Müller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Müller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Müller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Müller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Müller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Müller glia in young mice, where the Ascl1-expressing Müller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Müller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Müller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1.reprogramming | glia | regeneration | neurogenesis | eye

SummaryThe activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. Here we show that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate of hippocampal stem cells, and inactivating Ascl1 blocks quiescence exit completely, rendering them unresponsive to activating stimuli. Ascl1 promotes the proliferation of hippocampal stem cells by directly regulating the expression of cell-cycle regulatory genes. Ascl1 is similarly required for stem cell activation in the adult subventricular zone. Our results support a model whereby Ascl1 integrates inputs from both stimulatory and inhibitory signals and converts them into a transcriptional program activating adult neural stem cells.

Highlights d LTi progenitors originate from early embryonic hemogenic endothelium, excluding yolk sac d The fetal liver harbors proliferating LTi precursor populations d LTi maturation occurs in the periphery into two LTi 4 populations d Embryonic LTi are replaced by HSC-derived LTi cells in adults

Neuronal nerve processes in the tumor microenvironment were highlighted recently. However, the origin of intra-tumoral nerves remains poorly known, in part because of technical difficulties in tracing nerve fibers via conventional histological preparations. Here, we employ three-dimensional (3D) imaging of cleared tissues for a comprehensive analysis of sympathetic innervation in a murine model of pancreatic ductal adenocarcinoma (PDAC). Our results support two independent, but coexisting, mechanisms: passive engulfment of pre-existing sympathetic nerves within tumors plus an active, localized sprouting of axon terminals into non-neoplastic lesions and tumor periphery. Ablation of the innervating sympathetic nerves increases tumor growth and spread. This effect is explained by the observation that sympathectomy increases intratumoral CD163+ macrophage numbers, which contribute to the worse outcome. Altogether, our findings provide insights into the mechanisms by which the sympathetic nervous system exerts cancer-protective properties in a mouse model of PDAC.

Perivascular macrophages (pvMs) are associated with cerebral vasculature and mediate brain drainage and immune regulation. Here, using reporter mouse models, whole brain and section immunofluorescence, flow cytometry, and single cell RNA sequencing, besides the Lyve1+F4/80+CD206+CX3CR1+ pvMs, we identify a CX3CR1– pvM population that shares phagocytic functions and location. Furthermore, the brain parenchyma vasculature mostly hosts Lyve1+MHCII– pvMs with low to intermediate CD45 expression. Using the double Cx3cr1GFP x Cx3cr1-Cre;RosatdT reporter mice for finer mapping of the lineages, we establish that CD45lowCX3CR1– pvMs are derived from CX3CR1+ precursors and require PU.1 during their ontogeny. In parallel, results from the Cxcr4-CreErt2;Rosa26tdT lineage tracing model support a bone marrow-independent replenishment of all Lyve1+ pvMs in the adult mouse brain. Lastly, flow cytometry and 3D immunofluorescence analysis uncover increased percentage of pvMs following photothrombotic induced stroke. Our results thus show that the parenchymal pvM population is more heterogenous than previously described, and includes a CD45low and CX3CR1– pvM population.

Background The innate lymphoid cell (ILC) family consists of NK cells, ILC type 1, 2, 3 and lymphoid tissue inducer cells. They have been shown to play important roles in homeostasis and immune responses and are generally considered tissue resident. Not much is known about the presence of ILC members within the central nervous system and whether they are tissue resident in this organ too. Therefore, we studied the presence of all ILC members within the central nervous system and after ischemic brain insult. Methods We used the photothrombotic ischemic lesion method to induce ischemic lesions within the mouse brain. Using whole-mount immunofluorescence imaging, we established that the ILCs were present at the rim of the lesion. We quantified the increase of all ILC members at different time-points after the ischemic lesion induction by flow cytometry. Their migration route via chemokine CXCL12 was studied by using different genetic mouse models, in which we induced deletion of Cxcl12 within the blood–brain barrier endothelium, or its receptor, Cxcr4, in the ILCs. The functional role of the ILCs was subsequently established using the beam-walk sensorimotor test. Results Here, we report that ILCs are not resident within the mouse brain parenchyma during steady-state conditions, but are attracted towards the ischemic stroke. Specifically, we identify NK cells, ILC1s, ILC2s and ILC3s within the lesion, the highest influx being observed for NK cells and ILC1s. We further show that CXCL12 expressed at the blood–brain barrier is essential for NK cells and NKp46+ ILC3s to migrate toward the lesion. Complementary, Cxcr4-deficiency in NK cells prevents NK cells from entering the infarct area. Lack of NK cell migration results in a higher neurological deficit in the beam-walk sensorimotor test. Conclusions This study establishes the lack of ILCs in the mouse central nervous system at steady-state and their migration towards an ischemic brain lesion. Our data show a role for blood–brain barrier-derived CXCL12 in attracting protective NK cells to ischemic brain lesions and identifies a new CXCL12/CXCR4-mediated component of the innate immune response to stroke.

Innate lymphoid cells (ILCs) have a function in homeostasis and immune responses, but their role in ischemic brain insult is unknown. Here, we report that ILCs are not resident within the brain parenchyma during steady-state conditions, but are attracted after ischemic stroke. Specifically, we identified NK cells, ILC1s, ILC2s and ILC3s within the lesion, the highest influx being observed for NK cells and ILC1s. We further show that Cxcl12 expressed at the blood-brain barrier is essential for NK cells and NKp46+ ILC3s to migrate toward the lesion. Complementary, Cxcr4-deficiency in NK cells prevented NK cells from entering the infarct area. The lack of NK cell migration resulted in a higher neurological deficit in the beam-walk sensorimotor test. Our data show a new role for blood-brain barrier-derived Cxcl12 in attracting protective NK cells to ischemic brain lesions and identifies a new Cxcl12/ Cxcr4-mediated component of the innate immune response to stroke.