Marlene M. Hao

The vertebrate nervous system develops into functionally distinct domains along the body axis ¹ . Developmental studies implicate multiple signaling pathways in coordinating this process, but the order of specification events, sufficiency of individual signals, and molecular basis of competency remain unclear ^2,3 , leaving the field without a unified model of neuraxis patterning. Enteric nervous system (ENS) and midbrain dopaminergic (mDA) progenitors have reached clinical and preclinical testing ^4–11 , yet neither is produced at full purity, and yields vary markedly across human pluripotent stem cell (hPSC) lines, reflecting incomplete control of axial identity. Here we use single-cell genetic fate mapping to demonstrate that hPSCs can commit to anterior or posterior cranial fate within the first 24 hours of differentiation, before neural induction. Building on this finding, we show that the duration of a single saturating WNT pulse is sufficient to specify three “first-order” domains: anterior cranial (0 h), posterior cranial (24 h) and trunk (48 h), each pre-neural and competent for CNS, neural crest and epidermal lineages. Nested “second-order” domains are specified within first-order domains in response to WNT, FGF, and retinoic acid. Mechanistically, an anterior gene regulatory network repressing WNT targets is irreversibly shut down during the transition to posterior identities. Applying this binary WNT patterning logic, we generate mDA neurons and ENS progenitors robustly across hPSC lines and establish an assembloid model combining first-order progenitors to produce all major regions of the nervous system.

Tumor-associated macrophages (TAMs) are key mediators of tumor immunosuppression, yet the factors governing their polarization remain poorly understood, especially in highly immunosuppressive cancers, including cancers affecting the central nervous system. This study investigates the molecular pathways underlying TAM polarization in glioblastoma, one of the most immunosuppressive cancer types. Using a multi-omics approach integrating spatial proteomics, RNA-sequencing, and proteomic profiling of tumor cells and macrophages, we demonstrate that circulating monocytes polarize toward an immunosuppressive state when they exit tumor blood vessels, in response to glioblastoma cell-secreted cytokines. In situ and in vitro data shows that macrophage polarization is regulated via the cAMP-CREB signaling-transcription axis.

The enteric nervous system (ENS), the largest division of autonomic nervous system, is a tantalizing frontier in neuroscience. With the advent of single-cell transcriptomics, the ENS has been increasingly well-characterized. Precise functional mapping of enteric neuron diversity is critical for understanding ENS biology and disease, but technical barriers remain. We used different approaches to compare and contrast functional annotations of several independently-reported ENS datasets. Differential module scoring, co-expression and correlation analysis, unbiased biological function hierarchical clustering, data integration and label transfer highlighted substantial discrepancies stemming from an overreliance on transcriptomics data without adequate tissue validations. For understanding enteric neurons’ functional identity, it is imperative to expand tissue sources and incorporate technologies such as multiplexed imaging, electrophysiology, spatial transcriptomics, as well as comprehensive epigenome, proteome, and metabolome profiling. Harnessing human pluripotent stem cell models provides unique opportunities for ENS lineage tracing and offers unparalleled scalability and amenability to genetic and functional screens. We encourage a paradigm shift in our comprehension of ENS cellular and functional complexity by calling for large-scale collaborations and research investments.

Neuronal activity in the enteric nervous system changes during the 24-h day/night cycle, with increased neuronal function detected at night when mice are feeding and active. However, following an acute fast, neuronal sensitivity becomes more pronounced during the day. These changes in neuronal function did not correlate with changes in neurotransmitter receptor gene expression levels.

Enteric glial cells (EGCs) are an essential component of the enteric nervous system (ENS) and play key roles in gastrointestinal development, homeostasis, and disease. Derived from neural crest cells, EGCs undergo complex differentiation processes regulated by various signalling pathways. Being among the most dynamic cells of the digestive system, EGCs react to cues in their surrounding microenvironment and communicate with various cell types and systems within the gut. Morphological studies and recent single cell RNA sequencing studies have unveiled heterogeneity among EGC populations with implications for regional functions and roles in diseases. In gastrointestinal disorders, including inflammatory bowel disease (IBD), infections and cancer, EGCs modulate neuroplasticity, immune responses and tumorigenesis. Recent evidence suggests that EGCs respond plastically to the microenvironmental cues, adapting their phenotype and functions in disease states and taking on a crucial role. They exhibit molecular abnormalities and alter communication with other intestinal cell types, underscoring their therapeutic potential as targets. This review delves into the multifaceted roles of EGCs, particularly emphasizing their interactions with various cell types in the gut and their significant contributions to gastrointestinal disorders. Understanding the complex roles of EGCs in gastrointestinal physiology and pathology will be crucial for the development of novel therapeutic strategies for gastrointestinal disorders.

Glutamate is the major excitatory neurotransmitter in the central nervous system, and there is evidence that Group-I metabotropic glutamate receptors (mGlu1 and mGlu5) have established roles in excitatory neurotransmission and synaptic plasticity. While glutamate is abundantly present in the gut, it plays a smaller role in neurotransmission in the enteric nervous system. In this study, we examined the roles of Group-I mGlu receptors in gastrointestinal function. We investigated the expression of Grm1 (mGlu1) and Grm5 (mGlu5) in the mouse myenteric plexus using RNAscope in situ hybridization. Live calcium imaging and motility analysis were performed on ex vivo preparations of the mouse colon. mGlu5 was found to play a role in excitatory enteric neurotransmission, as electrically-evoked calcium transients were sensitive to the mGlu5 antagonist MPEP. However, inhibition of mGlu5 activity did not affect colonic motor complexes (CMCs). Instead, inhibition of mGlu1 using BAY 36-7620 reduced CMC frequency but did not affect enteric neurotransmission. These data highlight complex roles for Group-I mGlu receptors in myenteric neuron activity and colonic function.