The results describe development in embryonic and early postnatal mouse tissue and do not establish the same lineage architecture in humans. The study, published in Nature Neuroscience, examined the embryonic mouse thalamus from embryonic day 11.5, near the onset of thalamic neurogenesis, through embryonic day 16.5 and selected postnatal stages. To obtain temporally restricted developmental cohorts, the team injected a fluorescent tracer into the third ventricle of embryos and collected the labeled tissue 12 hours later.

These short-chase cohorts included apical progenitors, intermediate progenitors and recently generated postmitotic neurons rather than serving as a strict birthdating measure. Paired single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing produced 25,477 high-quality profiles, while spatial transcriptomics at embryonic days 12 and 17 assigned molecular populations to anatomical territories. The combined datasets were intended to connect gene expression, regulatory accessibility, physical position and clonal history within the same developmental framework.

At embryonic day 11.5, progenitor populations were already partly segregated along the thalamic–prethalamic axis. A pan-radial glia-like population and three more specialized progenitor classes could be distinguished by their marker genes. One expressed Tcf7l2 and was assigned as a caudal glutamatergic progenitor population; another expressed Nkx2.2 and Arx and corresponded to prethalamic GABAergic progenitors; a third expressed Shh, Pitx2 and Sim2 and corresponded to progenitors of the zona limitans intrathalamica and rostral GABAergic territories.

Spatial transcriptomics placed these populations in partially separated domains. The glutamatergic and GABAergic programs therefore already showed an early transcriptional and spatial bias. Trajectory analysis divided the thalamic glutamatergic compartment into two major branches.

The first originated from one caudal thalamic progenitor subclass and was enriched for molecular signatures associated with sensory thalamic nuclei, including genes such as Gbx2, Ntng1, Kirrel3, Rora and Robo2. The second originated from another progenitor subclass and carried signatures associated with motor and associative nuclei, including Klhl1, Msi2, Kcnma1, Ebf1 and Robo1. Spatial analysis at embryonic day 17 confirmed anatomical separation: the sensory-related branch corresponded to ventrolateral domains containing the dLG, ventral posteromedial nucleus, ventral medial geniculate nucleus and ventral posterolateral nucleus, as well as signatures associated with anterior nuclei.

The motor-associative branch mapped to territories containing the ventral anterior, ventral lateral and ventromedial nuclei, together with the mediodorsal and central medial nuclei. Thus, the major functional division of the thalamus was linked to distinct progenitor origins and spatially restricted molecular identities rather than emerging only after neuronal maturation. The branches also differed in their developmental timing.

At embryonic day 11.5, both contained substantial progenitor and intermediate-precursor populations. At later stages, however, sensory-related cells shifted predominantly toward postmitotic neuronal states, while motor-associative cells retained comparatively large progenitor and intermediate populations. Higher inferred transcription-factor activity in the motor-associative branch was consistent with a comparatively immature specification state.

Integrating chromatin accessibility with gene expression revealed distinct regulatory modules: the sensory branch included Hes1, Mef2a, Rora and Rorb, whereas the motor-associative branch included Onecut1, Onecut2, Nr2f2, Foxp2, Ebf1 and Neurod1. Computational simulations that represented transcription-factor knockouts predicted lineage-specific effects of Hes1 and Neurod1 and changes in developmental velocity. Those results were model-based predictions, not outcomes from experimental knockouts, and the broader study did not directly test those two factors in vivo.

Within the sensory-related branch, the researchers found a second level of organization involving first-order and higher-order thalamic identities. Multimodal trajectory reconstruction showed a bifurcation from shared progenitors into transcriptionally distinct terminal states, and barcoded clonal tracing was used to test whether these identities had common origins. Labeling of apical progenitors at embryonic day 11.5 and analysis of 9,794 nuclei from the caudal thalamus at postnatal day 2 showed that many clones were transcriptionally unipotent, but a substantial minority contained cells from more than one state.

