Glial progenitor heterogeneity and key regulators revealed by single-cell RNA sequencing provide insight to regeneration in spinal cord injury.

Recent studies have revealed the heterogeneous nature of astrocytes; however, how diverse constituents of astrocyte-lineage cells are regulated in adult spinal cord after injury and contribute to regeneration remains elusive. We perform single-cell RNA sequencing of GFAP-expressing cells from sub-chronic spinal cord injury models and identify and compare with the subpopulations in acute-stage data. We find subpopulations with distinct functional enrichment and their identities defined by subpopulation-specific transcription factors and regulons. Immunohistochemistry, RNAscope experiments, and quantification by stereology verify the molecular signature, location, and morphology of potential resident neural progenitors or neural stem cells in the adult spinal cord before and after injury and uncover the populations of the intermediate cells enriched in neuronal genes that could potentially transition into other subpopulations. This study has expanded the knowledge of the heterogeneity and cell state transition of glial progenitors in adult spinal cord before and after injury.

Transcriptome of rat subcortical white matter and spinal cord after spinal injury and cortical stimulation.

Spinal cord injury disrupts ascending and descending neural signals causing sensory and motor dysfunction. Neuromodulation with electrical stimulation is used in both clinical and research settings to induce neural plasticity and improve functional recovery following spinal trauma. However, the mechanisms by which electrical stimulation affects recovery remain unclear. In this study we examined the effects of cortical electrical stimulation following injury on transcription at several levels of the central nervous system. We performed a unilateral, incomplete cervical spinal contusion injury in rats and delivered stimulation for one week to the contralesional motor cortex to activate the corticospinal tract and other pathways. RNA was purified from bilateral subcortical white matter and 3 levels of the spinal cord. Here we provide the complete data set in the hope that it will be useful for researchers studying electrical stimulation as a therapy to improve recovery from the deficits associated with spinal cord injury.

Pancreatic Cell Fate Determination Relies on Notch Ligand Trafficking by NFIA.

Notch is activated globally in pancreatic progenitors; however, for progenitors to differentiate into endocrine cells, they must escape Notch activation to express Neurogenin-3. Here, we find that the transcription factor nuclear factor I/A (NFIA) promotes endocrine development by regulating Notch ligand Dll1 trafficking. Pancreatic deletion of NFIA leads to cell fate defects, with increased duct and decreased endocrine formation, while ectopic expression promotes endocrine formation in mice and human pancreatic progenitors. NFIA-deficient mice exhibit dysregulation of trafficking-related genes including increased expression of Mib1, which acts to target Dll1 for endocytosis. We find that NFIA binds to the Mib1 promoter, with loss of NFIA leading to an increase in Dll1 internalization and enhanced Notch activation with rescue of the cell fate defects after Mib1 knockdown. This study reveals NFIA as a pro-endocrine factor in the pancreas, acting to repress Mib1, inhibit Dll1 endocytosis and thus promote escape from Notch activation.

Glia-specific enhancers and chromatin structure regulate NFIA expression and glioma tumorigenesis.

Long-range enhancer interactions critically regulate gene expression, yet little is known about how their coordinated activities contribute to CNS development or how this may, in turn, relate to disease states. By examining the regulation of the transcription factor NFIA in the developing spinal cord, we identified long-range enhancers that recapitulate NFIA expression across glial and neuronal lineages in vivo. Complementary genetic studies found that Sox9-Brn2 and Isl1-Lhx3 regulate enhancer activity and NFIA expression in glial and neuronal populations. Chromatin conformation analysis revealed that these enhancers and transcription factors form distinct architectures within these lineages in the spinal cord. In glioma models, the glia-specific architecture is present in tumors, and these enhancers are required for NFIA expression and contribute to glioma formation. By delineating three-dimensional mechanisms of gene expression regulation, our studies identify lineage-specific chromatin architectures and associated enhancers that regulate cell fate and tumorigenesis in the CNS.

Temporal Profiling of Astrocyte Precursors Reveals Parallel Roles for Asef during Development and after Injury.

Lineage development is a stepwise process, governed by stage-specific regulatory factors and associated markers. Astrocytes are one of the principle cell types in the CNS and the stages associated with their development remain very poorly defined. To identify these stages, we performed gene-expression profiling on astrocyte precursor populations in the spinal cord, identifying distinct patterns of gene induction during their development that are strongly correlated with human astrocytes. Validation studies identified a new cohort of astrocyte-associated genes during development and demonstrated their expression in reactive astrocytes in human white matter injury (WMI). Functional studies on one of these genes revealed that mice lacking Asef exhibited impaired astrocyte differentiation during development and repair after WMI, coupled with compromised blood-brain barrier integrity in the adult CNS. These studies have identified distinct stages of astrocyte lineage development associated with human WMI and, together with our functional analysis of Asef, highlight the parallels between astrocyte development and their reactive counterparts associated with injury. SIGNIFICANCE STATEMENT: Astrocytes play a central role in CNS function and associated diseases. Yet the mechanisms that control their development remain poorly defined. Using the developing mouse spinal cord as a model system, we identify molecular changes that occur in developing astrocytes. These molecular signatures are strongly correlated with human astrocyte expression profiles and validation in mouse spinal cord identifies a host of new genes associated with the astrocyte lineage. These genes are present in reactive astrocytes in human white matter injury, and functional studies reveal that one of these genes, Asef, contributes to reactive astrocyte responses after injury. These studies identify distinct stages of astrocyte lineage development and highlight the parallels between astrocyte development and their reactive counterparts associated with injury.

