A team led by Jonathan M. Werner, Hamsini Suresh, Leon French, and Jesse Gillis, publishing in iScience, set out to determine whether the primate Pax6 interneuron subclass is a genuine primate-specific innovation or simply a mouse cell type hiding in plain sight under a different label. Their strategy was a sweeping meta-analysis of caudal ganglionic eminence (CGE) and medial ganglionic eminence (MGE) interneuron subclasses, drawing on data from the Allen Institute, the Broad Institute, Yale, and UT Southwestern. The collection spanned human, chimpanzee, gorilla, macaque, and marmoset alongside mouse, covering both 10x and Smart-seq platforms, single-cell and single-nucleus preparations, and 18 mouse and 3 primate brain regions. Using replicability-based cross-dataset alignment, the researchers re-evaluated subclass identity and then tested the resulting homologous groups against electrophysiological and spatial data.

The first clue came from an unexpected direction: the gene SNCG, the namesake of the Sncg interneuron subclass. In mouse, SNCG is a strong and reliable marker for the Sncg subclass. In primates, however, the team found that SNCG shows positive differential expression in MGE-derived rather than CGE-derived subclasses, a striking inversion confirmed independently in both the Yale and UT Southwestern primate datasets. Meanwhile, the mouse Sncg subclass showed mild positive differential expression of PAX6 itself. Taken together, these patterns hinted not at a lost mouse lineage but at a misannotation: the labels themselves may have drifted across species, even as the underlying cell types remained conserved.

To test that idea rigorously, the researchers examined how many marker genes are actually needed to anchor a subclass identity across species. Individual markers, they found, can be treacherous. Top single markers for subclasses such as Pvalb, Lamp5, Lamp5 Lhx6, and Sncg showed weak or inconsistent differential expression in their expected mouse counterparts. But when markers were aggregated, the picture changed dramatically: sets of just ten primate subclass markers in aggregate reliably recovered the expected trends in mouse. Most tellingly, primate Pax6 markers were more enriched in the mouse Sncg subclass than primate Sncg markers were, a result that directly supports a cross-species misannotation rather than a true loss of Pax6-like interneurons in the rodent brain.

The decisive step was a one-vs-best MetaNeighbor analysis, a stringent test that maps each mouse cluster to its best and next-best primate subclass matches and quantifies how well the cluster distinguishes its top match. The result was unambiguous: robust one-to-one matches emerged between mouse clusters and all nine primate interneuron subclasses, including Pax6. These homologous matches were supported by all eight mouse datasets and spanned all 18 brain regions examined. When the researchers compared the original mouse labels to their primate-aligned identities, the story crystallized: nearly all previously annotated mouse Sncg clusters actually align with the primate Pax6 subclass, along with subsets of Lamp5 and Vip clusters. Conversely, the mouse clusters most similar to the primate Sncg subclass were largely annotated as mouse Vip clusters. The homologous mouse Pax6 subclass, in other words, is largely composed of what had been called the mouse Sncg subclass, plus a PAX6-expressing subset of Lamp5 cells.

Crucially, the reassignment is not merely a relabeling exercise; it recovers real transcriptional structure. Under the homologous annotations, Pax6-labeled mouse clusters form a coherent, well-separated group, and the Vip and Lamp5 clusters that remap to Pax6 are more similar to each other than to the remaining Vip and Lamp5 clusters. The same held true for the homologous Sncg subclass, which forms a distinct outgroup among Vip clusters. The team also identified new marker genes that better capture subclass identity across species: FREM1 emerged as a high-performing marker for the homologous mouse Pax6 subclass, while SORCS3, MAML3, and CDH24 proved to be conserved Pax6 markers across primates and the combined primate-mouse analysis. For the Sncg subclass, CXCL14 was identified as the optimal cross-species marker, though its lack of specificity, showing strong signal for the Pax6 subclass as well, underscores how poorly conserved Sncg-defining markers really are.

The researchers then asked whether the reassigned Pax6 cells show distinctive properties beyond their transcriptomes. Using a large mouse Patch-seq dataset, they found that homologous labels explain slightly more electrophysiological variance than the original labels, though the improvement is modest compared to the near-global increase in explained gene-expression variance. Electrophysiological similarity alone collapsed onto a Vip-Lamp5 continuum, failing to cleanly organize Pax6, Sncg, or Lamp5-Lhx6 cells, a reminder that transcriptomic measurements currently outresolve electrophysiology for subclass-level distinctions. Still, one consistent signature emerged: Pax6 cells were distinguished from all other CGE subclasses by repolarization-related features, specifically lower rheobase spike width and a lower rheobase upstroke-downstroke ratio, indicating a slightly faster firing pattern. Spatial analyses of a whole-brain mouse atlas added further context, showing that MGE subclasses preferentially localize with deep-layer cell types and CGE subclasses with superficial-layer types, with both lineages including cortical and hippocampal clusters, though no subclass showed a sharply specific spatial niche.

