Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants

ABSTRACT Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated owing to lack of appropriate genetic tools. Here, we report a Split GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, through their dynamic control of the Notch ligand Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems.

Transcriptional Control of Gene Expression and the Heterogeneous Cellular Identity of Erythroblastic Island Macrophages

During definitive erythropoiesis, maturation of erythroid progenitors into enucleated reticulocytes requires the erythroblastic island (EBI) niche comprising a central macrophage attached to differentiating erythroid progenitors. Normally, the macrophage provides a nurturing environment for maturation of erythroid cells. Its critical physiologic importance entails aiding in recovery from anemic insults, such as systemic stress or acquired disease. Considerable interest in characterizing the central macrophage of the island niche led to the identification of putative cell surface markers enriched in island macrophages, enabling isolation and characterization. Recent studies focus on bulk and single cell transcriptomics of the island macrophage during adult steady-state erythropoiesis and embryonic erythropoiesis. They reveal that the island macrophage is a distinct cell type but with widespread cellular heterogeneity, likely suggesting distinct developmental origins and biological function. These studies have also uncovered transcriptional programs that drive gene expression in the island macrophage. Strikingly, the master erythroid regulator EKLF/Klf1 seems to also play a major role in specifying gene expression in island macrophages, including a putative EKLF/Klf1-dependent transcription circuit. Our present review and analysis of mouse single cell genetic patterns suggest novel expression characteristics that will enable a clear enrichment of EBI subtypes and resolution of island macrophage heterogeneity. Specifically, the discovery of markers such as Epor, and specific features for EKLF/Klf1-expressing island macrophages such as Sptb and Add2, or for SpiC-expressing island macrophage such as Timd4, or for Maf/Nr1h3-expressing island macrophage such as Vcam1, opens exciting possibilities for further characterization of these unique macrophage cell types in the context of their critical developmental function.

Mid-lateral cerebellar complex spikes encode multiple independent reward-related signals during reinforcement learning

Although the cerebellum has been implicated in simple reward-based learning recently, the role of complex spikes (CS) and simple spikes (SS), their interaction and their relationship to complex reinforcement learning and decision making is still unclear. Here we show that in a context where a non-human primate learned to make novel visuomotor associations, classifying CS responses based on their SS properties revealed distinct cell-type specific encoding of the probability of failure after the stimulus onset and the non-human primate’s decision. In a different context, CS from the same cerebellar area also responded in a cell-type and learning independent manner to the stimulus that signaled the beginning of the trial. Both types of CS signals were independent of changes in any motor kinematics and were unlikely to instruct the concurrent SS activity through an error based mechanism, suggesting the presence of context dependent, flexible, multiple independent channels of neural encoding by CS and SS. This diversity in neural information encoding in the mid-lateral cerebellum, depending on the context and learning state, is well suited to promote exploration and acquisition of wide range of cognitive behaviors that entail flexible stimulus-action-reward relationships but not necessarily motor learning.

TAMI-74. SPATIOTEMPORAL MULTI-OMIC LANDSCAPE OF HUMAN MEDULLOBLASTOMA AT SINGLE CELL RESOLUTION

Medulloblastoma is the most common malignant childhood tumor type with distinct molecular subgroups. While advances in the comprehensive treatment have been made, the mortality in the high-risk group is still very high, driven by an incomplete understanding of cellular diversity. Here we use single-nucleus RNA expression, chromatin accessibility and spatial transcriptomic profiling to generate an integrative multi-omic map in 40 human medulloblastomas spanning all molecular subgroups and human postnatal cerebella, which is supplemented by the bulk whole genome and RNA sequences across 300 cases. This approach provides spatially resolved insights into the medulloblastoma and cerebellum transcriptome and epigenome with identification of distinct cell-type in the tumor microenvironment. Medulloblastoma exhibited three tumor subpopulations including the quiescent, the differentiated, and a stem-like (proliferating) population unique to cancer, which localized to an immunosuppressive-vascular niche. We identified and validated mechanisms of stem-like to differentiated process among the malignant cells that drive tumor progression. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing stem-like malignant cells as a hub for intercellular communication. Multiple features of potential immunosuppression and angiogenesis were observed, including Treg cells and endothelial cells co-localization in compartmentalized tumor stroma. Collectively, our study provides an integrative molecular landscape of human medulloblastoma and represents a reference to advance mechanistic and therapeutic studies of pediatric neuro-oncological disease.

Identification of Adeno-Associated Virus Variants for Gene Transfer Into Human Neural Cell Types by Parallel Capsid Screening

Human brain cells generated by in vitrocell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant Adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, weharnessed a customizable parallel screening approach to assessa panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types.We identified common lead candidates suited for the transduction of directly converted,early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neuralprogenitors,as well as differentiated astrocytic and mixed neuroglial cultures.We then selected a subsetof these candidates for functional validation in iNSCs and iPSC-derived astrocytes, usingshRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview ofthe susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussionof the assets and limitations of the specific AAV capsidscreening approach.

