that define these populations. We find that proximal astrocytes show differences in lipid shuttling, with enriched expression of Apoe and Fabp5. Our datasets provide a resource for understanding the roles of astrocytes in stroke and showcase the utility of tDISCO for hypothesis-driven, spatially resolved single-cell experiments.