Perhaps the most provocative twist of the study concerns the Sncg subclass itself. While Pax6 turned out to be conserved and mislabeled, Sncg showed the opposite profile: highly replicable across primate datasets from Yale and UT Southwestern, yet weakly matched in mouse, where its best homolog is a Vip outgroup. The pattern extends beyond rodents. In tree shrew, an evolutionary intermediate between rodents and primates, interneurons showed clear enrichment for primate Vip and Pax6 markers but only mouse, not primate, Sncg markers. Rat cortex told the same story, with the Sncg homolog corresponding to a Vip outgroup. Ortholog and paralog analyses added an evolutionary mechanism: human Vip outgroup clusters switch from expressing more Vip orthologs in non-human primates to more Sncg orthologs in non-primates, while human Vip and Sncg clusters converge on Vip paralogs relative to mouse. The authors propose that the primate Sncg subclass evolved from an ancestral Vip outgroup precursor, making it a primate-specific elaboration rather than a conserved mammalian subtype, a hypothesis that assessments of more distant species will need to confirm.

The broader message reaches well beyond one misplaced label. Cellular identity, the authors argue, is distributed across the transcriptome, and small marker gene sets that work well within a species can degrade rapidly with evolutionary distance. Very few genes maintain both strong signal-to-noise and broad coverage across multiple mammalian species, meaning individual genes can shift specificity without disrupting the broader transcriptional program that preserves a cell type. Aggregate transcriptional similarity, benchmarked by replicability across technologies, datasets, brain regions, and above all species, offers a far more stable foundation for defining and comparing cell types. The study also highlights a humbling gap: neither electrophysiology nor spatial mapping currently recapitulates the fine structure of transcriptomically defined CGE subclasses, and the authors argue that internal transcriptomic consistency should be the first benchmark for recognizing a cell type, with other modalities layered on afterward. As single-cell atlases balloon in scale, the misplaced mouse Pax6 interneuron stands as a vivid warning that names can mislead, and that the deepest truths about brain cell diversity may only emerge when species are allowed to speak to one another through their full transcriptomes.

Subject of Research: Cross-species transcriptomic reassignment of the Pax6 and Sncg cortical interneuron subclasses in mouse and primates

Article Title: The misplaced mouse Pax6 interneuron subclass: A cross-species transcriptomic reassignment

Article References: The misplaced mouse Pax6 interneuron subclass: A cross-species transcriptomic reassignment. (n.d.). https://doi.org/10.1016/j.isci.2026.117632

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117632

Keywords: Pax6, interneurons, single-cell transcriptomics, cross-species comparison, cell-type taxonomy, Sncg, caudal ganglionic eminence, marker genes, brain evolution, Patch-seq, spatial transcriptomics, mouse brain atlas

The research, led by Justin Waletich, Danielle de Jong, and Christine E. Schnitzler of the University of Florida’s Whitney Laboratory for Marine Bioscience and Department of Biology, set out to interrogate eight newly identified marker genes drawn from a previously generated single-cell atlas of the organism. Their central finding is striking: i-cells do not constitute a homogeneous population spread evenly across the colony. Instead, they exhibit both transcriptional and morphological variation that shifts depending on where cells reside in the animal and what biological situation the animal faces, whether routine growth, tissue maintenance, or the dramatic demands of head regeneration.

Hydractinia occupies a special place in the modern study of adult stem cells. Unlike the stem cell compartments of mammals, which are typically restricted to producing specific tissue types, i-cells are definitively pluripotent, capable of generating both somatic lineages such as nerve cells and nematocytes, the stinging cells characteristic of cnidarians, as well as the germline. Earlier single-cell RNA sequencing work had produced a cellular atlas of the organism and revealed two distinct clusters of cells carrying i-cell signatures: one branch leading to somatic cell fates and another committed to germ cell fate. That atlas provided the roadmap, but the identity and behavior of specific genes distinguishing these clusters remained largely uncharted territory.