Congenital hypothyroidism impairs spine growth of dentate granule cells by downregulation of CaMKIV

Congenital hypothyroidism (CH), a common neonatal endocrine disorder, can result in cognitive deficits if delay in diagnose and treatment. Dentate gyrus (DG) is the severely affected subregion of the hippocampus by the CH, where the dentate granule cells (DGCs) reside in. However, how CH impairs the cognitive function via affecting DGCs and the underlying mechanisms are not fully elucidated. In the present study, the CH model of rat pups was successfully established, and the aberrant dendrite growth of the DGCs and the impaired cognitive behaviors were observed in the offspring. Transcriptome analysis of hippocampal tissues following rat CH successfully identified that calcium/calmodulin-dependent protein kinase IV (CaMKIV) was the prominent regulator involved in mediating deficient growth of DGC dendrites. CaMKIV was shown to be dynamically regulated in the DG subregion of the rats following drug-induced CH. Interference of CaMKIV expression in the primary DGCs significantly reduced the spine density of dendrites, while addition of T3 to the primary DGCs isolated from CH pups could facilitate the spine growth of dendrites. Insights into relevant mechanisms revealed that CH-mediated CaMKIV deficiency resulted in the significant decrease of phosphorylated CREB in DGCs, in association with the abnormality of dendrites. Our results have provided a distinct cell type in hippocampus that is affected by CH, which would be beneficial for the treatment of CH-induced cognitive deficiency.

Single Cell Atlas of Human Putamen Reveals Disease Specific Changes in Synucleinopathies: Parkinson's Disease and Multiple System Atrophy

Understanding disease biology at a cellular level from disease specific tissues is imperative for effective drug development for complex neurodegenerative diseases. We profiled 87,086 nuclei from putamen tissue of healthy controls, Parkinson's Disease (PD), and Multiple System Atrophy (MSA) subjects to construct a comprehensive single cell atlas. Although both PD and MSA are manifestations of alpha-synuclein protein aggregation, we observed that both the diseases have distinct cell-type specific changes. We see a possible expansion and activation of microglia and astrocytes in PD compared to MSA and controls. Contrary to PD microglia, we found absence of upregulated unfolded protein response in MSA microglia compared to controls. Differentially expressed genes in major cell types are enriched for genes associated with PD-GWAS loci. We found altered expression of major neurodegeneration associated genes, SNCA, MAPT, LRRK2, and APP, at cell-type resolution. We also identified disease associated gene modules using a network biology approach. Overall, this study creates an interactive atlas from synucleinopathies and provides major cell-type specific disease insights.

Excitatory cholecystokinin neurons of the midbrain integrate diverse temporal responses and drive auditory thalamic subdomains

The central nucleus of the inferior colliculus (ICC) integrates information about different features of sound and then distributes this information to thalamocortical circuits. However, the lack of clear definitions of circuit elements in the ICC has limited our understanding of the nature of these circuit transformations. Here, we combine virus-based genetic access with electrophysiological and optogenetic approaches to identify a large family of excitatory, cholecystokinin-expressing thalamic projection neurons in the ICC of the Mongolian gerbil. We show that these neurons form a distinct cell type, displaying uniform morphology and intrinsic firing features, and provide powerful, spatially restricted excitation exclusively to the ventral auditory thalamus. In vivo, these neurons consistently exhibit V-shaped receptive field properties but strikingly diverse temporal responses to sound. Our results indicate that temporal response diversity is maintained within this population of otherwise uniform cells in the ICC and then relayed to cortex through spatially restricted thalamic subdomains.

Reversible switching of leukemic cells to a drug-resistant, stem-like subset via IL-4 mediated cross-talk with mesenchymal stroma

Chemoresistance of leukemic cells has largely been attributed to clonal evolution secondary to accumulating mutations. Here, we show that a subset of leukemic blasts in contact with the mesenchymal stroma undergo cellular conversion into a distinct cell type that exhibits a stem cell-like phenotype and chemoresistance. These stroma-induced changes occurred in a reversible and stochastic manner driven by cross-talk, whereby stromal contact induces IL-4 in leukemic cells that in turn targets the mesenchymal stroma to facilitate the development of new subset. This mechanism was dependent on IL-4 mediated up-regulation of vascular cell adhesion molecule-1 in mesenchymal stroma, causing tight adherence of leukemic cells to mesenchymal progenitors for generation of new subsets. Together, our study reveals another class of chemoresistance in leukemic blasts via functional evolution through stromal cross-talk, and demonstrates dynamic switching of leukemic cell fates that could cause a non-homologous response to chemotherapy in concert with the patientspecific microenvironment.

Use of DREADD Technology to Identify Novel Targets for Antidiabetic Drugs

G protein–coupled receptors (GPCRs) form a superfamily of plasma membrane receptors that couple to four major families of heterotrimeric G proteins, Gs, Gi, Gq, and G12. GPCRs represent excellent targets for drug therapy. Since the individual GPCRs are expressed by many different cell types, the in vivo metabolic roles of a specific GPCR expressed by a distinct cell type are not well understood. The development of designer GPCRs known as DREADDs (designer receptors exclusively activated by a designer drug) that selectively couple to distinct classes of heterotrimeric G proteins has greatly facilitated studies in this area. This review focuses on the use of DREADD technology to explore the physiological and pathophysiological roles of distinct GPCR/G protein cascades in several metabolically important cell types. The novel insights gained from these studies should stimulate the development of GPCR-based treatments for major metabolic diseases such as type 2 diabetes and obesity.