Thalamic NRXN1-mediated input to human cortical progenitors drives excitatory neurogenesis

The human cerebral cortex develops through coordinated signals from within the cortex and from other brain regions, including the thalamus. However, how thalamic neuronal projections influence early human cortical development remains less well-understood. In this study, we fused cortical and thalamic organoids to investigate how thalamic input shapes the maturation of human cortical cells. Using single-nuclei RNA-sequencing and cellular imaging, we found that thalamic input increases the production of cortical excitatory neurons. We identify neurexin-1 (NRXN1) as a mediator of physical contact between thalamic axons and cortical outer radial glia. Genetic knockout of thalamic NRXN1 reduced these contacts and attenuated the production of upper-layer excitatory neurons. These findings reveal a mechanism by which thalamic input regulates human cortical progenitors and shapes excitatory neuron production during development. , Editor’s summary The mechanisms underlying the formation of cortical layers during cerebral cortex development (corticogenesis) remain to be fully elucidated. Nguyen et al . used human corticothalamic assembloids to determine the role of extrinsic inputs from thalamic neurons in corticogenesis. The authors suggest that thalamic axons promote the proliferation of outer radial glial cells and increase the number of excitatory neurons generated in cortical organoids compared with organoids grown without thalamic inputs. They also provide evidence that this process is mediated by neurexin 1, a cell-adhesion molecule with genetic variation that is associated with neurodevelopmental conditions. The results indicate that extrinsic inputs might be essential for the proper development of the human cerebral cortex. —Mattia Maroso , INTRODUCTION Thalamocortical afferents (TCAs) influence cortical arealization, laminar specification, and maturation, but how extrinsic thalamic inputs shape human cortical neurogenesis remains poorly understood. In primates, cortical development includes expanded progenitor populations such as outer radial glia (oRG), early thalamic innervation, and prolonged upper-layer excitatory neurogenesis. Because these features are difficult to disentangle in vivo, the mechanisms by which thalamic signals regulate progenitor behavior and cortical cell fate have remained unclear. RATIONALE Human cortical development offers an extraordinary opportunity to study how TCAs influence neurogenesis because projections arrive during earlier stages of corticogenesis than they do in rodents. This is a period when human cortical progenitors, including oRG, are still actively shaping cortical expansion and upper-layer neurogenesis. This temporal overlap suggests that thalamic input may have a direct role in regulating progenitor behavior in humans. However, these early developmental interactions are difficult to dissect in vivo, and even in animal models, severing TCAs is not always complete. Human stem cell–derived assembloids provide a tractable platform to model these events. This system enables controlled comparison of cortical tissue with or without thalamic input and allows manipulations difficult to perform in vivo, including timed fusion, severance of axonal connections, lineage tracing, and targeted genetic perturbation. RESULTS Thalamic fusion increased cortical excitatory neuron production, with expansion of upper-layer special AT-rich sequence-binding protein 2 (SATB2+), cut-like homeobox 1 (CUX1+), and POU class 3 homeobox 2 (BRN2+) populations and modest increases in deep-layer neuronal markers. These changes were accompanied by early increases in progenitor proliferation and, by week 12, a reciprocal reduction in HOPX+ oRG, suggesting enhanced neurogenic output. Single-nuclei RNA-sequencing confirmed increased upper- and deep-layer excitatory neuron abundance and showed that thalamic input induced programs associated with neuronal maturation, laminar specification, and excitatory differentiation, while radial glia exhibited signatures consistent with increased neurogenic commitment. Temporal severance experiments demonstrated that early thalamic input was sufficient to preserve the upper-layer neurogenesis phenotype, indicating a critical window during which thalamic signals durably alter cortical trajectories. 5-ethynyl-2′-deoxyuridine (EdU) birth dating showed that increased SATB2+ neuron numbers were not explained by earlier neuronal birth, supporting a change in progenitor fate behavior rather than timing. Lineage tracing further showed that thalamic input increased clonal partnerships between oRG and excitatory neurons, indicating that thalamic afferents bias oRG toward excitatory neurogenesis. Mechanistically, receptor-ligand analysis identified thalamic neurexin-1 (NRXN1) and cortical neuroligin-1 (NLGN1) as mediators of physical thalamocortical-progenitor interactions. Super-resolution imaging and analysis of primary mid-gestation human cortex demonstrated NRXN1-NLGN1–associated contact between thalamic afferents and HOPX+ oRG in the outer subventricular zone. Transcellular tracing showed communication from thalamic neurons to cortical progenitors, including oRG. Genetic loss of NRXN1 in thalamic organoids reduced transcellular contact with oRG and upper-layer neuron production but increased radial glia abundance and shifted radial glial gene programs away from neurogenic maturation, establishing thalamic NRXN1 as a mediator of cortical progenitor fate. CONCLUSION Our study demonstrates how a physical connection between thalamocortical afferents and cortical radial glia promote shifts in oRG cell fate specification, opening numerous future directions for further interrogation of broader neuronal-progenitor interactions in the developing human cortex and in neurodevelopmental disorders. Thalamocortical (CX-TH) assembloids were compared to corticocortical controls (CX-CX) to interrogate extrinsic thalamic signaling on cortical fate specification during development. We found that thalamic input is sufficient to drive excitatory neurogenesis. This phenotype is partly driven by noncanonical NRXN1-mediated interactions between thalamic axons and cortical radial glia. Expansion of upper-layer neurons was retained when assembloids were severed during mid-neurogenesis, but both deep and upper layers were attenuated by thalamic NRXN1 knockout.