Daam2-PIP5K is a regulatory pathway for Wnt signaling and therapeutic target for remyelination in the CNS.

Wnt signaling plays an essential role in developmental and regenerative myelination of the CNS; however, contributions of proximal regulators of the Wnt receptor complex to these processes remain undefined. To identify components of the Wnt pathway that regulate these processes, we applied a multifaceted discovery platform and found that Daam2-PIP5K comprise a novel pathway regulating Wnt signaling and myelination. Using dorsal patterning of the chick spinal cord we found that Daam2 promotes Wnt signaling and receptor complex formation through PIP5K-PIP2. Analysis of Daam2 function in oligodendrocytes (OLs) revealed that it suppresses OL differentiation during development, after white matter injury (WMI), and is expressed in human white matter lesions. These findings suggest a pharmacological strategy to inhibit Daam2-PIP5K function, application of which stimulates remyelination after WMI. Put together, our studies integrate information from multiple systems to identify a novel regulatory pathway for Wnt signaling and potential therapeutic target for WMI.

Astrocyte form and function in the developing central nervous system.

Astrocytes have long been forgotten entities in our quest to understand brain function. Over the last few decades, there has been an exponential increase in our knowledge of central nervous system (CNS) function, and, consequently, astrocytes have emerged as key figures in CNS physiology and disease. Indeed, several pediatric neurologic disorders have recently been linked to astrocyte dysregulation, including leukodystrophies, autism spectrum disorders, and epilepsy. Given that pediatric disorders are rooted in developmental processes, the goal of this review is to catalog what we know about astrocyte development and function in the developing CNS. Moreover, we highlight current challenges and questions that remain in the field about astrocyte development. Our hope is that this review illuminates the potential of astrocytes and their associated developmental and physiological functions as potential therapeutic targets for the treatment of neurologic disorders.

Expression profiling of Aldh1l1-precursors in the developing spinal cord reveals glial lineage-specific genes and direct Sox9-Nfe2l1 interactions.

Developmental regulation of gliogenesis in the mammalian CNS is incompletely understood, in part due to a limited repertoire of lineage-specific genes. We used Aldh1l1-GFP as a marker for gliogenic radial glia and later-stage precursors of developing astrocytes and performed gene expression profiling of these cells. We then used this dataset to identify candidate transcription factors that may serve as glial markers or regulators of glial fate. Our analysis generated a database of developmental stage-related markers of Aldh1l1+ cells between murine embryonic day 13.5-18.5. Using these data we identify the bZIP transcription factor Nfe2l1 and demonstrate that it promotes glial fate under direct Sox9 regulatory control. Thus, this dataset represents a resource for identifying novel regulators of glial development.

Developmental origins of astrocyte heterogeneity: the final frontier of CNS development.

Astrocytes are the most abundant cell type in the central nervous system, have diverse physiological roles in both health and disease, and exhibit phenotypic heterogeneity. In spite of the overwhelming evidence that astrocytes are a diverse population, there has been relatively little consideration of their molecular heterogeneity. In this review, we will summarize what is known about the heterogeneity of astrocytes and outline challenges that have limited studies understanding their molecular diversity. Approaches that have sought to overcome these limitations will be discussed, with an emphasis on recent progress in the field of developmental gliogenesis, which has revealed that positional identity during embryogenesis is an organizing feature of astrocyte diversity. These recent findings, coupled with emerging technologies that allow for direct isolation of astrocyte populations, have led us to propose that approaches rooted in astrocyte development may be the key to unlocking this immense, untapped diversity.

Label-free analysis of breast tissue polarity by Raman imaging of lipid phase.

The formation of the basoapical polarity axis in epithelia is critical for maintaining the homeostasis of differentiated tissues. Factors that influence cancer development notoriously affect tissue organization. Apical polarity appears as a specific tissue feature that, once disrupted, would facilitate the onset of mammary tumors. Thus, developing means to rapidly measure apical polarity alterations would greatly favor screening for factors that endanger the breast epithelium. A Raman scattering-based platform was used for label-free determination of apical polarity in live breast glandular structures (acini) produced in three-dimensional cell culture. The coherent anti-Stokes Raman scattering signal permitted the visualization of the apical and basal surfaces of an acinus. Raman microspectroscopy subsequently revealed that polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca(2+)-chelator EGTA. This method overcame variation between different cultures by tracking the status of apical polarity longitudinally for the same acini. Therefore, the disruption of apical polarity by a dietary breast cancer risk factor, ω6 fatty acid, could be observed with this method, even when the effect was too moderate to permit a conclusive assessment by the traditional immunostaining method.

Factors necessary to produce basoapical polarity in human glandular epithelium formed in conventional and high-throughput three-dimensional culture: example of the breast epithelium.

BACKGROUND: Basoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated. RESULTS: We demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity. CONCLUSION: These results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.