Ependyma have been proposed as adult neural stem cells that provide the majority of newly proliferated scar-forming astrocytes that protect tissue and function after spinal cord injury (SCI). This proposal was based on small, midline stab SCI. Here, we tested the generality of this proposal by using a genetic knock-in cell fate mapping strategy in different murine SCI models. After large crush injuries across the entire spinal cord, ependyma-derived progeny remained local, did not migrate and contributed few cells of any kind and less than 2%, if any, of the total newly proliferated and molecularly confirmed scar-forming astrocytes. Stab injuries that were near to but did not directly damage ependyma, contained no ependyma-derived cells. Our findings show that ependymal contribution of progeny after SCI is minimal, local and dependent on direct ependymal injury, indicating that ependyma are not a major source of endogenous neural stem cells or neuroprotective astrocytes after SCI.

The stem cell source of neural and glial progenitors in the periventricular regions of the adult forebrain has remained uncertain and controversial. Using a cell specific genetic approach we rule out Foxj1+ ependymal cells as stem cells participating in neurogenesis and gliogenesis in response to acute injury or stroke in the mouse forebrain. Non stem-and progenitor-like responses of Foxj1+ ependymal cells to injury and stroke remain to be defined and investigated.Past reports have suggested that adult ependymal cells (ECs), or a subpopulation thereof, have endogenous stem cell potential with the ability to generate new neurons for the olfactory bulbs (OBs) and in response to stroke in the mouse forebrain. In one study, intraventricular injections of adeno or lenti viruses driving expression of reporters downstream of the human FOXJ1 promoter resulted in labeling of new cells generated from transduced cells only after induction of stroke but not in naïve adult mice 1 . The same human promoter element was used in a subsequent study leading the authors to postulate substantial plasticity in the EC lineage and their relationship to nearby astrocytes 2 . The same promoter was also cloned into a reporter piggyback vector and electroporated into the rat brain, resulting in lineage-traced cells in the olfactory bulbs after 6 or 12 weeks in both healthy and stroke-induced brains through medial cerebral artery occlusions (MCAO) 3 . In other studies of the spinal cord, similar lineage tracing approaches were utilized to show that a substantial portion of the glial scar in damaged spinal cords come from ECs 4-6 presumably due to their extensive proliferation 7 .Concerned that the human promoter element utilized in the past studies (a ~ 1 kb upstream human FOXJ1 locus) was resulting in ectopic expression patterns, we generated a knock-in Foxj1 creERT2::GFP mouse to lineage-trace potential EC progeny from the endogenous locus. This line has been characterized 8 and was used in a recent study illustrating that spinal cord injury fails to induce Foxj1+ ECs to proliferate or to substantially contribute new cells to the glial scar 9 . To test the possibility that damage or stroke in the forebrain may contribute to the reported transformation of ependyma into neurogenic or gliogenic progenitors, a stab injury and three distinct stroke models were employed. ResultsRecombination was induced by tamoxifen administration (TAM) in naïve and experimental mice, and cre-dependent expression of tdTomato (tdTom) was quantified using the well-established Ai9 reporter allele. In experimental animals, TAM was administered daily at postnatal day 39 (P39) for five days in young adult mice, stab injuries were inflicted in the motor cortex on the forth day of TAM administration (at P42), followed by perfusion and analysis two weeks later at P56 (Fig. 1a). Two weeks post-injury is a well-established time line for neurogenic and gliogenic responses to injury and stroke based on numerous past studies [10][11][12] . Sectioning and microscopic analysis...

Angiogenesis is a key feature of central nervous system injury. A neovessel-derived signal mediated by prostacyclin triggers axonal sprouting and functional recovery in a mouse model of inflammatory spinal cord injury (pages 1658–1664). Are such angiocrine signals relevant to neurovascular remodeling and recovery in other neurological contexts?

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA-sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically up-regulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

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