To fill that gap, the team selected eight marker genes for detailed characterization. Five of them, Pcna, Nop58, Mcm4, Ubr7, and Uhrf1, showed expression in both i-cell clusters, suggesting roles that are broadly shared across the stem cell compartment. The remaining three genes were more selective: Pter and FoxQ2-like were associated specifically with the somatic i-cell cluster, while Zcwpw1 marked the germ i-cell cluster. Several of these genes have well-known functions in other systems. Pcna and Mcm4 are canonical components of the cell cycle machinery, involved in DNA replication and proliferation, while Uhrf1 is a chromatin-associated protein linked to DNA methylation maintenance, hinting at epigenetic regulation within the stem cell pool.

Characterizing where and when these genes are active required a combination of spatial and temporal approaches. The researchers mapped expression patterns across multiple tissue locations within the colony, from the feeding polyps that capture prey to the stolons, the cable-like structures that connect individual polyps and allow the colony to grow. They then extended the analysis to a demanding biological challenge: head regeneration in feeding polyps following decapitation. By tracking marker expression at different time points after amputation, measured in hours post-decapitation, the team could watch how different subsets of i-cells responded to injury and whether specific subpopulations were recruited into the regenerative blastema, the mass of proliferating cells that rebuilds the lost head.

The results painted a nuanced picture of context dependence. Certain marker genes were expressed consistently in the stolons of young colonies, while the same genes showed variable expression across different developmental stages of feeding polyps, from budding polyps through immature to fully mature individuals. During head regeneration, some markers were clearly expressed within the blastema and the body column, whereas others were conspicuously absent from the regenerating tissue. This divergence indicates that the two i-cell clusters identified in the atlas are not merely computational artifacts but correspond to biologically meaningful subpopulations with distinct spatial distributions and distinct responses to injury.

Morphology added yet another layer of complexity. The researchers examined cell diameters of cells marked by Histone H1.1 expression across feeding polyp developmental stages and in the stolons of young colonies, and found statistically significant differences among these contexts. In other words, i-cells do not just differ in what genes they express; they also differ in physical form depending on where they are and what stage the surrounding tissue is in. This pairing of transcriptional and morphological heterogeneity strengthens the conclusion that the stem cell compartment of Hydractinia is a mosaic of specialized states rather than a single interchangeable population.

The implications of this work extend well beyond a single unusual organism. Adult pluripotent stem cells are rare in the animal kingdom, found most prominently in invertebrates with exceptional regenerative abilities. Understanding how such cells are organized, how they maintain pluripotency, and how subsets are channeled toward somatic versus germline fates speaks to fundamental questions of developmental biology and stem cell biology more broadly. By providing a set of validated marker genes that discriminate between somatic and germ i-cell clusters, the study hands future researchers precise molecular tools to isolate, track, and manipulate specific stem cell subpopulations in living animals.

There are also potential echoes for regenerative medicine. Mammalian stem cells, whether in the intestine, skin, or blood, are increasingly recognized as heterogeneous, with subpopulations occupying distinct niches and displaying distinct transcriptional states that influence their behavior in homeostasis and injury repair. The Hydractinia data reinforce a growing principle: stem cell identity is not fixed but is shaped by spatial location and biological context. Although no one expects to transplant cnidarian i-cells into human patients, the mechanistic lessons about how a pluripotent compartment is maintained and deployed during regeneration could inform strategies for engineering regenerative responses in clinically relevant tissues.

The authors emphasize that their data provide important nuances for understanding Hydractinia’s remarkable adult stem cell population and a solid foundation for future research. The study was supported by the National Institutes of Health through grant R35GM138156 awarded to Christine Schnitzler. As open-access research, the work, including its supplementary figures, gene tables, and phylogenetic analyses, is freely available to the scientific community. What began as an effort to validate new markers has ended up redrawing the map of a stem cell system, replacing the comforting image of a uniform pluripotent pool with something richer and more intriguing: a living landscape of stem cell states, each tuned to its place in the colony and to the tasks the animal must perform.

Subject of Research: Transcriptional heterogeneity of adult pluripotent interstitial stem cells in the colonial cnidarian Hydractinia symbiolongicarpus

Article Title: Context-dependent transcriptional heterogeneity of new markers expands our understanding of stem cells in Hydractinia

Article References: Waletich, J., de Jong, D., & Schnitzler, C. E. (2026). Context-dependent transcriptional heterogeneity of new markers expands our understanding of stem cells in Hydractinia. BMC Biology. https://doi.org/10.1186/s12915-026-02736-y

Image Credits: AI Generated

DOI: 10.1186/s12915-026-02736-y

Keywords: Hydractinia, cnidarians, stem cells, i-cells, pluripotency, regeneration, single-cell RNA sequencing, marker genes, transcriptional heterogeneity, head regeneration, germline, BMC Biology