Marcus Hooper

. Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.

Regenerative neuroscience aims to stimulate endogenous repair in the nervous system to replace neurons lost from degenerative diseases. Recently, we reported that overexpressing the transcription factor Ascl1 in Müller glia (MG) is sufficient to stimulate MG to regenerate functional neurons in the adult mouse retina. However, this process is inefficient, and only a third of the Ascl1-expressing MG generate new neurons. Here, we test whether proneural transcription factors of the Atoh1/7 class can further promote the regenerative capacity of MG. We find that the combination of Ascl1:Atoh1 is remarkably efficient at stimulating neurogenesis, even in the absence of retinal injury. Using electrophysiology and single-cell RNA sequencing (scRNA-seq), we demonstrate that Ascl1:Atoh1 generates a diversity of retinal neuron types, with the majority expressing characteristics of retinal ganglion cells. Our results provide a proof of principle that combinations of developmental transcription factors can substantially improve glial reprogramming to neurons and expand the repertoire of regenerated cell fates.

Müller glia (MG) serve as sources for retinal regeneration in non-mammalian vertebrates. We find that this process can be induced in mouse MG, after injury, by transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are generated only from a subset of MG. Identifying factors that limit Ascl1-mediated MG reprogramming could make this process more efficient. In this study, we test whether injury-induced STAT activation hampers the ability of Ascl1 to reprogram MG into retinal neurons. Single-cell RNA-seq shows that progenitor-like cells derived from Ascl1-expressing MG have a higher level of STAT signaling than do those cells that become neurons. Ascl1-ChIPseq and ATAC-seq show that STAT potentially directs Ascl1 to developmentally inappropriate targets. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of MG to generate neurons.

The innate immune system plays key roles in tissue regeneration. For example, microglia promote neurogenesis in Müller glia in birds and fish after injury. Although mammalian retina does not normally regenerate, neurogenesis can be induced in mouse Müller glia by Ascl1, a proneural transcription factor. We show that in mice, microglia inhibit the Ascl1-mediated retinal regeneration, suggesting that the innate immune system limits the regenerative response to injury.

Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in nonmammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. Here, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in Müller glia, including Ascl1, Pou4f2, Islet1, and Atoh1. Using immunohistochemistry, single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing, and electrophysiology, we find that retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental retinal ganglion cell TFs.