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Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies. Single-cell and single-nucleus RNA sequencing are used to construct a cellular atlas of the human heart that will aid further research into cardiac physiology and disease.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates 1 , 2 . Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development 3 . Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells 4 . We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning 5 in the hypothalamus. In particular, loss of SLIT–ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life. Single-cell RNA sequencing reveals molecular determinants of the developmental programs that orchestrate the intermingling of neuronal subtypes in the hypothalamus.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood–brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50–100% of mice. Our study identifies a promising multi-targetable PTP4A–ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.

The SLITs (SLIT1, SLIT2, and SLIT3) are a family of secreted proteins that mediate positional interactions between cells and their environment during development by signaling through ROBO receptors (ROBO1, ROBO2, ROBO3, and ROBO4). The SLIT/ROBO signaling pathway has been shown to participate in axonal repulsion, axon guidance, and neuronal migration in the nervous system and the formation of the vascular system. However, the role of the SLIT/ROBO pathway has not been thoroughly clarified in tumor development. The SLIT/ROBO pathway can produce both beneficial and detrimental effects in the growth of malignant cells. It has been confirmed that SLIT/ROBO play contradictory roles in tumorigenesis. Here, we discuss the tumor promotion and tumor suppression roles of the SLIT/ROBO pathway in tumor growth, angiogenesis, migration, and the tumor microenvironment. Understanding these roles will help us develop more effective cancer therapies.

Angiogenesis is a critical, fine-tuned, multi-staged biological process. Tip-stalk cell selection and shuffling are the building blocks of sprouting angiogenesis. Accumulated evidences show that tip-stalk cell selection and shuffling are regulated by a variety of physical, chemical and biological factors, especially the interaction among multiple genes, their products and environments. The classic Notch-VEGFR, Slit-Robo, ECM-binding integrin, semaphorin and CCN family play important roles in tip-stalk cell selection and shuffling. In this review, we outline the progress and prospect in the mechanism and the roles of the various molecules and related signaling pathways in endothelial tip-stalk cell selection and shuffling. In the future, the regulators of tip-stalk cell selection and shuffling would be the potential markers and targets for angiogenesis.

Slit/Robo signals were initially found to play an essential role in nerve development as axonal guidance molecules. In recent years, with in-depth study, the role of Slit/Robo in other life activities, such as tumor development, angiogenesis, cell migration, and bone homeostasis, has gradually been revealed. Bone is an organ with an active metabolism. Bone resorption and bone formation are closely related through precise spatiotemporal coordination. There is much evidence that slit, as a new bone coupling factor, can regulate bone formation and resorption. For example, Slit3 can promote bone formation and inhibit bone resorption through Robo receptors, which has excellent therapeutic potential in metabolic bone diseases. Although the conclusions of some studies are contradictory, they all affirm the vital role of Slit/Robo signaling in regulating bone metabolism. This paper reviews the research progress of Slit/Robo signaling in bone metabolism, briefly discusses the contradictions in the existing research, and puts forward the research direction of Slit/Robo in the field of bone metabolism in the future.

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions. VEGF-A/VEGFR2 signaling is a key driver of endothelial cell migration during sprouting angiogenesis. Here Genet et al. show that endophilin A2 regulates these processes by mediating clathrin-independent VEGFR2 internalization.

During nervous system development, growing axons find their targets with the help of guidance cues. These cues, which can be secreted molecules provided by neighbouring cells or transmembrane proteins mediating cell–cell contacts with the growing axons, act as either chemoattractants or chemorepellents. Over the last decades, several axon guidance molecules have been identified. One of the classical guidance cues is the Slit protein. Slit is a secreted protein, initially identified in a genetic screen in the fruit fly Drosophila melanogaster but later shown to be present in other organisms including vertebrates. Slit was originally classified as a repellent guidance cue, but nowadays it is recognized as a promoter of axonal growth in some contexts. Slit action is mediated mainly by the Roundabout (Robo) family of single pass transmembrane proteins, although it has been shown more recently that other proteins can also function as Slit receptors. In this review, we describe the main aspects of Slit–Robo signalling during development of the nervous system. We start with a historical view of the discovery of these proteins, followed by a description of their main molecular characteristics. We then explore specific examples that describe the functions and signal transduction mechanisms of this signalling pathway.

Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1 Cre ;Robo2 F/F ) show increased activation of Robo1 + myofibroblasts and induction of TGF-β and Wnt pathways. During pancreatitis, Pdx1 Cre ;Robo2 F/F mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-β inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2 low ;ROBO1 high patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-β inhibitors or other stroma /immune modulating agents. SLIT-ROBO alterations arise in pancreatic ductal adenocarcinoma (PDAC), but their role in the pancreas is unclear. Here, the authors use mouse models to show that loss of epithelial Robo2 activates the neighbouring stroma via TGF-β signalling; findings  are relevant to PDAC patients, where ROBO expression correlates with survival outcomes.

The formation of the neocortex relies on intracellular and extracellular signaling molecules that are involved in the sequential steps of corticogenesis, ranging from the proliferation and differentiation of neural progenitor cells to the migration and dendrite formation of neocortical neurons. Abnormalities in these steps lead to disruption of the cortical structure and circuit, and underly various neurodevelopmental diseases, including dyslexia and autism spectrum disorder (ASD). In this review, we focus on the axon guidance signaling Slit-Robo, and address the multifaceted roles of Slit-Robo signaling in neocortical development. Recent studies have clarified the roles of Slit-Robo signaling not only in axon guidance but also in progenitor cell proliferation and migration, and the maturation of neocortical neurons. We further discuss the etiology of neurodevelopmental diseases, which are caused by defects in Slit-Robo signaling during neocortical formation.