Higher-order and first-order identities were strongly coupled: 107 clones spanned higher-order and first-order VPM identities, and 57 spanned higher-order and first-order dLG identities. Only 19 clones contributed to both first-order types without also including a higher-order contribution. This pattern supports passage through a broadly permissive higher-order-like state rather than a precursor committed in advance to one first-order modality.

The timing of the sensory programs matched that lineage model. Embryonic day 11.5 cells showed little enrichment for either first-order- or higher-order-associated signatures. By embryonic day 14.5, higher-order-like signatures predominated, while first-order-like features appeared in 2.1% of the examined cohort.

By embryonic day 16.5, that fraction had increased to 17.6%, although higher-order-like programs remained prevalent. Clonal relationships also connected the mixed higher-order state with the lateral posterior, posterior, dLG and VPM territories. Direct coupling between the two first-order identities was rare, and defined lateral posterior and posterior populations had links to both first-order outcomes.

The findings therefore support a hierarchy in which an early higher-order-like program is established first and later resolves into separate visual and somatosensory trajectories, with additional regulatory changes consolidating each branch. Regulatory-network analysis identified different transcription-factor activities along those two sensory trajectories. Sp9, Zic1 and Zic4 were enriched along the visual branch leading to the dLG, whereas Nr2f1, Tshz1 and Rorb predominated along the somatosensory branch leading to the VPM.

Sp9 was initially detected in early thalamic progenitors and later became restricted to the developing dLG. Its promoter was accessible in developing dLG neurons but remained inaccessible in VPM neurons. The inferred network placed Sp9 upstream of genes including Gbx2, Zic1, Zic4, Pou2f2 and Lhx9, linking it to a visual-specific transcriptional program.

The investigators tested this candidate regulator by delivering a CRISPR interference construct targeting Sp9 into the embryonic thalamus at embryonic day 11.5 and examining the tissue at postnatal day 0. Compared with controls, Sp9-targeted embryos had a smaller dLG, fewer GFP-positive electroporated cells within the nucleus, a lower overall density of DAPI-positive cells and fewer Sp9-positive, GFP-positive cells per square millimeter. The reported unpaired two-tailed Student’s t-test P values were 0.01206 for dLG area, 0.0005356 for cell density and 0.001493 for the density of Sp9-positive targeted cells.

Single-nucleus RNA sequencing of sorted targeted cells showed a depletion of dLG neurons and a broader shift from first-order toward higher-order-associated programs. In separately microdissected dLG tissue, the remaining cells showed reduced expression of programs associated with RNA processing, protein translation and synapse organization, a pattern consistent with delayed maturation. Because perturbation did not directly target every cell in the nucleus, detectable Sp9 expression remained in the tissue, likely reflecting contributions from early-born neurons outside the targeted population.

Taken together, the mouse experiments identify Sp9 as a lineage-restricted regulator required for normal dLG formation and the consolidation of visual first-order identity, while the lineage relationships and functional consequences of the broader thalamic organization remain to be determined in other systems.

Subject of Research: Embryonic mouse thalamic lineage specification and sensory-motor regionalization

Article Title: Early lineage divergence segregates sensory and motor-associative identities in the thalamus

Article References: Puche-Aroca, L., Andrés-Bayón, B., Wilson, E. S., Javed, A., Torregrosa-Mira, A., Rodríguez-Malmierca, L. M., López-Atalaya, J. P., & López-Bendito, G. (2026). Early lineage divergence segregates sensory and motor-associative identities in the thalamus. Nature Neuroscience. https://doi.org/10.1038/s41593-026-02485-9

Image Credits: AI Generated

DOI: 10.1038/s41593-026-02485-9

Keywords: thalamus development, mouse neurodevelopment, lineage tracing, single-cell multiomics, spatial transcriptomics, sensory thalamus, motor thalamus, chromatin accessibility, Sp9 regulation, visual thalamus, Early, lineage